Engineering Extracellular Vesicle Production through Magnetic Ion Channel Activation for Bone Regeneration

Bone disorders represent a significant global health challenge. Extracellular vesicles (EVs) are emerging as a promising nanotherapeutic approach for bone regeneration, addressing the translation barriers associated with cell-based therapies. Despite their immense potential, the clinical application of EVs is limited by low production yields and inconsistent quality. Magnetic Ion Channel Activation (MICA) utilises remote magnetic fields to stimulate mechano-sensitive ion channels through magnetic nanoparticles (MNPs). This study explores the potential of utilising MICA to enhance the production yield and therapeutic efficacy of EVs for bone regeneration. The findings demonstrate that MICA significantly increased the production yield of EVs from MC3T3 pre-osteoblasts compared to magnetic stimulation or TREK1 functionalised graphene oxide-MNP particles alone. The obtained EVs exhibited typical size distribution, morphology, and EV protein expression consistent with nano-sized vesicles. Furthermore, MICA/TREK EVs treatment considerably enhanced human bone marrow-derived mesenchymal stem cells osteogenic differentiation and mineralisation compared to EVs derived from MICA, TREK, or untreated groups. Proteomics analysis revealed the enrichment of proteins involved in mechanotransduction and osteogenic differentiation within MICA/TREK EVs. In summary, these findings highlight the substantial potential of MICA as a platform to enhance the scalable production and therapeutic application of pro-regenerative EVs for bone augmentation strategies.

extracellular vesicles, magnetic nanoparticles, nanomedicine, mechanotransduction, osteogenesis, bioengineering (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint 1. Introduction Bone-related disorders, including traumatic injuries, osteoporosis and tumour resection defects play a significant clinical and socioeconomic burden globally. Osteoporosis is a highly prevalent disease and results in massive costs both to the individual and to society through associated fragility fractures. An estimated 10 million people over the age of 50 years have osteoporosis, and around 1.5 million fragility fractures occur in these patients each year[1]. Osteoporosis treatments aim to strengthen bones and reduce fracture risk, often involving medications like bisphosphonates, denosumab, or anabolic agents, alongside lifestyle changes like exercise and a calcium and vitamin D-rich diet. Current treatments often fail to fully restore bone mass and function and are focused on slowing down bone loss rather than stimulating new bone formation in critical areas such as the vertebrae. This highlights the urgent need for advanced therapeutic strategies that address restoring bone biological function following regeneration[2-4]. Increasing evidence has demonstrated the importance of cell-derived bioactive molecules in facilitating cellular communication and modulating diverse biological processes[5-7]. Among these bioactive factors, extracellular vesicles (EVs) have emerged as vital mediators of intracellular communication, playing pivotal roles in a variety of physiological and pathological processes including bone regeneration and immune modulation[8, 9]. EVs are cell-secreted lipid nanoparticles enriched with bioactive molecules such as proteins, nucleic acids and metabolites making them promising candidates for nanotherapeutic applications, especially in regenerative medicine[10, 11]. EV-based therapeutics offer significant advantages to traditional cell-based therapies including reduced immunogenicity, improved stability and the ability to cross biological membranes (i.e. blood-brain barrier)[12]. However, despite their potential, the translation of EV-based therapies to the clinical arena is (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint hindered due to their inherent therapeutic potency, low and variable production yield and unreliable manufacturing methods [13, 14]. Conventional EV production strategies including genetic modifications, application of external forces and the use of chemical reagents often suffer from limitations such as scalability issues, cellular stress and even compromised EV bioactivity[15, 16]. Hence, there is a significant unmet need to refine the culture conditions to enhance EV therapeutic potency and yield for bone augmentation strategies. Recent developments in mechanobiology and bioengineering have given new opportunities for EV production, especially with ion channel activation emerging as a potential target for modulating cellular behaviour[17] and EV production [18]. Ion channels play a crucial role in maintaining cellular haemostasis by mediating the ion flux across the cell membranes and hence regulating the intracellular signalling pathways[19]. Mechano-sensitive ion channels are responsive to external mechanical stimuli providing a unique opportunity to modulate cellular activity and EV production [20-22]. However, approaches using chemical inducers or random mechanical forces can often result in inconsistent cell stimulation and inducing cell stress, ultimately detrimentally impacting the production of therapeutic EVs[23, 24]. Therefore, a targeted and reproducible method for ion channel activation is critical for improving EV yield and its bioactivity[25-27]. Magnetic ion channel activation (MICA) represents an innovative approach to improve EV production. Magnetic nanoparticles (MNPs) under remote magnetic fields offer a non- invasive and controllable means of stimulating mechano-sensitive ion channels to trigger cellular processes from outside the body [26, 28]. MICA is well aligned with scalable platforms such as bioreactor technologies used for EV production. Previously, we reported that TREK1 functionalised Graphene oxide-MNPs (TGMNPs) resulted in enhanced MSc differentiation into bone cells with enhanced calcification and bone matrix production in vitro. In addition, MICA-induced enhancement of bone growth has been demonstrated in vivo (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint in rodent and sheep models[29]. MICA can be fine-tuned to deliver consistent stimuli across large cell populations, addressing one of the primary limitations of current EV production techniques. Moreover, this non-invasive approach also facilitates real-time monitoring and control ensuring high yields of functional EVs suitable for clinical applications. This study investigates the potential of harnessing MICA as a novel strategy to enhance the production and therapeutic potency of EVs for bone regeneration. TGMNPs in conjunction with MICA were used to culture pre-osteoblasts and EVs were obtained from the conditioned medium and then characterised. EVs from MICA/TREK stimulated osteoblasts were administered to human bone marrow-derived mesenchymal stem cells (hBMSCs) to evaluate their osteogenic potency (Figure 1). Moreover, proteomics analysis was conducted to elucidate the mechanisms by which the MICA EVs impart their pro-osteogenic function. By bridging the gap between mechanobiology and regenerative medicine, this approach has the potential to revolutionise the EV manufacturing process and pave the way for next-generation EV-based therapies. Figure 1. Experimental overview (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint 2. Materials and Methods Cell culture and reagents MC3T3 pre-osteoblasts and hBMSCs were obtained from American Type Culture Collection (A TCC, UK) and Lonza (Lonza, UK) respectively. Basal culture media consisted of minimal essential medium ( α -MEM; Sigma-Aldrich, UK) supplemented with 10% foetal bovine serum (FBS), 1% penicillin/streptomycin (Sigma-Aldrich, UK) and L-glutamine (Sigma- Aldrich, UK). hBMSCs were used in passage 4. The mineralisation medium was comprised of basal culture media supplemented with 10 mM β -glycerophosphate (Sigma-Aldrich, Gillingham, UK), 50 μ g/mL L-ascorbic acid (Sigma-Aldrich, Gillingham, UK) and 100 nM Dexamethasone (Sigma- Aldrich, Gillingham, UK). The culture medium utilised for EV isolation and dosing was depleted of FBS-derived EVs by ultracentrifugation at 120,000 g for 16 hr prior to use. GO-MNP synthesis and characterisation GO-MNPs were synthesised as described elsewhere[17]. The synthesised GO-MNPs were functionalised with TREK1 antibody (Almone labs, APC-047) as mentioned previously[30]. The prepared TGMNPs were modified with 1 μ L of DOTAP (1,2-Dioleoyl-3- trimethylammonium propane) to enhance the internalisation and avoid particle agglomeration[31]. TGMNPs (25 µg) were added to cells and to provide magnetic stimulation to the cells, a custom-designed MICA bioreactor (MICA Biosystems, West Midlands, UK) was kept in an incubator maintained at 37 /i4 °C with 5% CO /i4 . Additionally, control groups including those without MICA, without TGMNPs, and with TGMNPs alone were maintained under identical incubation conditions to study the MICA-mediated osteogenic inductive studies. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint The X-ray diffraction (XRD) characterisation of MNPs and GO-MNPs was assessed using Bruker D2 phaser equipped with a cobalt source of wavelengths Ka1 1.7890 and Ka2 1.7929, step size 0.02028 o. The Raman spectroscopy measurements were recorded using Renishaw inVia and the data was collected using a 633nm laser beam. The magnetic properties of the MNPs and GOMNPs were evaluated using a model 10vector VSM with a maximum field of 20kOe and sensitivity of 1x10 -6emu at room temperature. MICA-induced osteogenesis MC3T3 (4 x 10 4) cells were seeded in osteogenic media in 24 well plates to assess the osteogenic potential of the TGMNPs. After 24 h, TGMNPs (25 µg) were added to each well and the cells were subjected to magnetic stimulation for 1h every day with a media change every 2 days. Following 3 and 7 daily treatments of magnetic stimulation, the ALP enzymatic activity was evaluated using the Sensolyte ALP assay kit. Briefly, the cells were lysed using 0.1 % Triton X. After 10 min, the cells were gently scraped and centrifuged at 2500g for 10 min at 4°C to separate cellular debris from the enzymatic supernatant. Afterwards, 50 µL of supernatant and 50 μ l p-nitrophenyl phosphate solution was combined in each well of a 96- well plate and gently shaken for 30 s. Following 1h of incubation, the absorbance was measured at 405 nm using the microplate reader. The ALP concentration was then evaluated using a standard curve against known protein concentrations. Additionally, the total protein concentration was assessed using the BCA Protein Assay Kit (Thermo Scientific, USA). EV isolation Osteoblasts were cultured at scale in 6 well plates (Sarstedt, UK) and the medium was collected every two days. TGMNPs (25 µg) were added to cells and were subjected to magnetic stimulation for 1h every day. EVs were isolated from the conditioned medium from the following groups: Untreated cells (CTL EVs), MICA stimulated cells (MICA EVs), (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint TGMNPs stimulated cells (TREK EVs) and MICA stimulated TGMNPs cells (MICA/TREK EVs). EVs were obtained from the conditioned medium by differential ultracentrifugation as previously described [32]: 2000 g for 20 min, 10,000 g for 30 min and 120,000 g for 70 min to pellet EVs. The supernatant was removed, and the pellet was washed in sterile PBS and centrifuged at 120,000 g for 70 min and the resultant pellet was re-suspended in 200 μ l PBS. Ultracentrifugation was performed with the Sorvall WX Ultra Series Ultracentrifuge (Thermo Scientific, UK) and a Fiberlite, F50L-8×39 fixed angle rotor (Piramoon Technologies Inc., USA). EV Particle Size, Concentration and Tetraspanin Analysis To determine the EV particle size, concentration and tetraspanin content, flow cytometry was conducted as previously described [33]. A NanoAnalyzer U30 (SPCM APDs) was used for the detection of side scatter (SSC) and fluorescence of individual particles. Measurements were taken over a 1-minute interval at a sampling pressure of 1.0 kPa, maintained by an air- based pressure module. Particle count was diluted to remain within the optimal range of 2000 -12,000/min. The concentration of samples was determined by comparison to 250 nm silica nanoparticles of known concentration to calibrate the sample flow rate. EV isolates were sized according to standard operating procedures using a proprietary 4-modal silica nanosphere cocktail (NanoFCM Inc., S16M-Exo). Using the NanoFCM software (NanoFCM Profession V2.0), a standard curve was generated based on the side scattering intensity of the four different silica particle populations of 68, 91, 113 and 155 nm in diameter. The laser was set to 15 mW and 10% SSC decay. To assess the EV tetraspanin phenotype, the following antibodies were used: FITC- conjugated anti-human CD63 (BioLegend), FITC-conjugated anti-human CD9 (Abcam, (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Cambridge, UK) and FITC-conjugated anti-human CD81 (Abcam, Cambridge, UK). EVs were diluted to 1 × 10 10 particles/mL in PBS and 9 μ L was mixed with 1 μ L of conjugated antibody (single or mixed cocktail), before incubation for 30 min at room temperature. The incubation concentration ratio for single antibodies was 1:50 (in PBS) and 1:150 for the cocktail of 3 antibodies (1 μ L of 1:5 of mixed antibody cocktail). After incubation, the mixture was diluted in PBS to 1 × 10 8 - 1 × 10 9 particles/mL for analysis. Data processing was performed by the nFCM Professional Suite v2.0 software. The total EV protein concentration was determined using the Pierce Micro BCA Protein Assay Kit (Thermo Scientific, Paisley, UK). Transmission Electron Microscopy (TEM) The MNPs, GOMNPs and EVs were imaged using the JEOL JEM1400 transmission electron microscope coupled with an AMT XR80 digital acquisition system. For EV , the vesicles were physisorbed to 200-mesh carbon-coated copper formvar grids (Agar Scientific, Stansted, UK) and 1% uranyl acetate was used for negative staining. EV-Induced hBMSC Osteogenesis hBMSCs were seeded at a density of 21 × 10 3 cells/cm2 in the basal medium within 96-well plates (Nunc, Thermo Scientific, Paisley, UK). After 24 h, the medium was replaced with a mineralisation medium supplemented with EVs derived from untreated (CTL EVs), MICA stimulated (MICA EVs), TGMNP only (TREK EVs) and MICA with TGMNPs stimulated osteoblasts (MICA/TREK EVs) (10 μ g/mL) for 14 days. The EV-supplemented mineralisation medium changes were performed every 48 hrs. Cells cultured in the mineralising medium alone were used as the control. Collagen Production (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Picrosirius red staining was conducted to assess extracellular matrix collagen production. Briefly, cells were washed twice in PBS, fixed in 10% NBF for 30 min, and then stained with 0.1% Sirius red in saturated picric acid (Sigma-Aldrich, Gillingham, UK) for 60 min. Acetic acid (0.5 M) wash was used to remove the unbound dye, followed by a distilled water wash. Samples were left to air dry prior to imaging using light microscopy (EVOS XL Core, Invitrogen, Paisley, UK). Calcium Deposition Alizarin red staining was used to evaluate the extracellular matrix calcium deposition. Briefly, cells were washed twice in PBS and fixed in 10% NBF for 30 min. Samples were washed in distilled water and incubated with alizarin red solution (Sigma-Aldrich, Gillingham, UK) for 10 min. Distilled water was used to remove the unbound dye. Calcium deposition was visualised using light microscopy (EVOS XL Core, Invitrogen, Paisley, UK). LC-MS Sample Preparation and Analysis: Samples were heated to 95 °C for 5 minutes followed by sonication. Protein was extracted through the addition of 400 µL of ice-cold acetone and incubated at -80 ˚C for 1 hour before centrifugation at 14,000 xg for 10 minutes. Supernatant was discarded and the pellets air dried. A 0.1% RapiGest (Waters Corporation, Milford, MA, USA) solution was prepared in 50 mM Ammonium Bicarbonate (pH 7.8). 50 µL was added to each pellet prior to incubating at 80 °C for 45 minutes, followed by centrifugation at 14,000 xg for 10 minutes. Dithiothreitol (DTT, 5 mM) (Fisher Scientific, Loughborough, UK) was added and to the samples, which were heated to 65°C for 20 minutes for protein denaturation. Once cooled, 15 mM Iodoacetamide was added at room temperature and placed in the dark for 30 minutes. This was followed by incubation in 1 µg trypsin (ThermoFisher Scientific, UK) overnight at 37°C. Samples were acidified (0.5% v/v formic acid) and incubated at 37°C for 25 minutes. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Finally, samples were centrifuged at 21,000 xg for 20 minutes and the supernatant collected and stored at −80°C for liquid chromatography mass spectrometry (LC-MS) analysis. The ACQUITY M Class (Waters Corporation, Milford, MA, USA) with a Symmetry C18 5 μ m, 2 cm × 180 μ m pre-column and a High Strength Silica (HSS) T3 C18 1.7 μ m, 15 cm × 75 μ m analytical reversed-phase column (Waters Corporation, Milford, MA, USA) was utilised to perform one-dimensional nanoscale LC separation of tryptic peptides. The analytical column temperature was set to 35 ◦ C. MBV samples were transferred to the pre- column at 15 μ L/min for 2 minutes with mobile phase A; aqueous 0.1% (v/v) formic acid. Peptides were eluted and separated with a gradient of 3%–40% of mobile phase B (acetonitrile with 0.1% (v/v) formic acid) for 90 minutes at 400 nL/min. Lock mass solution was delivered to the reference sprayer at 1 μ L/min by the LC system auxiliary pump and sprayed with a frequency of 60 s. Mass spectrometric analysis was acquired using the SELECT SERIES™ Cyclic Ion Mobility Mass Spectrometer (Waters Corporation, Wilmslow, UK) in v-mode with a nominal resolution of 35,000 full width at half maximum (FWHM) in positive mode electrospray ionization (ESI). The ion source block temperature was at 100°C and capillary voltage at 3.2 kV. The time-of-flight analyser was externally calibrated with NaCsI from m/z 50 to 1990. The data were post-acquisition lock mass- corrected using the doubly charged monoisotopic ion of (Glu1)-Fibrinopeptide B (m/z 785.8426). Accurate mass LC-MS data were collected in a randomised order using the ion mobility-enabled, data-independent acquisition mode (HDMSE) for 0.5 seconds with a 0.02 second interscan delay. A low and elevated energy data cycle was acquired each second, where transfer collision energy was 6 eV (per unit charge) in low energy mode and was increased from 19 to 45 eV (per unit charge) in 0.5 seconds in elevated energy mode. Data Processing and Bioinformatics: (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Progenesis QI for Proteomics version 4.2 (Nonlinear Dynamics, Newcastle upon Tyne, UK) was used to process all acquired data. Protein identifications were obtained by the reviewed entries of a murine UniProt database (20,405 reviewed entries, release 2022_12). To detect and monitor protein and peptide identification error rates (1% FDR), decoy database strategies were utilised. Peptide and fragment ion tolerances were determined automatically, one missed cleavage site was allowed, as well as fixed modification carbamidomethylation of cysteine. Variable modifications were also specified, which included the oxidation of methionine and deamidation of asparagine and/or glutamine. From the abundance data obtained by Progenesis, linear regressions were plotted using Origin Lab 2020. The protein annotation through evolutionary relationship (PANTHER) classification systems (version 19.0) was used for gene ontology (GO) annotation of biological pathways, molecular mechanisms and cellular components of protein found to be significantly upregulated in MICA/TREK EVs. StringDB was used to generate a protein-protein interaction network of differentially expressed proteins [34]. Statistical analysis For all data, experiments were performed in triplicate. Statistical analysis was assessed using the IBM SPSS software (IBM Analytics, version 21). The Shapiro /i4 Wilk test was used to analyse the normality of data. Data that was proven to be normally distributed were analysed using parametric students' T /i4 test, one /i4 way ANOV A, or paired T /i4 test. Non /i4 normally distributed data were assessed using non /i4 parametric Mann /i4 Whitney t /i4 test or Kruskal/i4 Wallis ANOV A. P values equal to or lower than 0.05 was considered as significant. *P ≤ 0.05, **P ≤ 0.01 ***P ≤ 0.001. 3. Results and Discussion 3.1. Characterisation of Graphene Oxide Magnetic particles (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Previous studies have shown how GO demonstrates enhanced biocompatibility and surface area in comparison to pure MNPs, with higher functionalisation potential and excellent electrochemical properties[17] making it an ideal coating material for use with magnetic particles. GO-MNPs offer superior TREK1 binding efficiency due to their increased surface area, π -π stacking interactions and the presence of functional groups such as hydroxyl and carboxyl groups that can improve protein anchoring[17, 35]. These characteristics make GO- MNPs a promising platform for targeted mechanobiology applications. Monodispersed MNPs with uniform size (~25nm), shape and crystallinity were synthesised using the high-temperature thermal decomposition method as previously reported[17] and seen in the TEM image shown in Figure 2A. MNPs were amine functionalised with APTES through a ligand exchange mechanism and coupled with the carboxyl groups of GO sheets through an EDC/NHS binding followed by functionalisation with TREK1 antibody to enable the selective targeting. The morphology of the discreet GO-MNPs, shown in the TEM image (Figure 2B) clearly indicates the uniform distribution of MNPs over the GO surface and a distinct view of GO sheets along the border of the GO-MNP composite. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Figure 2. Characterisation of MNPs and GO-MNPs , TEM images of A) MNPs, B) GO- MNPs, C) XRD analysis, D) Raman spectroscopy, and E) VSM measurement of magnetic hysteresis loops of MNPs and GO-MNPs. The crystallinity and phase composition of the MNPs, GO and GO-MNPs were investigated through the XRD analysis. The XRD pattern of the synthesised MNPs showed the characteristic XRD diffraction peaks at 2 θ = 21.3o, 35.2o, 42.5o, 51.2o, 56.9o, 62.0o and 74.5o indicating the cubic inverse spinel structure of Fe 3O4 (Figure 2C). Meanwhile, GO showed the characteristic XRD peak around 2 θ = 12 o indicating the (002) plane with an interlayer (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint distance of approximately 0.84nm. This increased interlayer distance is an indication of oxygen-containing functional groups such as hydroxyl and carboxyl groups on the GO[36, 37]. The obtained XRD pattern is in accordance with the previously reported values hence confirming the successful synthesis of GO [38]. The XRD spectra of GO-MNP (Figure 2C) showed all the key peaks of both MNP and GO confirming the formation of a highly crystalline GO-MNP composite with MNPs well integrated into the GO sheets. Moreover, the absence of any unwanted peaks indicates the purity of the synthesised GO-MNPs with no detectable secondary phases or byproducts. The peak broadening observed in the GO-MNP compared to MNPs and GO also indicates a nanoscale crystallite size further confirming the uniform distribution of MNPs on GO sheets. Raman spectroscopy is a powerful tool for characterising the structural and electronic properties of GO. The Raman spectra of GO revealed two prominent peaks at 1340 cm -1 and 1601 cm -1 indicating D-band and G-band respectively. Here the D-band at 1340 cm -1 is associated with the defects and disorder in the sp 2 carbon network and the G-band at 1601 is linked to the in-plane stretching of sp 2 carbon bonds[39]. As seen in Figure 2D, the Raman spectra of GO-MNP showed peaks around 1331 cm -1 (D-band) and 1597 cm -1 (G-band) indicating the presence of both MNPs and GO in the GO-MNP composite. The observed peak shifts in GO-MNP from the GO-only sample in the D-band indicated the possible charge transfer between GO and MNPs[40]. Similarly, the shift in the G-band suggests the influence of MNP-induced strain or electronic interactions affecting the GO-structure further confirming the formation of GO-MNP composite while retaining the basic structural integrity of GO[41]. The superparamagnetic behaviour of GO-MNP can be validated through the VSM as it is essential for the controlled and non-invasive magnetic stimulation of mechanosensitive ion channels. The hysteresis loop revealed that both MNPs and GO-MNPs possess a (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint superparamagnetic behaviour with no evidence of coercivity and remanence (Figure 2E). The saturation magnetisation values of MNPs and GO-MNPs were observed to be 44.7 emu/g and 30.4 emu/g respectively. The observed lower saturation magnetisation for GO-MNP can be due to the presence of GO sheets which are non-magnetic in nature. This non-magnetic nature of GO may interfere with the magnetic alignment of MNPs, thereby leading to a decrease in the magnetic saturation of the GO-MNP composite. 3.2. MICA/ TGMNPs-induced osteogenic differentiation of osteoblasts To evaluate the osteoinductive potential of the TGMNPs under MICA stimulation, ALP activity was measured as a key early-stage marker of osteogenesis. After 3 and 7 days of treatment with TGMNPs, ALP activity in the MICA-treated groups was significantly enhanced compared to non-MICA groups at days 3 and 7 (Figure 3A). Additionally, the ALP on day 7 is higher than that of day 3. The obtained results signify the enhanced osteogenic potential of the MICA-treated groups, highlighting their ability to promote early-stage osteoblast differentiation more effectively than the non-MICA treatments. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Figure 3. Osteogenic differentiation potential of MICA /TGMNPs using MC3T3 cells. A) ALP activity after 3 and 7 days culture with/without MICA stimulation. B) Quantitative analysis of Alizarin red staining using CPC after 14 and 21 days of treatment with/without MICA. C) Representative optical microscopy images of MC3T3 after Alizarin Red staining (Scale 200 µm). Alizarin Red staining was performed to assess extracellular matrix mineralisation, a hallmark of late-stage osteogenesis, in both MICA and non-MICA-treated groups after 14 and 21 days of treatment. Our findings showed that TGMNPs treatment alone increased MC3T3s calcium deposition when compared to the untreated control (Fig 3B, C). MICA stimulation in conjunction with TGMNPs treatment further enhanced the mineralisation potential of these cells compared to TGMNPs alone and the untreated control, consistent with the ALP activity results. Isolation and characterisation of MICA/TREK-EVs EVs were then isolated from the conditioned medium of osteoblasts tagged with TGMNPs and stimulated with/without MICA (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint over a 2-week culture period using differential ultracentrifugation. EVs were isolated from the conditioned medium from the following groups: Untreated cells (CTL EVs), MICA stimulated cells (MICA EVs), TGMNPs stimulated cells (TREK EVs) and MICA stimulated TGMNPs cells (MICA/TREK EVs). TEM imaging showed the obtained EVs exhibited a typical spherical morphology indicative of nano-sized vesicles (Figure 4A), consistent with previous studies [32]. The nano-flow cytometry analysis detected particles with an average diameter of 65.32 ± 0.46 nm, 71.36 ± 0.60 nm, 71.53 ± 0.15 nm, and 67.92 ± 0.22 nm for the CTL EVs, MICA EVs, TREK EVs and MICA/TREK EVs respectively (Figure 4B), corroborating with the TEM images. The positive staining percentages of CD9, CD63 and CD81 for the CTL EVs were 66.63%, 36.77% and 27.77%; MICA EVs were 55.70%, 40.97% and 31.33%; TREK EVs were 57.00%, 39.80% and 32.57%; MICA/TREK EVs were 32.63%, 27.13% and 15.50%. When assessing triple-positive staining, 69.17%, 60.20%, 60.13% and 43.17% of all particles stained positive for the CTL EVs, MICA EVs, TREK EVs and MICA/TREK EVs respectively (Figure 4C). EV protein quantification showed that EVs acquired from the MICA/TREK, MICA, and TREK stimulated groups exhibited a 3.53-fold, 2.65-fold, and 1.62-fold enhanced protein content when compared to EVs obtained from the untreated cells (Figure 4D) (P < 0.001). These findings confirmed that magnetic stimulation and the addition of TGMNPs during osteoblast mineralisation enhanced the EV protein yield during culture. The influence of mechanotransduction on EV production yield has been reported in the literature. For example, the use of 3D-printed bone-mimetic titanium scaffolds significantly enhanced the production of osteoblast-derived EVs when compared to cells cultured on tissue culture plastic [42]. Mechanotransduction induced by hydrostatic pressure significantly increased the production of EVs from cartilage microtissues [43]. Importantly, our data showed superior (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint EV production yield combining the synergistic effects of magnetic stimulation and TGMNPs, highlighting the potential of harnessing this platform technology for the scalable manufacture of EVs. Figure 4. Characterization of EVs isolated from MICA/ TGMNPs stimulated and untreated mineralising osteoblasts. A) TEM images of EVs obtained from MICA/TREK stimulated and untreated osteoblasts. Scale bar = 50 nm, B) Flow cytometry analysis depicting the size distribution of particles. C) Single-particle phenotyping of osteoblast- derived EVs. EVs were fluorescently labelled with APC-conjugated antibodies specific to CD9, CD63 and CD81. D) Protein quantification of isolated EVs. Data are expressed as mean ± SD (n = 3). 3.3. MICA/TREK-EVs Enhanced hBMSCs Extracellular Matrix Mineralization (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint In this study, hBMSCs were treated with osteoblast-derived EVs manufactured under MICA/TREK stimulation. Extracellular matrix production was assessed by Picrosirius Red staining to evaluate collagen production. Picrosirius red staining can selectively bind to collagen fibers thus enabling the visualisation of extracellular matrix formation [44, 45]. Our findings showed EVs acquired from MICA/TGMNPs stimulated osteoblasts exhibited greater hBMSCs collagen production when compared to those cells treated with EVs from MICA, TGMNPs, or untreated groups (Figure 5A). The extent of extracellular matrix mineralisation was evaluated via Alizarin Red staining to detect calcium-rich deposits. Our findings show that MICA/TREK-EV treatment substantially increased calcium deposition when compared to the treatment with EVs derived from the MICA, TREK, or untreated groups (Figure 5B), consistent with the collagen production results. Previous studies demonstrated that MICA/TREK stimulated MG63 osteosarcoma cells and immortalised MSCs mineralisation [46], suggesting the influence of MICA/TREK on enhancing the production of osteoinductive EVs propagating the enhanced osteogenic phenotype. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Figure 5. Osteogenic differentiation of hBMSCs treated with osteoblast-derived EVs manufactured with/without MICA/TREK stimulation . A) Picrosirius red staining for collagen production, and B) Alizarin red staining for calcium deposition following 14 days of osteogenic culture. 3.4. Proteomics analysis of MICA/TREK EVs Having confirmed the striking improvement of synergistic MICA/TREK stimulation of osteoblast EV osteoinductive potential, vesicle protein content was profiled to further elucidate their possible mechanism of action. The proteomes of the CTL EVs and MICA/TREK EVs were compared for three independent sample preparations using a label- free MS-LC/LC approach. The use of stringent criteria only permitted the inclusion of proteins identified in a least two biological replicates, with > 2 spectral counts in at least one repeat. The volcano plot shows the variation of protein expression between the untreated and MICA/TREK EVs. Protein database searching resulted in the identification of a total of 995 proteins. Of these, 507 proteins were significantly upregulated in MICA/TREK EVs, 182 upregulated in CTL EVs, and 287 shared proteins. The differentially expressed proteins are identified in Table S1. To provide an overview of the principal processes, mechanisms and cellular locations of proteins significantly upregulated in MICA/TREK EVs, GO analysis was performed. Significantly enriched MICA/TREK EVs proteins were found to be associated with GO functional annotation of molecular functions (i.e. structural molecule activity, protein binding), cellular components (i.e. extracellular vesicles, cytoskeleton) and biological processes i.e. organelle organization, cytoskeleton organization). A String DB network was constructed to further investigate any potential interactions between proteins associated with EVs (Figure X) revealing a significant degree of protein-protein interaction (P /i4 </i4 10-16). (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Figure 6. A) Analysis of differentially expressed proteins from CTL EVs and MICA/TREK EVs. B) Volcano plot displaying Log2 values for the proteins fold-change against Log10 FDR. Proteins with a Log2 fold difference below 1 and a statistical value of > 0.05 were not considered to be statistically significant (vertical and horizontal lines respectively). The red points in the plot represent the significantly upregulated MICA/TREK EV proteins, and the green points represent significantly upregulated CTL EV proteins. C) Venn diagram comparing proteins differentially expressed from EVs. A total of 287 shared proteins; 507 proteins upregulated in MICA/TREK EVs and 182 proteins upregulated in CTL EVs. Top ten GO prediction scores covering the domains of D) cellular components, E) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint biological processes and F) molecular function of proteins significantly upregulated in MICA/TREK EVs. Figure 7. String DB network illustrating interactions between proteins in the MICA/TREK EVs, with a significant degree of protein-protein interaction (p < 10-16). (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Our findings showed that the MICA/TREK EVs were enriched with pro-osteogenic proteins. Among them were the calcium channelling annexin proteins, where several proteins of this family were significantly upregulated within the MICA/TREK EVs (i.e. Annexin A2, A4, A5, A6, A11). These transmembrane proteins are known to play critical roles in the binding to the extracellular matrix [47, 48]. Moreover, these proteins are involved in transporting extracellular calcium into the lumen of EVs, resulting the mineral nucleation and ECM mineralization [49]. This highlights the possible role of the MICA/TREK EVs in stimulating recipient hBMSCs ECM mineralization observed in this study. Moreover, it provides indications of the role of MICA/TREK stimulation in producing EVs with superior ECM binding and mineralization potential. The proteomics analysis identified the enrichment of several Rab proteins within the MICA/TREK EVs. Rab proteins are small GTPases that act as key regulators of intracellular membrane trafficking, from the formation of transport vesicles to their fusion with membranes [50, 51]. This function is essential for osteoblasts to produce and secrete the

needed for bone formation [52]. RAB1A regulates vesicular protein transport from the endoplasmic reticulum (ER) to the Golgi compartment and onto the cell surface and plays a role in IL-8 and growth hormone secretion [53]. Moreover, it was reported that inhibiting Rab32 with the miR-124a, impaired EV secretion in lung cancer cells [54]. Studies have also suggested the role Rab proteins play in coordinating early osteogenesis [55]. Thus, the upregulation of several members of the Rab family within MICA/TREK EVs, indicates the influence of MICA on stimulating intracellular membrane trafficking, likely contributing to the enhanced EV production and osteoinductivity observed in this study. Heat shock proteins (HSPs) play a crucial role in bone formation. Primarily known for their role in protecting cells from stress, HSPs also act as molecular chaperones, assisting in the proper folding and assembly of proteins essential for bone development [56-58]. Particularly, (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint the MICA/TREK EVs were significantly upregulated with the HSP70 protein. Several studies have reported the influence of HSP70 in regulating osteogenesis. Li et al. highlighted the importance of HSP70 in MSC osteogenic induction, where following HSP70 knockdown, they observed significantly reduced MSC osteogenic marker expression [59]. Chen et al. conducted microarray and pathway analyses revealing that HSP70 promotes MSC osteogenesis via the activation of the ERK signalling pathway [60]. Moreover, studies have reported the enrichment of HSP within EVs during bone homeostasis [61]. Thus, the MICA- induced cellular stress likely upregulated the production of HSP70 in MC3T3s, leading to their enrichment within secreted EVs for autocrine/paracrine signalling. It has been reported that calcium signalling plays an important role in the synthesis and release of EVs. Our findings show that the MICA-TREK EVs were enriched with the V oltage-Sensitive Calcium Channel (VSCC) protein. VSCCs have been shown to play an important role in bone cell regulation and are important regulators of intracellular calcium signalling in skeletal tissues[62]. The key role of VOCCs in mechanotransduction has been elucidated and it has been shown that mechanical force induces Ca 2+-dependent contractions of the osteocyte cell membrane mediating EV release and demonstrates that EVs are another mechanism by which VSCCs influence the secretion of bioactive molecules within bone [62, 63]. EV signalling plays an important role in regulating bone remodelling during mechanical stimulation [64]. Considering the role that VSCCs have in differentiation and mechanically induced responses, Ca +2 influx via VSCCs could modulate EV secretion in the skeleton to regulate bone remodelling. Thus, the enrichment of VSCC proteins in MICA/TREK EVs indicates the role of these nanoparticles in stimulating Ca 2+ signalling and subsequent EV release in recipient hBMSCs. Proteomics analysis also highlighted the enriched transcriptional regulating proteins within the MICA/TREK EVs. There has been growing evidence regarding the role of epigenetic (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint regulation in controlling lineage-specific differentiation [65, 66]. Our findings show that the MICA/TREK EVs were enriched with the epigenetic modifying protein histone acetyltransferase - KAT6A. This protein is involved in the addition of acetyl groups to histone proteins, ultimately enhancing the chromatin's transcriptional activity [67]. Studies have shown that increasing acetylation within MSCs through the inhibition of histone deacetylase (HDAC) by HDAC inhibitors, significantly enhances osteogenic differentiation [68-70]. Previous studies have reported that HDAC inhibition in osteoblasts significantly enriched their EVs with epigenetic modifying proteins and microRNAs, which contributed to enhancing the vesicle's osteoinductive potency [32]. Moreover, it has been reported that KAT6A plays a crucial role in maintaining the stemness of MSCs through regulation of the Nrf1/ARE signalling pathway, thus inhibiting ROS accumulation [71]. The MICA/TREK EVs were also enriched in lysine-specific histone demethylase 1A - KDM1A. Lysine demethylases control the process of histone methylation, which contributes to controlling chromatin structure and gene expression [72]. Studies have reported the importance of KDM1A in osteoblast differentiation, where Rummukainen et al. reported that KDM1A knockdown in MSCs led to a reduction in osteoblast activity and disrupted bone formation in vivo [73]. Moreover, the induction of hypomethylation within MSCs resulted in the production of EVs with enhanced osteoinductive potency [33]. Together, these findings demonstrate that MICA/TREK stimulation significantly enriched osteoblast EVs with epigenetic regulating proteins, which likely contributed to augmenting the transcriptional activity in recipient hBMSCs, enhancing osteogenic differentiation. It is important to note that due to the diverse biological cargo of EVs, is it likely that the osteoinductive capacity of MICA/TREK EVs is a combination of changes across all EV components (i.e. metabolites, lipids, proteins, RNA species etc.), although this would require further investigation. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint 4. Conclusion In conclusion, these findings demonstrate that enhancing the osteogenic differentiation of osteoblasts via MICA stimulation, significantly increased EV production yield and their osteoinductive potency. Furthermore, proteomics profiling revealed that the MICA/TREK EVs were enriched with proteins involved in ECM binding, osteogenic differentiation, EV signalling, and transcriptional regulation. These findings showcase the considerable utility of harnessing MICA as a novel engineering approach to enhance the scalable production of EVs as an acellular tool for bone repair. To our knowledge, this is the first study to promote the production yield and therapeutic potency of EVs for bone regenerative strategies through MICA.

This research has been funded by ERC DYNACEUTICS (789119) Remote control healing: Next generation mechano-nano-therapeutics Conflict of Interest The authors declare no conflict of interest ; AEH is a Founder/Director of MICA Biosystems, Ltd which is commercialising the MICA platform for cell therapy. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint

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[63] A.E. Mo rrell, G.N. B r own, S . T . R obin s on, R.L . S a ttler , A . D . Ba ik, G. Zh en , X. Cao , L.F . Bonewald , W . J i n , L . C . K a m , X . E . G u o , M e c h a n i c a l l y i n d u c e d C a ( 2 + ) o s c i l l a t i o n s i n o s t e o c y t e s r e l e a s e e x t r a c e l l u l a r v e s ic l es and e nha nc e bone f orm at io n , Bo ne R e s 6 (2018) 6. [64] J.X. R en, R. C . Y u, J . Y . X ue, Y .Q . T ang , S .H . S u, C .X. Lia o , Q. Y . Guo, W .M . Guo , J .X . Zhe ng , How Do Ex t r ac ellu lar V e s i cl es Play a K ey R ole in t he Ma i n tenanc e of Bon e Homeo st a s i s a nd R e g e nera tion ? A Comprehe n s iv e R ev iew of Lit erature, I nt J N an om ed 17 (2 022) 5375 - 5 389. [65] K. Man , N. Mek hil eri, K . L im, L .H. Jiang , T . W oodfi eld, X .B . Y ang , MI192 induce d epi g e netic r eprogr ammi ng enhanc e s th e therapeu t ic ef fic ac y of hum an bon e marr o ws s t r oma l cells for bone r egen era ti on, Bon e (2021) 11 6138. [66] K . A. Ma n, C .; M ekhil er i , N. V .; L im, K. S.; J i ang , L .-H.; W oo d f i eld , T .B. F . ; Y ang , X.B , G elMA Hy d r og e l R ein f or c ed wi t h 3 D Pri n ted PEG T /P B T Sc af fold s f o r S upp ort i ng Epi gene tic a lly -Ac t i va ted Human Bon e Marr ow S t roma l C ell s f or Bo ne R ep air , J . Func t . Biom at er . 13(2) (2022) . (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint [67] V . V . L un y ak , M. G. R o s e n f el d, Epig e netic r egul a tio n o f st em cel l fa te, Hum Mol Genet 1 7( R1) (2008 ) R28 -36. [68] K. Ma n, H . J o ukhdar , X.D . Manz , M . Y . Br un et, L.H. Jiang , J. Rnjak - Kov ac ina, X. B. Y an g , Bone tis s u e eng ineer ing u sing 3D s il k sca f f old s a nd h uman de n tal pulp str oma l cell s epige n etic r e pr ogr amme d wi t h t he s e l ective hi stone de a cetyl a s e in hibit o r MI192 , Cell Ti s s ue Re s 3 88(3 ) (20 22) 5 65 - 581 . [69] K.L . Ma n, L .; Jiang , L . -H .; Y ang , X. B , The Selec tiv e Hi stone De ac etyla se Inhi bi t or MI1 92 Enh anc e s the Ost e og eni c Di ff erenti at i on E fficac y o f Human D e n t a l Pul p St r omal Cell s, I n t J Mol Sc i 22 ( 1 0) (2021 ) 1-17 . [70] H . J i n , J . Y . Park, H . Choi , P . H . Choun g , H D AC Inhi bi t o r T r ic ho st ati n A P r omo tes Pr oli f era tion an d Odont o bla s t Di ff er e n ti a t i on o f Human D ent al Pu lp S t e m Ce ll s, Ti ssu e Eng Pt A 19 (5-6) (20 13) 613 - 624. [71] D .D. F ei , Y .Z. W a ng , Q.M. Z hai, X. G . Zha ng , Y . Z hang , Y . W ang , B. Li, Q . T . W a n g , KA T6A r eg ul at e s st emn e ss of agi ng bone marr ow-de r i ved mese nc h ymal st em ce lls t h r oug h N rf2 / ARE s ig naling pa thwa y , Stem Ce ll Re sea r c h & Therapy 12(1) (2021 ). [72] A. D ud ak ov ic, F .H . X u, E. C amill er i , M. Mc Gee -Law r e nce, E . L ew a llen, S . Rie s t er , J .R . Hawse, G . St ei n , M. Mo n teci no , J . W e stendor f , A . V an W i jnen, E pi g ene tic co n t rol o f s k ele t al dev el opme n t by t h e hist o ne meth y ltr a n s f e r a s e E ZH2, J Bone M i ner R e s 29 (201 4 ) S40-S 40 . [73] P . Rummuk a in en, K. T arkk one n, A . D udak ovic , R. Al- Majidi, V . N i emin en- Pi hal a, C. V alen si si, R .D. Hawk ins, A. J . V a n Wijne n, R . K ivir an t a , L y sin e-S p ecifi c De meth yla s e 1 (LS D1 ) e pi g ene t i call y c ontr ol s osteobla st di ff e r e n ti ation, Plo s O ne 17 (3) (2022 ). (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Supplementary information Supplementary Table 1. Differentially enriched proteins within the MICA/TREK EVs. Protein Name Gene Name Log2fold Anova (p) Nucleotide-binding oligomerization domain-containing protein 2 NOD2 10.04 6.21E-11 Fragile X mental retardation protein 1 FMRP 3.62 1.44E-04 Collagen alpha-3(VI) chain COL6A3 2.94 5.45E-06 Centromere-associated protein E CENPE 5.03 9.91E-07 Histone H4 H4 4.94 6.16E-07 MDS1/EVI1 MECOM 3.79 7.82E-07 Nucleolar protein 4 NOL4 3.58 6.31E-07 Tetraspanin-16 TSPAN16 5.49 3.08E-07 Malate dehydrogenase_ cytoplasmic MDH1 6.25 3.75E-07 Bromodomain-containing protein 9 BRD9 3.07 8.75E-06 Annexin A4 ANXA4 2.71 9.80E-06 Neurofilament heavy polypeptide NEFH 6.14 2.27E-04 Fibrillin-1 FBN1 5.70 2.55E-07 FERM and PDZ domain-containing protein 3 FRMD3 2.76 1.97E-06 Fibronectin FN1 4.88 2.91E-07 Zinc finger protein 614 ZNF614 3.87 2.02E-02 Keratin_ type I cuticular Ha7 KRT37 3.20 2.34E-05 Zinc finger protein 518A ZNF518A 4.29 2.96E-06 Tubulin beta-2A chain TUBB2A 10.13 1.81E-07 Protein Daple CCDC88C 1.96 6.09E-02 Teneurin-1 TEN1 7.30 6.36E-07 Ras-related protein Rap-1b RAP1B 3.69 7.60E-07 POTE ankyrin domain family member E POTEE 3.04 1.57E-05 14-3-3 protein theta YWHAQ 5.09 3.19E-06 Moesin MSN 2.52 3.40E-06 Annexin A5 ANXA5 2.32 5.00E-05 Mitogen-activated protein kinase kinase kinase 13 MAP4K3 4.69 4.54E-07 Basement membrane-specific heparan sulfate proteoglycan core protein HSPG2 3.04 5.75E-06 Tau-tubulin kinase 1 TTBK1 4.26 2.88E-06 Transcription regulator protein BACH2 BACH2 3.03 3.90E-05 Semaphorin-3D SEMA3D 2.53 1.13E-03 Amyloid beta A4 protein APP 2.72 4.91E-03 Myosin-10 MYO10 2.80 9.00E-05 Nuclear receptor corepressor 2 NCOR2 6.59 4.39E-07 (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Ubiquitin-conjugating enzyme E2 H UBE2H 3.64 2.17E-06 Annexin A6 ANXA6 2.95 8.81E-06 Alpha-2-HS-glycoprotein AHSG 2.57 9.54E-05 Protein Red IK 3.02 1.99E-05 Proteasome-associated protein ECM29 homolog ECPAS 3.46 1.22E-04 Nesprin-2 NESP2 3.11 3.38E-06 Nephronectin NPNT 3.34 2.66E-06 Collagen alpha-2(VI) chain COL6A2 7.61 4.76E-06 Matrin-3 MATR3 2.06 5.56E-04 60S acidic ribosomal protein P2 RPLP2 3.93 7.73E-06 Phospholipid-transporting ATPase ATP 7.35 4.37E-03 Unconventional prefoldin RPB5 interactor 1 URI 9.31 1.90E-05 Vinculin VCL 6.15 1.66E-03 Nuclear autoantigenic sperm protein NASP 5.95 3.26E-06 Metalloproteinase inhibitor 2 TIMP2 3.47 2.49E-06 Nucleosome-remodeling factor subunit BPTF BPTF 4.37 2.23E-04 Cleavage stimulation factor subunit 3 CSTF3 5.38 1.23E-05 Sperm flagellar protein 2 SPEF2 5.87 3.53E-03 Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas GNAS 9.92 2.83E-07 Putative heat shock protein HSP 90-beta 2 HSP90B1 1.73 1.28E-04 60S ribosomal protein L7 RPL7 3.89 4.00E-03 Testis-expressed sequence 2 protein TEX2 8.33 9.99E-05 Sema domain_ transmembrane domain (TM)_ and cytoplasmic domain_6A_ isoform CRA_d SEMA6B 1.96 1.76E-03 Protein disulfide-isomerase A5 PDIA5 5.44 5.77E-06 Heat shock protein HSP 90-alpha A2 HSP90AA2P 5.91 2.96E-05 Metallothionein-1E MT1E 3.89 1.87E-04 Metallothionein-1X MT1X 4.77 6.81E-04 Golgi-associated plant pathogenesis-related protein 1 GLIPR2 2.25 1.64E-05 Prothymosin alpha PTMA 5.75 1.32E-05 60S acidic ribosomal protein P2 RPLP2 3.97 5.10E-06 14-3-3 protein epsilon YWHAE 3.05 8.18E-03 DNA endonuclease RBBP8 RBBP8 4.29 5.52E-04 Elongation factor 1-gamma EEF1G 1.83 1.14E-05 Periostin POSTN 1.93 6.35E-04 Nuclear pore complex protein Nup98-Nup96 NUP98 1.09 1.05E-03 RAS protein activator like-3 RASAL3 6.41 6.10E-05 Beta-arrestin-1 ARRB1 3.68 8.63E-06 Myocyte-specific enhancer factor 2B MEF2B 4.81 8.66E-05 Microtubule-associated protein 2 MAP2 2.80 6.06E-05 EGF-like repeat and discoidin I-like domain-containing protein 3 EDIL3 3.77 1.35E-06 AF4/FMR2 family member 3 AFF3 4.19 9.50E-07 Prelamin-A/C LMNA 4.47 1.02E-06 Protocadherin FAT3 3.94 1.04E-04 Serine/threonine-protein kinase PLK1 8.31 6.39E-07 Fermitin family homolog 2 FERMT2 7.35 2.33E-05 Centrosomal protein of 192 kDa CEP192 2.13 3.20E-04 Ras-related protein Rab-15 RAB15 5.63 9.20E-05 (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint E3 ubiquitin-protein ligase TTC3 2.69 5.29E-05 Lymphocyte antigen 75 LY75 1.40 1.27E-05 MORC family CW-type zinc finger protein 3 MORC3 4.15 6.86E-05 14-3-3 protein eta YWHAH 2.63 4.56E-03 Putative protein FAM10A4 ST13P4 3.13 2.88E-05 Hsc70-interacting protein ST13 3.13 2.88E-05 Galactokinase GALK1 4.61 8.85E-04 Protein kinase C and casein kinase substrate in neurons protein 3 PACSIN3 10.23 8.22E-06 Zinc finger and SCAN domain-containing protein 5A ZSCAN5A 11.80 2.95E-08 Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit beta PIK3C2B 3.10 5.28E-03 Hormone-sensitive lipase LIPE 4.06 3.27E-06 60S ribosomal protein L3 RPL3 2.45 7.21E-04 Talin-1 TLN1 3.16 9.98E-05 UV-stimulated scaffold protein A UVSSA 3.02 1.44E-04 Alpha-crystallin B chain CRYAB 3.06 1.66E-06 Cortactin-binding protein 2 CTTNBP2 2.75 1.56E-03 Dermcidin DCD 2.95 2.02E-05 Keratin_ type I cuticular Ha3-II KRT33B 3.19 6.93E-06 EF-hand calcium-binding domain-containing protein 2 EFCAB2 1.16 1.35E-03 116 kDa U5 small nuclear ribonucleoprotein component EFTUD2 3.99 2.54E-07 Zinc finger protein 366 ZNF366 5.51 9.02E-05 Keratin_ type II cytoskeletal 74 KRT74 5.95 3.12E-04 Zinc finger ZZ-type and EF-hand domain-containing protein 1 ZZEF1 7.73 2.04E-05 Testis- and ovary-specific PAZ domain-containing protein 1 TOPAZ1 5.47 3.17E-04 Clavesin-2 CLVS2 1.39 3.35E-02 High mobility group protein B1 HMGB1 4.21 1.03E-04 Heat shock 70 kDa protein 1-like HSPA1L 1.90 7.92E-04 Solute carrier family 44 member 2 SLC44A2 3.84 1.86E-03 Mitogen-activated protein kinase kinase kinase 3 MAP3K3 1.62 3.06E-04 Serine/threonine-protein phosphatase PP1-alpha catalytic subunit PPP1CA 4.90 1.44E-04 Vitamin K-dependent protein S PROS1 2.23 1.12E-05 Zinc finger protein 827 ZNF827 6.35 1.92E-04 Monocarboxylate transporter 4 SLC16A3 6.38 6.81E-04 Male-specific lethal 3 homolog MSL3 5.57 3.05E-06 Phosphatidylinositol 3-kinase catalytic subunit type 3 PIK3C3 9.14 3.46E-05 Histone H1oo H1FOO 2.17 9.64E-08 Sodium/potassium-transporting ATPase subunit alpha-1 ATP1A1 11.64 1.08E-05 Baculoviral IAP repeat-containing protein 1 NAIP 8.03 8.58E-05 Guanine nucleotide-binding protein G(t) subunit alpha-1 GNAT1 2.09 3.99E-03 G patch domain-containing protein 8 GPATCH8 4.07 6.61E-03 Chloride intracellular channel protein 4 CLIC4 2.53 2.98E-04 Nascent polypeptide-associated complex subunit alpha NACA 4.70 2.98E-05 Cyclic nucleotide-gated cation channel beta-3 CNGB3 7.84 3.76E-05 Zinc finger protein 521 ZNF521 4.75 1.05E-04 Dihydropyrimidinase-related protein 2 DPYSL2 3.91 2.85E-05 Cyclin-dependent kinase 13 CDK13 5.30 1.37E-04 Coagulation factor X F10 5.18 1.66E-05 High mobility group nucleosome-binding domain-containing protein 5 HMGN5 4.14 1.22E-04 (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Leucine zipper putative tumor suppressor 2 LZTS2 6.77 1.60E-07 Keratin_ type II cytoskeletal 3 KRT3 4.44 9.60E-04 Voltage-dependent T-type calcium channel subunit alpha-1I CACNA1I 1.84 2.00E-06 Protein shisa-7 SHISA7 16.61 4.18E-05 Exocyst complex component 8 EXOC8 3.60 4.62E-03 Vacuolar protein sorting-associated protein 18 homolog VPS18 3.81 1.95E-03 60S ribosomal protein L5 RPL5 2.28 5.01E-02 Lebercilin-like protein LCA5L 2.54 6.23E-06 Proliferation-associated protein 2G4 PA2G4 2.22 8.61E-05 WD repeat and FYVE domain-containing protein 3 WDFY3 3.04 1.09E-03 Ankyrin-2 ANK2 7.24 1.30E-03 Nucleoporin NUP188 homolog NUP188 1.42 1.09E-04 Ectonucleotide pyrophosphatase/phosphodiesterase family member 2 ENPP2 2.73 3.25E-03 HLA class I histocompatibility antigen_ A-80 alpha chain HLA-A 5.58 1.68E-06 Keratin_ type II cytoskeletal 4 KRT4 4.27 7.14E-05 Guanine nucleotide-binding protein G(olf) subunit alpha GNAL 3.13 4.42E-04 Zinc finger protein 710 ZNF710 4.23 6.07E-06 Ubiquitin carboxyl-terminal hydrolase 48 USP48 3.09 3.85E-02 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 GNB1 2.53 3.43E-04 Myosin-7B MYH7B 2.78 1.60E-03 DnaJ homolog subfamily B member 6 DNAJB6 7.24 7.55E-05 Fibroblast growth factor receptor 3 FGFR3 6.54 5.00E-04 Disintegrin and metalloproteinase domain-containing protein 10 ADAM10 3.78 1.12E-03 Histone acetyltransferase KAT6A KAT6A 1.24 4.65E-04 Desmoglein-1 DSG1 2.12 1.21E-07 HLA class I histocompatibility antigen_ A-3 alpha chain HLA-A 6.19 4.79E-03 BTB/POZ domain-containing protein KCTD17 KCTD17 6.90 3.02E-02 Keratin_ type II cytoskeletal 5 KRT5 4.44 9.79E-05 Pseudouridylate synthase 7 homolog PUS7 1.67 2.12E-05 Heat shock protein HSP 90-alpha HSP90AA1 7.17 2.83E-03 Keratin_ type I cuticular Ha1 KRT31 2.78 5.00E-06 Protein S100-A11 S100A11 7.64 3.15E-04 Deleted in lung and esophageal cancer protein 1 DLEC1 3.94 2.59E-06 Keratin_ type II cuticular Hb4 KRT84 6.49 1.05E-04 Heterogeneous nuclear ribonucleoprotein H HNRNPH1 5.89 4.28E-02 Protein 4.1 EPB41 1.31 2.65E-03 Tubulin beta-2B chain TUBB2B 7.63 1.80E-03 Sodium/potassium-transporting ATPase subunit alpha-2 ATP1A2 3.79 3.04E-05 Transforming protein RhoA RHOA 4.66 6.00E-05 Keratin_ type I cytoskeletal 25 KRT25 1.38 1.60E-02 CWF19-like protein 2 CWF19L2 1.08 1.03E-03 Calpain-7 CAPN7 6.70 4.30E-07 Putative heat shock protein HSP 90-beta-3 HSP90AB3P 10.49 2.24E-04 MORC family CW-type zinc finger protein 1 MORC1 3.98 8.06E-04 Ras-related protein Rab-3B RAB3B 1.79 1.01E-02 Elongator complex protein 3 ELP3 4.19 4.19E-07 Profilin-1 PFN1 4.83 1.28E-03 Laminin subunit beta-2 LAMB2 4.53 9.04E-05 (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint LIM and senescent cell antigen-like-containing domain protein 1 LIMS1 3.30 4.04E-05 Probable aminopeptidase NPEPL1 7.15 1.43E-03 Coagulation factor V F5 2.58 2.06E-08 Syntaxin-8 STX8 6.28 1.38E-04 Keratin_ type II cytoskeletal 6B KRT6B 2.81 6.70E-06 60S ribosomal protein L18a RPL18A 2.76 1.16E-04 Zinc finger FYVE domain-containing protein 26 ZFYVE26 1.27 1.74E-04 Lactadherin MFGE8 3.03 7.67E-05 DNA excision repair protein ERCC6 2.51 1.50E-02 DNA-directed RNA polymerase I subunit RPA34 CD3EAP 1.52 1.36E-05 Radixin RDX 7.25 3.45E-04 Galectin-1 LGALS1 4.49 3.50E-06 Eukaryotic translation initiation factor 3 subunit A EIF3A 4.89 6.98E-06 60S ribosomal protein L4 RPL4 3.12 7.52E-05 Ferritin heavy chain FTH1 5.38 3.78E-03 Protein bassoon BSN 1.07 1.72E-04 Ras-related protein Rab-4B RAB4B 8.51 1.31E-04 Peptidyl-prolyl cis-trans isomerase PPIE 1.63 2.03E-04 Polyamine-modulated factor 1-binding protein 1 PMFBP1 2.63 2.21E-04 Vascular endothelial growth factor receptor 1 FLT1 1.70 1.24E-07 Collagen alpha-1(I) chain COL1A1 4.95 1.32E-05 STE20-like serine/threonine-protein kinase SLK 6.30 5.14E-04 Alpha-actinin-4 ACTN4 6.76 1.24E-04 Zinc finger and SCAN domain-containing protein 32 ZSCAN32 4.37 8.27E-04 Ras-related protein Rab-39A RAB39A 4.15 2.21E-04 Annexin A2 ANXA2 7.81 1.51E-04 Lactotransferrin LTF 2.48 1.63E-04 Keratin_ type I cytoskeletal 9 KRT9 1.59 3.26E-06 Fibrinogen gamma chain FGG 2.97 3.63E-05 Coiled-coil domain-containing protein 183 CCDC183 3.69 2.81E-04 CCAAT/enhancer-binding protein zeta CEBPZ 4.29 4.48E-04 Serine/threonine-protein kinase PRP4 homolog PRPF4B 1.04 4.35E-04 T-lymphoma invasion and metastasis-inducing protein 2 TIAM2 1.85 3.59E-05 L-lactate dehydrogenase C chain LDHC 2.20 7.86E-05 Coiled-coil domain-containing protein 157 CCDC157 2.01 4.85E-04 Guanine nucleotide-binding protein G(i) subunit alpha-1 GNAI1 3.05 3.29E-05 Kalirin KALRN 8.00 6.30E-03 Zinc finger protein Aiolos IKZF3 1.64 1.75E-03 t-SNARE domain containing 1 TSNARE1 3.06 2.58E-04 Prothrombin F2 6.45 3.03E-03 Nucleotide-binding oligomerization domain-containing protein 1 NOD1 1.37 3.16E-04 Fibulin-1 FBLN1 4.63 1.07E-04 Pleiotrophin PTN 2.30 2.63E-03 Ribosomal protein L19 RPL19 3.62 8.67E-04 Programmed cell death 6-interacting protein PDCD6IP 4.43 4.30E-04 Transient receptor potential cation channel subfamily M member 3 TRPM3 2.51 2.60E-04 Calcium/calmodulin-dependent protein kinase kinase 1 CAMKK1 6.08 1.72E-07 Exportin-6 XPO6 14.45 6.73E-02 (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Stromelysin-1 MMP3 4.43 2.50E-03 General transcription factor 3C polypeptide 1 GTF3C1 5.42 6.67E-04 Protein S100-A10 S100A10 3.39 2.11E-04 14-3-3 protein sigma SFN 6.19 1.17E-04 DNA repair and recombination protein RAD54-like RAD54L 1.27 2.31E-03 Alpha-enolase ENO1 1.13 2.29E-03 Keratin_ type II cytoskeletal 1 KRT1 1.35 6.45E-05 KIAA0100_ isoform CRA_a KIAA0100 1.20 4.46E-06 Mitogen-activated protein kinase kinase kinase MLT ZAK 4.48 8.96E-03 Serine/threonine-protein phosphatase 6 regulatory subunit 3 PPP6R3 3.87 8.03E-02 Prostaglandin E synthase 3 PTGES3 3.25 5.43E-04 Alpha-actinin-2 ACTN2 1.50 1.39E-02 DNA topoisomerase 2-alpha TOP2A 1.90 1.08E-03 Guanine nucleotide-binding protein G(i) subunit alpha-2 GNAI2 1.64 4.47E-05 Dynein heavy chain 7_ axonemal DNAH7 4.89 8.96E-04 RB1-inducible coiled-coil protein 1 RB1CC1 4.37 2.43E-04 Nucleosome assembly protein 1-like 4 NAP1L4 5.27 1.52E-03 DPH3 homolog DPH3 3.95 3.91E-04 OTU domain-containing protein 7A OTUD7A 5.62 6.62E-06 Transferrin receptor protein 1 TFRC 4.55 5.63E-07 Msx2-interacting protein SPEN 2.04 3.47E-05 Lysine-specific demethylase 5C KDM5C 4.85 2.49E-02 Dedicator of cytokinesis protein 4 DOCK4 1.30 1.10E-04 Inter-alpha-trypsin inhibitor heavy chain H2 ITIH2 3.07 3.46E-05 Heat shock 70 kDa protein 6 HSPA6 6.44 6.24E-05 Keratin_ type II cytoskeletal 80 KRT80 4.51 1.47E-03 Coiled-coil domain-containing protein 137 CCDC137 3.93 2.96E-05 Rabphilin-3A RPH3A 6.02 1.52E-03 Nipped-B-like protein NIPBL 5.16 4.35E-03 Spermatogenesis-associated protein 7 SPATA7 7.33 4.33E-04 Peptidyl-prolyl cis-trans isomerase F PPIF 1.66 4.17E-04 Keratin_ type II cytoskeletal 75 KRT75 1.66 9.01E-04 Growth arrest-specific protein 6 GAS6 4.32 1.34E-03 Nucleosome assembly protein 1-like 1 NAP1L1 11.58 2.89E-05 N-acetyl-beta-glucosaminyl-glycoprotein 4-beta-N-acetylgalactosaminyltransferase 1 B4GALNT4 7.83 9.58E-03 RAS guanyl-releasing protein 1 RASGRP1 1.92 9.24E-03 Vacuolar protein sorting-associated protein 13A VPS13A 3.11 1.16E-05 Bromodomain-containing protein 4 BRD4 4.38 2.45E-06 Rho GTPase-activating protein 20 ARHGAP20 5.39 2.82E-04 U6 snRNA-associated Sm-like protein LSm1 LSM1 1.46 9.67E-05 GMP reductase GMPR2 8.69 9.97E-03 Myosin light chain 6B MYL6B 2.03 3.91E-02 Phosphoglycerate mutase 1 PGAM1 2.01 9.64E-03 Guanine nucleotide-binding protein G(k) subunit alpha GNAI3 2.91 9.47E-02 Mast/stem cell growth factor receptor Kit KIT 1.46 1.31E-04 Ubiquitin-40S ribosomal protein S27a RPS27A 1.73 1.73E-03 Ubiquitin-60S ribosomal protein L40 UbUBA52 5.08 1.73E-03 (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Macrophage-stimulating protein receptor MST1R 5.08 9.92E-03 Tyrosine-protein kinase Fer FER 5.26 6.00E-07 Hemoglobin subunit gamma-2 HBG2 7.94 1.86E-05 AMP deaminase 3 AMPD3 6.75 5.09E-04 Bcl-2/adenovirus E1B 19 kDa-interacting protein 2-like protein BNIPL 5.34 4.91E-06 Zinc finger protein 423 ZNF423 2.34 1.72E-06 Huntingtin HTT 5.67 2.01E-06 Histone-lysine N-methyltransferase SUV420H1 8.04 3.41E-04 Zinc finger CCCH domain-containing protein 7A ZC3H7A 2.27 1.78E-02 Myosin-3 MYH3 1.22 5.17E-04 Nucleolin NCL 4.48 2.78E-04 Sorcin SRI 3.35 1.19E-04 Growth arrest-specific protein 8 GAS8 4.81 3.22E-04 Histone H2A type 1-B/E HIST1H2AB 1.83 6.83E-06 NEDD4-binding protein 2 N4BP2 1.81 1.13E-03 Integrin beta-3 ITGB3 1.29 1.46E-03 POTE ankyrin domain family member F POTEF 2.40 9.58E-02 Mitogen-activated protein kinase kinase kinase 12 MAP3K12 1.24 1.20E-04 T-lymphoma invasion and metastasis-inducing protein 1 TIAM1 3.47 5.76E-05 Glutamine--tRNA ligase QARS 2.47 4.13E-02 Unconventional myosin-XIX MYO19 1.67 5.20E-05 Putative Ras-related protein Rab-1C RAB1C 5.12 5.24E-04 Plasminogen PLG 1.17 2.69E-03 Collagen alpha-1(XII) chain COL12A1 4.27 4.89E-06 Secreted phosphoprotein 24 SPP2 3.34 1.25E-04 Zinc finger protein 283 ZNF283 1.58 1.09E-05 Heterogeneous nuclear ribonucleoprotein U HNRNPU 1.61 1.23E-04 Rod cGMP-specific 3'_5'-cyclic phosphodiesterase subunit beta PDE6B 3.53 5.18E-03 Serine protease HTRA1 5.48 5.54E-05 Jouberin AHI1 3.57 4.65E-03 Heat shock-related 70 kDa protein 2 HSPA2 5.48 1.77E-02 Fibrous sheath-interacting protein 1 FSIP1 2.30 4.12E-03 Integrin-linked protein kinase ILK 3.43 1.65E-03 Heat shock cognate 71 kDa protein HSPA8 1.43 1.03E-02 Filamin A FLNA 2.78 4.33E-03 Serine protease 23 PRSS23 1.89 1.03E-02 Transformation/transcription domain-associated protein TRRAP 1.33 1.11E-05 Unconventional myosin-Ic MYO1C 3.12 1.30E-04 Tumor necrosis factor ligand superfamily member 13 TNFSF13 5.49 2.99E-03 Integrin beta-1 ITGB1 2.13 3.68E-05 Protein SSX1 SSX1 5.24 3.70E-04 Keratin_ type II cytoskeletal 7 KRT7 9.44 2.77E-03 Apolipoprotein E APOE 1.69 1.26E-06 Intraflagellar transport protein 172 homolog IFT172 2.21 3.74E-02 Latent-transforming growth factor beta-binding protein 3 LTBP3 1.21 2.59E-02 Catenin alpha-1 CTNNA1 4.02 2.80E-02 Eukaryotic initiation factor 4A-II EIF4A2 1.18 1.98E-05 Serine/threonine-protein kinase 24 STK24 4.97 2.35E-02 (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Myomesin-2 MYOM2 2.97 9.62E-02 Protein kinase C alpha type PRKCA 2.01 3.21E-05 Tropomyosin alpha-4 chain TPM4 5.43 8.70E-05 Collagen alpha-1(VI) chain COL6A1 3.96 9.95E-06 Cell division control protein 42 homolog CDC42 3.51 1.58E-03 E3 ubiquitin-protein ligase TRIM37 1.80 1.66E-03 Dynein light chain 1_ cytoplasmic DYNLL1 1.22 6.73E-08 Keratin_ type II cytoskeletal 72 KRT72 11.90 4.76E-03 Dedicator of cytokinesis protein 9 DOCK9 3.31 6.58E-03 Fermitin family homolog 3 FERMT3 3.61 1.16E-03 72 kDa type IV collagenase MMP2 2.06 1.29E-04 Tyrosine-protein kinase receptor UFO AXL 6.73 5.40E-03 Kinesin-like protein KIF16B KIF16B 3.13 3.10E-04 Ephrin type-A receptor 4 EPHA4 2.82 1.20E-03 Intron-binding protein aquarius AQR 2.20 5.60E-04 Pro-neuregulin-1_ membrane-bound isoform NRG1 4.69 9.71E-05 Glyceraldehyde-3-phosphate dehydrogenase GAPDH 1.67 2.38E-02 14-3-3 protein gamma YWHAG 2.42 2.66E-03 CAP-Gly domain-containing linker protein 3 CLIP3 1.79 1.63E-05 Myosin-14 MYH14 7.85 4.72E-06 40S ribosomal protein S3a RPS3A 3.62 4.23E-03 FYVE and coiled-coil domain-containing protein 1 FYCO1 1.06 1.25E-02 DENN domain-containing protein 4B DENND4B 1.34 2.25E-03 Chloride intracellular channel protein 1 CLIC1 4.77 1.72E-02 Dystrophin DMD 4.92 7.40E-03 C-type lectin domain family 1 member B CLEC1B 3.69 8.63E-07 Syntenin-1 SDCBP 5.77 5.94E-05 Coiled-coil domain-containing protein 144B CCDC144B 2.60 3.45E-02 Hemoglobin subunit alpha HBA1 2.98 3.43E-04 LIM/homeobox protein Lhx2 LHX2 1.31 8.79E-05 A-kinase anchor protein 9 AKAP9 1.99 1.72E-05 Elongation factor 1-alpha 1 EEF1A1 4.15 3.70E-04 Beta-2-glycoprotein 1 APOH 2.19 7.17E-03 BICD1 protein BICD1 5.10 3.93E-06 Ribosomal protein S6 kinase RPS6KA1 4.68 1.59E-04 Laminin subunit gamma-1 LAMC1 1.82 7.34E-04 Immunoglobulin lambda-like polypeptide 1 IGLL1 6.77 4.50E-06 Metallothionein-1G MT1G 8.90 6.89E-03 Ribosomal protein S6 kinase alpha-3 RPS6KA3 15.60 2.49E-04 High affinity cAMP-specific and IBMX-insensitive 3'_5'-cyclic phosphodiesterase 8B PDE8B 6.11 2.85E-04 Laminin subunit alpha-2 LAMA2 1.92 2.92E-04 Coiled-coil domain-containing protein 144A CCDC144A 2.75 2.21E-02 Dedicator of cytokinesis protein 1 DOCK1 5.63 8.75E-03 AP-3 complex subunit beta-1 AP3B1 5.10 1.43E-06 Heat shock protein 75 kDa_ mitochondrial TRAP1 14.02 4.59E-02 Keratin_ type II cytoskeletal 2 epidermal KRT2 1.97 1.15E-06 Heterogeneous nuclear ribonucleoprotein H2 HNRNPH2 4.29 3.65E-02 (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Tropomyosin alpha-3 chain TPM3 1.80 3.29E-05 Hemoglobin subunit delta HBD 6.67 1.50E-05 Keratin_ type I cuticular Ha5 KRT35 4.85 2.55E-04 Heat Shock Protein 90 Beta Family Member 1 HSP90B1 1.89 5.56E-05 Echinoderm microtubule-associated protein-like 5 EML5 1.64 3.16E-03 60S ribosomal protein L30 RPL30 6.93 1.27E-06 Myosin-7 MYH7 7.40 1.12E-03 Ras-related protein Rab-3C RAB3C 5.19 1.15E-03 Laminin subunit alpha-4 LAMA4 3.89 9.86E-05 Tubulin alpha-4A chain TUBA4A 2.01 4.58E-06 Centriolin CNTRL 5.97 4.81E-04 Keratin_ type I cuticular Ha6 KRT36 4.81 6.31E-04 Hemoglobin subunit beta HBB 1.94 8.72E-05 Fibrinogen beta chain FGB 5.05 3.71E-04 Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 NUCKS1 4.78 6.73E-04 Prolargin PRELP 8.32 9.13E-03 Centrosomal protein of 57 kDa CEP57 3.76 2.22E-04 MADS box transcription enhancer factor 2_ polypeptide C_ isoform CRA_e MEF2C 4.92 6.81E-05 Guanine nucleotide-binding protein subunit alpha-11 GNA11 11.00 8.31E-07 Myosin-6 MYH6 5.25 1.57E-03 Gamma-aminobutyric acid type B receptor subunit 1 GABBR1 3.62 5.02E-05 Tyrosine-protein phosphatase non-receptor type 12 PTPN12 10.65 4.86E-03 DEP domain-containing protein 5 DEPDC5 4.45 3.67E-05 Vitamin D-binding protein GC 4.17 1.61E-04 Myosin-1 MYH1 5.91 1.76E-02 Keratin_ type I cytoskeletal 20 KRT20 3.19 1.86E-02 Vascular endothelial growth factor receptor 3 FLT4 3.88 2.36E-06 HLA class I histocompatibility antigen_ alpha chain G HLA-G 3.68 2.41E-02 Unconventional myosin-IXb MYO9B 1.27 2.26E-02 Dynein heavy chain 1_ axonemal DNAH1 4.81 8.11E-05 Filamin-B FLNB 4.36 9.03E-05 Pre-rRNA-processing protein TSR1 homolog TSR1 6.31 1.04E-03 Histone H2B type 1-K HIST1H2BK 4.98 3.89E-03 Bromodomain adjacent to zinc finger domain protein 2B BAZ2B 2.79 6.98E-02 Ezrin EZR 1.80 1.65E-02 Acidic leucine-rich nuclear phosphoprotein 32 family member E ANP32E 4.15 6.90E-06 Myosin-8 MYH8 7.03 3.10E-03 TATA box-binding protein-associated factor RNA polymerase I subunit B TAF1B 2.28 4.53E-02 40S ribosomal protein S8 RPS8 2.30 2.96E-03 AF4/FMR2 family member 4 AFF4 1.37 5.23E-04 EF-hand calcium-binding domain-containing protein 5 EFCAB5 8.78 7.35E-02 Ras-related protein Rap-1A RAP1A 2.23 7.75E-03 Pyruvate kinase PKM PKM 1.14 2.92E-06 Putative beta-actin-like protein 3 POTEKP 2.68 6.25E-03 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 GNB2 3.37 6.70E-04 Centrosome and spindle pole-associated protein 1 CSPP1 3.21 2.44E-05 Transcription factor 20 TCF20 6.55 7.17E-06 Collagen type IV alpha-3-binding protein COL4A3BP 4.44 6.45E-04 (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Alpha-actinin-1 ACTN1 4.55 3.11E-03 Ninein-like protein NINL 2.36 2.03E-04 Calmodulin CALM1 3.86 8.28E-04 Protein S100-A4 S100A4 3.95 1.81E-06 Kelch-like protein 1 KLHL1 4.23 1.40E-02 Lysine-specific histone demethylase 1A KDM1A 6.04 2.57E-04 Tripartite motif-containing protein 26 TRIM26 2.80 1.22E-04 L-lactate dehydrogenase A-like 6A LDHAL6A 2.63 1.25E-02 Transketolase TKT 1.97 2.22E-02 Probable E3 ubiquitin-protein ligase DTX2 2.01 1.64E-02 Nucleoside diphosphate kinase B NME2 2.81 1.66E-03 DNA replication licensing factor MCM4 2.38 1.08E-07 Period circadian protein homolog 2 PER2 11.93 2.78E-05 T-complex protein 1 subunit theta CCT8 6.30 4.42E-04 Tektin-1 TEKT1 7.02 8.39E-02 Ankyrin and armadillo repeat-containing protein ANKAR 2.07 5.38E-03 14-3-3 protein beta/alpha YWHAB 3.95 2.28E-04 AT-rich interactive domain-containing protein 4A ARID4A 2.60 2.95E-04 Apolipoprotein B-100 APOB 1.48 9.29E-06 Synaptic vesicle membrane protein VAT-1 homolog VAT1 2.75 6.29E-03 Keratin_ type I cytoskeletal 16 KRT16 3.47 9.04E-06 Annexin A11 ANXA11 3.78 1.85E-03 Transmembrane protein 98 TMEM98 3.17 2.09E-06 Tubulin alpha-1C chain TUBA1C 8.12 4.34E-05 Sister chromatid cohesion protein PDS5 homolog A PDS5A 7.17 2.54E-04 Vacuolar protein sorting-associated protein 13C VPS13C 7.07 7.23E-03 Myosin-4 MYH4 2.91 1.02E-02 Rho GTPase-activating protein 18 ARHGAP18 2.63 3.16E-06 Actin_ cytoplasmic 1 ACTB 1.19 6.23E-06 Zinc finger protein 687 ZNF687 2.76 9.67E-03 Ribosomal protein L15 RPL15 3.03 4.60E-03 Adenosylhomocysteinase AHCY 1.90 2.28E-04 Reticulon-4 RTN4 3.38 5.59E-04 Teneurin-3 TENM3 8.74 2.14E-02 40S ribosomal protein S9 RPS9 2.38 3.44E-02 Ras-related protein Rab-5B RAB5B 2.07 2.23E-02 Ubiquitin carboxyl-terminal hydrolase USP36 1.58 5.29E-05 Tubulin polyglutamylase TTLL5 4.25 1.49E-03 Ras-related protein Rab-11B RAB11B 3.49 2.10E-04 Long-chain-fatty-acid--CoA ligase ACSBG2 1.86 3.50E-03 Band 4.1-like protein 2 EPB41L2 5.24 3.74E-05 Terminal uridylyltransferase 4 ZCCHC11 4.87 3.35E-04 Pleckstrin homology domain-containing family J member 1 PLEKHJ1 1.31 2.78E-03 mRNA-capping enzyme RNGTT 6.56 5.83E-02 Guanine nucleotide-binding protein subunit beta-2-like 1 GNB2L1 3.70 6.19E-03 Rabenosyn-5 RBSN 6.11 1.05E-07 Receptor protein-tyrosine kinase TYRO3 9.76 1.49E-03 Ubiquitin-conjugating enzyme E2 D3 UBE2D3 1.29 3.05E-03 (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint UPF0505 protein C16orf62 1.78 2.27E-03 Signal transducer and activator of transcription 6 STAT6 4.85 1.40E-04 Rho-related GTP-binding protein RhoB RHOB 2.52 2.37E-03 Unconventional myosin-If MYO1F 3.49 1.33E-02 Ras-related protein Rab-7a RAB7A 1.89 2.59E-05 Ras-related protein Rab-10 RAB10 2.19 1.04E-06 Inosine-5'-monophosphate dehydrogenase 1 IMPDH1 5.88 6.77E-02 Probable ATP-dependent RNA helicase DDX20 2.33 6.69E-04 HLA class I histocompatibility antigen_ Cw-2 alpha chain HLA-C 7.49 9.27E-06 Histone H3.1t HIST3H3 6.27 1.56E-04 Plexin-D1 PLXND1 1.22 5.88E-02 Zinc finger protein 16 ZNF16 4.52 5.28E-04 Acetyl-CoA carboxylase 1 ACACA 4.67 1.13E-04 Ribosomal protein S6 kinase beta-2 RPS6KB2 2.63 4.51E-06 Mediator of RNA polymerase II transcription subunit 24 MED24 2.97 3.89E-05 Katanin p60 ATPase-containing subunit A-like 2 KATNAL2 3.35 5.77E-02 HCG1745306_ isoform CRA_a HBA2 1.78 2.04E-05 TBC1 domain family member 1 TBC1D1 2.31 2.31E-05 Alpha-2-macroglobulin A2M 3.31 1.65E-04 Cofilin-2 CFL2 1.35 1.83E-02 E3 ubiquitin-protein ligase UBR5 UBR5 3.07 6.56E-02 Putative heat shock protein HSP 90-alpha A5 HSP90AA5P 2.00 4.66E-05 Ras-related protein Rab-30 RAB30 3.43 2.28E-02 Rab GDP dissociation inhibitor beta GDI2 1.19 8.44E-02 Attractin-like protein 1 ATRNL1 1.98 3.06E-04 Coiled-coil domain-containing protein 146 CCDC146 3.50 4.90E-06 Coagulation factor IX F9 3.82 1.26E-05 Coagulation factor XIII A chain F13A1 3.63 4.52E-06 E3 ubiquitin-protein ligase CBL CBL 8.42 7.72E-02 Guanine nucleotide-binding protein subunit alpha-12 GNA12 2.56 1.97E-02 Semaphorin-4A SEMA4A 1.83 2.19E-06 WD repeat-containing protein 60 WDR60 8.42 1.47E-05 Claspin CLSPN 5.46 1.62E-02 Brefeldin A-inhibited guanine nucleotide-exchange protein 2 ARFGEF2 5.15 5.96E-05 Keratin_ type I cytoskeletal 18 KRT18 1.06 1.95E-05 Ubiquitin-like modifier-activating enzyme ATG7 2.66 3.04E-05 L-lactate dehydrogenase B chain LDHB 4.50 3.69E-03 Acidic leucine-rich nuclear phosphoprotein 32 family member A ANP32A 1.66 4.45E-02 GTP-binding nuclear protein Ran RAN 2.88 8.43E-03 Triosephosphate isomerase TPI1 2.28 5.37E-06 Dynein heavy chain 14_ axonemal DNAH14 3.85 1.10E-02 Ribonuclease H1 RNASEH1 1.58 1.03E-07 60S acidic ribosomal protein P0 RPLP0 6.78 6.34E-04 Elongation factor 2 EEF2 2.70 8.34E-02 Kinesin-like protein KIF28P KIF28P -11.2987 2.31E-08 Dihydropyrimidinase-related protein 1 CRMP1 -2.37685 5.21E-06 Epididymis luminal protein 189 DKFZp686J1372 -1.24396 1.24E-05 (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Wings apart-like protein homolog WAPAL -4.96751 2.36E-07 Gamma-aminobutyric acid receptor subunit rho-1 GABRR1 -2.23479 4.17E-06 NFX1-type zinc finger-containing protein 1 ZNFX1 -4.92847 2.60E-06 H2.0-like homeobox protein HLX -2.05725 5.02E-06 NADPH:adrenodoxin oxidoreductase_ mitochondrial FDXR -7.2734 2.01E-07 Zinc finger protein 40 HIVEP1 -5.34564 0.000668 Tyrosine-protein kinase CSK CSK -3.04899 7.65E-06 DNA topoisomerase I_ mitochondrial TOP1MT -13.8424 1.22E-06 Zinc finger protein 626 ZNF626 -7.94775 5.70E-07 RING finger protein 207 RNF207 -11.2952 8.02E-06 Kinectin KTN1 -3.91703 9.12E-08 DNA-dependent protein kinase catalytic subunit PRKDC -4.40111 1.60E-05 Receptor protein-tyrosine kinase FGFR4 -13.8654 6.37E-06 Bromodomain testis-specific protein BRDT -1.22532 0.000405 Ankyrin repeat domain-containing protein 6 ANKRD6 -3.6318 0.001243 Zinc finger protein 671 ZNF671 -3.56031 0.000204 Keratin_ type II cytoskeletal 78 KRT78 -3.14652 1.03E-05 Cytoplasmic dynein 2 light intermediate chain 1 DYNC2LI1 -13.4216 5.29E-06 Cytoplasmic phosphatidylinositol transfer protein 1 PITPNC1 -5.43888 3.99E-05 DNA replication licensing factor MCM7 MCM7 -4.80057 2.90E-05 Histidine triad nucleotide-binding protein 1 HINT1 -1.37455 0.009339 Bridging integrator 3 BIN3 -8.48248 0.000317 Vitronectin VTN -2.86258 4.56E-05 Major vault protein MVP -2.08711 2.73E-05 Heat shock 70 kDa protein 1B HSPA1B -3.69879 1.25E-06 Keratin_ type II cytoskeletal 1b KRT77 -3.10162 0.003618 Synaptotagmin-1 SYT1 -7.46074 1.53E-06 Transforming growth factor-beta receptor-associated protein 1 TGFBRAP1 -1.58673 2.31E-05 E3 ubiquitin-protein ligase BRE1A RNF20 -13.9309 0.000103 ADP-ribosylation factor 5 ARF5 -2.53397 0.003917 Erythrocyte band 7 integral membrane protein STOM -3.37638 0.000177 Fibulin-1 FBLN1 -10.4008 0.000231 Obscurin OBSCN -7.47334 6.76E-07 Deleted in malignant brain tumors 1 protein DMBT1 -3.00563 5.24E-06 Putative heat shock protein HSP 90-alpha A4 HSP90AA4P -1.0604 0.000472 Thrombospondin-1 THBS1 -2.15287 0.000151 Probable G-protein-coupled receptor 179 GPR179 -16.8088 2.51E-07 Golgin subfamily A member 4 GOLGA4 -2.09717 9.78E-05 Inactive carboxypeptidase-like protein X2 CPXM2 -3.80255 3.99E-05 Serine/threonine-protein kinase Nek3 NEK3 -6.72427 6.26E-05 Alpha-internexin INA -1.58251 0.000936 ATP synthase subunit beta_ mitochondrial ATP5B -3.27813 0.030472 Four and a half LIM domains protein 2 FHL2 -9.54326 4.45E-07 Myosin-11 MYH11 -3.96298 1.19E-06 Prohibitin PHB -2.54788 0.003068 Guanine nucleotide-binding protein subunit beta-4 GNB4 -1.85066 4.89E-05 Neurexin-1-beta NRXN1 -3.44744 9.88E-06 Minor histocompatibility antigen H13 HM13 -1.3988 0.001545 (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Complement component C9 C9 -1.16842 0.002187 LINE-1 type transposase domain-containing protein 1 L1TD1 -1.05498 0.013525 Caveolin CAV1 -2.02597 0.060923 Histone-lysine N-methyltransferase SETD1B SETD1B -1.33365 0.000143 Angiopoietin-related protein 2 ANGPTL2 -4.25013 0.000148 HLA class I histocompatibility antigen_ Cw-6 alpha chain HLA-C -3.5302 0.00077 Ras GTPase-activating-like protein IQGAP2 IQGAP2 -1.48829 9.41E-05 E3 ubiquitin-protein ligase HUWE1 HUWE1 -2.07311 0.000134 Solute carrier family 12 member 5 SLC12A5 -8.26886 0.0004 Guanine nucleotide-binding protein G(z) subunit alpha GNAZ -2.71028 0.000685 Lysine--tRNA ligase KARS -3.51893 2.00E-05 Glutamate decarboxylase 1 GAD1 -2.61975 0.014592 Procollagen C-endopeptidase enhancer 1 PCOLCE -2.47267 0.002743 Ras-related protein Rab-1A RAB1A -1.02024 0.00133 Mucolipin 2_ isoform CRA_a MCOLN2 -3.02693 0.003282 Dedicator of cytokinesis protein 11 DOCK11 -4.48341 0.001357 Alpha-fetoprotein AFP -1.0593 0.001168 ADP-ribosylation factor 3 ARF3 -1.25338 0.016323 Signal-induced proliferation-associated 1-like protein 1 SIPA1L1 -4.26698 0.00404 Protein NDNF NDNF -3.5861 0.056094 ADP-ribosylation factor 6 ARF6 -1.72927 0.006607 Nucleolin NCL -3.87146 0.001743 Troponin T_ slow skeletal muscle TNNT1 -3.28594 6.52E-05 Ras GTPase-activating-like protein IQGAP1 IQGAP1 -4.98883 3.11E-06 Collagen alpha-2(I) chain COL1A2 -4.21187 1.04E-05 Apolipoprotein A-I APOA1 -1.29911 0.001044 Zinc finger CCHC domain-containing protein 8 ZCCHC8 -2.99466 0.032464 Keratin_ type II cytoskeletal 73 KRT73 -3.42258 0.006581 Periplakin PPL -1.3092 0.001671 Peptidyl-prolyl cis-trans isomerase A PPIA -2.65242 3.49E-05 Casein kinase I isoform gamma-3 CSNK1G3 -3.4264 0.000363 Semaphorin-3D SEMA3D -3.9987 0.000728 Histone-lysine N-methyltransferase ASH1L ASH1L -4.74311 4.62E-05 Transient receptor potential cation channel subfamily M member 6 TRPM6 -3.05741 1.47E-06 Zinc finger protein GLI4 GLI4 -7.7467 0.00389 Adipocyte enhancer-binding protein 1 AEBP1 -2.45873 0.000524 E3 ubiquitin-protein ligase NEDD4 NEDD4 -11.7909 4.84E-05 Tetranectin CLEC3B -3.08162 0.00044 Endogenous retrovirus group K member 10 Pol protein ERVK-10 -2.73701 0.056818 Disks large-associated protein 3 DLGAP3 -3.8619 0.009872 Junctional protein associated with coronary artery disease KIAA1462 -4.06526 4.34E-06 FACT complex subunit SSRP1 SSRP1 -7.64625 4.95E-07 Probable E3 ubiquitin-protein ligase MARCH10 Mar/10 -5.09228 4.49E-05 Retinoblastoma-like protein 1 RBL1 -1.96557 0.00026 Retrotransposon-like protein 1 RTL1 -2.75611 0.034298 Heterogeneous nuclear ribonucleoprotein C-like 2 HNRNPCL2 -7.05187 1.51E-06 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-3 GNB3 -2.82008 0.006753 Teneurin-4 TENM4 -3.30487 0.00487 (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Mismatch repair endonuclease PMS2 PMS2 -10.024 1.15E-05 Matrix-remodeling-associated protein 5 MXRA5 -3.94513 7.92E-06 Latent-transforming growth factor beta-binding protein 3 LTBP3 -2.82119 0.000273 Ras-related protein Rab-14 RAB14 -6.3603 0.000134 Pigment epithelium-derived factor SERPINF1 -3.87614 0.002238 Serotransferrin TF -2.1585 0.002069 Ras-related protein Rab-5C RAB5C -2.93361 0.002162 Annexin ANXA3 -4.23808 0.00413 Serine/arginine repetitive matrix protein 2 SRRM2 -7.42019 0.001257 Serine/arginine-rich-splicing factor 7 SRSF7 -5.39971 4.87E-05 Beta-defensin 112 DEFB112 -4.45576 0.000135 MAGUK p55 subfamily member 4 MPP4 -3.62936 0.005681 Latrophilin-1 LPHN1 -3.88671 7.41E-05 Exocyst complex component 2 EXOC2 -4.78587 0.017349 Hemoglobin subunit zeta HBZ -4.25156 0.000513 Trafficking kinesin-binding protein 1 TRAK1 -1.69456 0.001442 Keratin_ type I cytoskeletal 17 KRT17 -2.17874 0.000122 Hydroxyacylglutathione hydrolase_ mitochondrial HAGH -6.35235 1.94E-05 Protocadherin gamma-C4 PCDHGC4 -8.53722 9.52E-07 Putative Polycomb group protein ASXL3 ASXL3 -1.67402 0.007715 Tubulin polyglutamylase TTLL7 TTLL7 -3.24259 0.004981 Cyclin-L1 CCNL1 -7.83696 2.58E-05 Tubulin beta-1 chain TUBB1 -4.04964 1.08E-05 Zinc finger protein 638 ZNF638 -11.2987 0.032204 T-complex protein 1 subunit eta CCT7 -2.26364 0.000603 T-complex protein 1 subunit beta CCT2 -6.38681 0.011023 Gamma-aminobutyric acid receptor subunit gamma-3 GABRG3 -1.67603 0.057783 Transient receptor potential cation channel subfamily M member 2 TRPM2 -3.15304 0.003987 Zinc finger protein 320 ZNF320 -10.2461 0.000117 Dual specificity mitogen-activated protein kinase kinase 3 MAP2K3 -13.0719 1.79E-07 Peroxiredoxin-1 PRDX1 -4.8246 9.44E-07 Putative tRNA pseudouridine synthase Pus10 PUS10 -1.82421 0.050015 Brefeldin A-inhibited guanine nucleotide-exchange protein 1 ARFGEF1 -8.57428 6.08E-06 Pleckstrin homology-like domain family B member 2 PHLDB2 -4.76837 6.25E-06 Serine/threonine-protein kinase 25 STK25 -6.7863 0.002037 Isovaleryl-CoA dehydrogenase_ mitochondrial IVD -7.61084 3.66E-05 Amine oxidase [flavin-containing] A MAOA -5.72259 0.022826 Transmembrane protein 131-like KIAA0922 -2.10775 0.057208 Zinc finger protein 432 ZNF432 -6.40177 0.005427 AF4/FMR2 family member 2 AFF2 -1.40982 0.089088 Glyoxalase domain-containing protein 5 GLOD5 -2.73731 0.000757 Glutamyl aminopeptidase ENPEP -3.87776 0.015569 Reticulocalbin-3 RCN3 -3.86025 0.014171 Ras-related protein Rab-39B RAB39B -3.89586 0.000833 Terminal uridylyltransferase 7 ZCCHC6 -2.53704 0.036366 Transcription factor TFIIIB component B'' homolog BDP1 -2.32497 2.05E-05 Vesicle-fusing ATPase NSF -3.43592 0.019775 Apolipoprotein M APOM -1.66837 0.06297 (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint Vimentin VIM -1.71933 0.050469 Transitional endoplasmic reticulum ATPase VCP -5.04267 0.065533 Collagen alpha-1(III) chain COL3A1 -3.34153 0.009644 Neurofibromin NF1 -4.66771 2.53E-05 Alpha-actinin-3 ACTN3 -1.942 1.48E-05 Dedicator of cytokinesis protein 2 DOCK2 -4.68863 9.23E-06 Choline transporter-like protein 1 SLC44A1 -1.05627 0.000259 Methylenetetrahydrofolate reductase MTHFR -3.30586 0.051116 Membrane-associated phosphatidylinositol transfer protein 2 PITPNM2 -8.58612 0.000208 Splicing factor_ arginine/serine-rich 19 SCAF1 -2.98593 0.002543 Dedicator of cytokinesis protein 10 DOCK10 -2.96087 0.062113 Thrombospondin type-1 domain-containing protein 7B THSD7B -3.34977 0.001198 Nuclear factor 1 A-type NFIA -9.3859 6.66E-06 Platelet glycoprotein Ib beta chain GP1BB -2.96207 0.004638 Histone H2A type 1-A HIST1H2AA -2.51795 0.000776 Guanine nucleotide-binding protein G(q) subunit alpha GNAQ -1.67953 0.004988 Creatine kinase B-type CKB -5.33995 0.088955 Centrosomal protein of 152 kDa CEP152 -8.92998 0.000155 Spindlin interactor and repressor of chromatin-binding protein C11orf84 -8.00452 3.94E-06 General vesicular transport factor p115 USO1 -1.06531 0.02802 Phosphatidylinositol 4-kinase alpha PI4KA -1.74599 0.095309 HCG1995540_ isoform CRA_b RAB4B -3.07752 3.12E-05 Nicotinamide N-methyltransferase NNMT -4.43479 0.087043 Keratin_ type II cuticular Hb6 KRT86 -3.23188 0.004474 60 kDa heat shock protein_ mitochondrial HSPD1 -3.41188 0.030222 Nucleophosmin NPM1 -1.59149 0.026481 Ras-related protein Rab-8A RAB8A -3.14814 0.019636 Rab GTPase-binding effector protein 1 RABEP1 -1.47151 0.00506 FERM_ RhoGEF and pleckstrin domain-containing protein 1 FARP1 -1.92728 0.037429 Anoctamin ANO3 -5.37346 0.000408 3-ketodihydrosphingosine reductase KDSR -6.30767 0.024137 Centrosomal protein of 135 kDa CEP135 -10.778 1.04E-06 Fascin FSCN1 -2.22732 0.047632 14-3-3 protein zeta/delta YWHAZ -2.87886 0.000181 Cilia- and flagella-associated protein 61 CFAP61 -3.34177 0.000234 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.07.669024doi: bioRxiv preprint