Tubercidin Enhances Apoptosis in Serum-Starved and Hypoxic Mouse Cardiomyocytes by Inducing Nuclear Speckle Condensation

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This study aims to clarify the molecular pathways of Tubercidin-induced cardiotoxicity, focusing on nuclear speckles (NSs) disruption in cardiomyocytes under serum deprivation and/or hypoxia. To simulate ischemic cardiomyopathy in vitro, we utilized FMC84 and HL-1 murine cardiomyocyte cell lines, exposing them to conditions of serum limitation and/or hypoxia to evaluate the cardiotoxic impact of Tubercidin and the contributing mechanisms. Apoptosis was quantified using flow cytometry, NSs condensation was visualized via immunofluorescence with an anti-SC35 antibody, and the expression levels of key apoptotic transcripts (RFFL, RIF1, and RNF144B) were analyzed by RT-PCR. Our findings revealed that Tubercidin significantly increased apoptosis in both HL-1 and FMC84 cell lines under conditions mimicking serum deprivation (21% O2 with 1% FBS), hypoxia (1% O2 with 10% FBS), or a combination of both. Furthermore, Tubercidin treatment led to a pronounced enlargement of NSs, as detected by immunofluorescence. Concurrently, we documented significant alterations in the expression of critical apoptotic regulatory genes, implying that Tubercidin may modulate the apoptotic pathway in stressed cardiomyocytes. It is hypothesized that Tubercidin induces NSs condensation, affecting alternative splicing of cell death genes, potentially worsening ischemic cardiomyocytes' damage. Therefore, a cautious clinical use of Tubercidin for ischemic cardiomyopathy patients is advised to reduce cardiotoxicity risks. Tubercidin Apoptosis Hypoxia Nuclear speckles Mouse Cardiomyocytes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Tubercidin, an adenosine analog derived from Streptomyces tubercidicus, possesses a broad spectrum of pharmacological activities, exerting a potent inhibitory influence on diverse microorganisms, including bacteria, fungi, viruses, and protozoa [ 1 ]. Moreover, this compound has demonstrated promise as a chemotherapeutic agent for tumor treatment [ 2 ]. Upon cellular uptake through nucleoside transporters, Tubercidin undergoes intracellular phosphorylation to generate mono-, di-, and triphosphate forms that competitively inhibit adenosine nucleotides, thereby disrupting polymerase activity and subsequently halting DNA replication, RNA transcription, and protein synthesis [ 3 ]. Additionally, structural modifications of Tubercidin have been shown to confer inhibitory effects on adenosine kinase [ 4 ]. The clinical deployment of Tubercidin is significantly limited by its considerable hepatotoxicity, nephrotoxicity, and cardiotoxicity [ 5 ], especially in individuals with cardiac comorbidities such as ischemic cardiomyopathy. Therefore, deciphering the molecular pathways leading to its cardiotoxic effects is essential for devising strategies to alleviate these adverse outcomes. In the context of schistosomiasis, combination therapies have been employed to ameliorate the toxicity profile and reduce patient side effects [ 6 ]. Furthermore, chemical modification of Tubercidin has been investigated as a means to diminish its toxicity [ 7 ]. Despite these efforts, a thorough understanding of Tubercidin's toxicity mechanisms remains critical for enhancing its clinical safety profile. Nuclear speckles (NSs) are RNA-binding protein-rich nuclear bodies, numbering approximately 20 to 30 per eukaryotic nucleus [ 8 ]. They are populated by a multitude of SR (Serine/Arginine-rich) proteins, which are pivotal in pre-mRNA splicing, mRNA transport, RNA stability, and translation [ 9 ]. Consequently, NSs serve as key assembly and storage sites for pre-mRNA splicing machinery [ 9 ]. During eukaryotic RNA splicing, SR proteins operate as essential splicing factors, in conjunction with snRNPs (small nuclear ribonucleoproteins) and other factors, to bind specific RNA sequences, identify splice sites, and facilitate both constitutive and alternative splicing [ 10 ]. The SR proteins are also instrumental in regulating apoptosis through the alternative splicing of genes associated with cell death [ 11 ], a process that is dependent on NSs [ 12 ]. To date, over ten SR protein varieties have been identified, with SRSF2 (Serine/Arginine-rich splicing factor 2), also known as SC35, being a critical member of the SR family and a central constituent of NSs [ 13 ]. Tubercidin has been observed to perturb the assembly of NSs and consequently disrupt mRNA processing. Following Tubercidin exposure, poly (A) + RNAs dispersed throughout the nucleoplasm undergo decay, whereas SC35-labeled NSs persist in a condensed state [ 14 ]. This suggests that Tubercidin specifically impairs mRNA processing within NSs without disassembling these condensed nuclear subcompartments. NSs are known to form under various stress conditions, including hypoxia and serum starvation, with their RNA processing events presumed to aid in cellular stress survival. Therefore, the disruption of these RNA processing events by Tubercidin, including mRNA splicing, is hypothesized to augment cardiomyocyte toxicity in individuals with ischemic heart diseases. Utilizing murine cardiomyocytes, this study provides evidence that Tubercidin induces the condensation of NSs and exacerbates apoptosis under hypoxic and/or serum-starved conditions. These findings suggest a cautious approach to the clinical use of Tubercidin in patients with ischemic cardiomyopathy. Materials and Methods Maintenance of Cardiomyocyte Cell Cultures The HL-1 cell line, derived from mouse atrial cardiomyocytes, was sourced from Cellcook (Guangzhou, China) and maintained in a customized Claycomb medium (Sigma-Aldrich). This medium was fortified with 10% fetal bovine serum (FBS; Gibco), 4 mM L-glutamine, 0.1 mM noradrenaline, and an antibiotic-antimycotic mix (100 U/mL penicillin and 100 µg/mL streptomycin) to support cell viability and prevent contamination. The FMC84 cell line, which models mouse ventricular cardiomyocytes, was provided by Jennio Biotech (GuangZhou, China) and cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM; Gibco) supplemented with the same additives as the HL-1 medium. Both cell lines were housed in a humidified incubator set at 37°C with a 5% CO2 atmosphere to maintain homeostatic culture conditions. Cellular experiments were performed during the exponential growth phase to ensure the cells exhibited consistent growth kinetics and biochemical responses. Quantification of Apoptosis Induced by Tubercidin Following exposure to Tubercidin for 1, 3, or 6 hours, cells were harvested, including supernatant fractions, and centrifuged at 1000 rpm for 5 minutes to pellet. Cells were washed twice with ice-cold PBS to remove culture medium components. Subsequently, cells were stained with Annexin V-FITC and PI according to the Cell Apoptosis Kit protocol (LIFE iLAB BIO, China), incubated in the dark for 15 minutes at room temperature, and analyzed using a flow cytometer (Invitrogen) to differentiate viable (Annexin V-FITC negative/PI negative), early apoptotic (Annexin V-FITC positive/PI negative), and late apoptotic or necrotic (Annexin V-FITC positive/PI positive) cells, thereby characterizing the apoptotic response over time. Immunofluorescence Assay Cells were cultured on coverslips in 24-well plates and treated with Tubercidin for 6 hours. After PBS rinses and 15-minute fixation with 4% PFA, cells were permeabilized with 0.5% Triton X-100. Following three PBST washes, cells were blocked with 10% FBS for 30 minutes and incubated overnight at 4°C with a 1:200 diluted mouse anti-SC35 primary antibody (Novus Biologicals, USA). The next day, following three PBST washes, cells received a 1-hour incubation with a 1:100 diluted Alexa 488-labeled goat anti-mouse IgG (ZSGD-BIO, China). Nuclei were stained with DAPI for 5 minutes, and coverslips were mounted using ProLong Gold Antifade Reagent (Thermo Fisher Scientific, USA) before imaging under a fluorescence microscope. Gene Expression Analysis by RT-PCR Total RNA was isolated from cells using TRIZOL Reagent (Sigma) and reverse-transcribed into cDNA (1 µg input) with the Reverse Transcription Kit (Biosharp, China). Subsequent RT-PCR employed M5 HiPer plus Taq HiFi PCR mix (Mei5 Biotechnology) for amplification. Specific primer pairs for GAPDH (5'-CGCCTGGAGAAACCTGC-3' and 5'-CGCCTGGAGAAACCTGC-3'), RNF144B (5'-CTCCAAGAGTGCCAGTGTATC-3' and 5'-CCATGTCAGGACAAGTGATAGG-3'), RIF1 (5'-CGTATGACTGGAGAAGAAGG-3' and 5'-GCCTACAATGAAGAAACCAAT-3'), and RFFL (5'-AGTACCTACTGAGGATGAGACC-3' and 5'-CGGTCAGGCCTTCAATGT-3') were used, with GAPDH serving as a normalizer. The PCR conditions were optimized as follows: an initial denaturation at 95°C for 3 minutes, followed by 30 cycles of 95°C for 1 minute, 60°C for 30 seconds, and 72°C for 30 seconds, with a final extension at 72°C for 10 minutes to ensure complete synthesis of the target amplicons. Statistical Analysis Experiments were conducted in triplicate, and data were subjected to statistical analysis using IBM SPSS Statistics 22.0. Differences among groups were assessed via one-way analysis of variance (ANOVA) followed by post hoc tests for pairwise comparisons, where appropriate. For two-group comparisons, an independent samples t-test was applied. A p-value of less than 0.05 was considered to denote statistical significance. Data visualization was performed using GraphPad Prism 8.0 to create informative and publication-quality graphs and charts. Results Enhanced Apoptosis in Mouse Cardiomyocytes Following Tubercidin Exposure Under Ischemic and Hypoxic Conditions This investigation revealed that exposure to Tubercidin significantly elevated apoptosis levels in FMC84 (Fig. 1 )and HL-1(Fig. 2 ) cardiomyocytes under conditions mimicking ischemia and hypoxia (Table 1 ). Under normoxic conditions with standard serum, Tubercidin (5 µg/mL) initiated apoptosis in FMC84 cells by 6 hours and consistently increased apoptosis in HL-1 cells throughout the 1 to 6-hour monitoring period. The apoptotic effect was markedly intensified under conditions of serum deprivation and hypoxia, with both cell lines exhibiting increased apoptosis at each monitored time point (1h, 3h, 6h). The concomitant presence of serum starvation and hypoxia was found to have a synergistic effect, significantly amplifying the cardiotoxic impact of Tubercidin and leading to a pronounced increase in apoptosis. These results underscore the heightened sensitivity of cardiomyocytes to the apoptotic effects of Tubercidin when subjected to combined stressors, providing insights into the mechanisms of drug-induced cardiotoxicity within the context of ischemic heart disease. S, P < 0.05; $ , Compared with 1% O 2 + 10% FBS, P < 0.05. (A) Apoptosis of FMC84 cells after 1 hour of Tubercidin treatment. (B) Apoptosis of FMC84 cells after 3 hours of Tubercidin treatment .(C) Apoptosis of FMC84 cells after 6 hours of Tubercidin treatment. (D) Percentage of apoptotic cells in each group. (*, Compared with Tubercidin (0 µg/ml), P < 0.05; #, Compared with 21% O2 + 10% FBS, P < 0.05; ∆, Compared with 21% O2 + 1% FBS, P < 0.05; $ , Compared with 1% O2 + 10% FBS, P < 0.05.) Table 1 The impact of Tubercidin on cell apoptosis of FMC84 and HL-1 cells under stress of serum starvation or/and hypoxia (Mean ± SD, n = 3) Groups Apoptotic rate(%) FMC84 HL-1 1h 3h 6h 1h 3h 6h 21% O 2 + 10% FBS Tubercidin (0 µg/ml) 4.52 ± 0.30 4.54 ± 0.43 5.89 ± 0.41 5.76 ± 0.35 6.49 ± 0.32 6.60 ± 0.27 Tubercidin (5 µg/ml) 4.62 ± 0.43 5.00 ± 0.36 7.53 ± 0.46* 6.89 ± 0.44* 8.00 ± 0.33* 8.08 ± 0.37* 21% O 2 + 1% FBS Tubercidin (0 µg/ml) 8.00 ± 0.48 # 8.60 ± 0.54 # 9.10 ± 0.51 # 6.93 ± 0.33 # 7.33 ± 0.36 # 7.71 ± 0.30 # Tubercidin (5 µg/ml) 14.33 ± 0.60* # 16.38 ± 0.58* # 19.37 ± 0.52* # 11.9 ± 0.41* # 12.41 ± 0.48* # 12.72 ± 0.39* # 1% O 2 + 10% FBS Tubercidin (0 µg/ml) 6.43 ± 0.46 # 6.42 ± 0.31 # 6.65 ± 0.39 # 6.60 ± 0.28 # 6.59 ± 0.29 7.17 ± 0.31 # Tubercidin (5 µg/ml) 11.59 ± 0.57* # 13.00 ± 0.46* # 16.54 ± 0.65* # 10.50 ± 0.45* # 11.10 ± 0.43* # 11.83 ± 0.47* # 1% O 2 + 1% FBS Tubercidin (0 µg/ml) 9.50 ± 0.40 #∆$ 9.12 ± 0.35 #∆$ 10.13 ± 0.46 #∆$ 8.22 ± 0.34 #∆$ 8.46 ± 0.38 #∆$ 9.71 ± 0.36 #∆$ Tubercidin (5 µg/ml) 18.98 ± 0.62* #∆$ 24.16 ± 0.48* #∆$ 41.90 ± 0.87* #∆$ 13.95 ± 0.36* #∆$ 14.39 ± 0.41* #∆$ 16.00 ± 0.48* #∆$ *, Compared with Tubercidin (0 µg/ml), P < 0.05; #, Compared with 21% O 2 + 10% FBS, P < 0.05. ∆, Compared with 21% O 2 + 1% FB Apoptosis of HL-1 cells after 1 hour of Tubercidin treatment. (B) Apoptosis of HL-1 cells after 3 hours of Tubercidin treatment .(C) Apoptosis of HL-1 cells after 6 hours of Tubercidin treatment. (D) Percentage of apoptotic cells in each group. (*, Compared with Tubercidin (0 µg/ml), P < 0.05; #, Compared with 21% O 2 + 10% FBS, P < 0.05; ∆, Compared with 21% O 2 + 1% FBS, P < 0.05; $ , Compared with 1% O 2 + 10% FBS, P < 0.05.) Tubercidin-Induced Condensation of SC35-Labeled Nuclear Speckles Under Ischemic and Hypoxic Stress The influence of Tubercidin on the structural integrity of nuclear speckles (NSs) was assessed through immunofluorescence staining with an anti-SC35 antibody. Our findings indicate that after 6 hours, a timepoint chosen based on the maximal condensation of NSs (19), cells exposed to both serum starvation and hypoxia exhibited a pronounced increase in NS size and a concomitant decrease in their number, suggesting a synergistic impact of these stressors on NSs assembly. Moreover, Tubercidin treatment under conditions of serum starvation or hypoxia, individually or in combination, consistently resulted in enlarged SC35-labeled NSs with a reduced count in FMC84 and HL-1 cells (Fig. 3 A & B). These observations imply that Tubercidin may induce condensation and fragmentation of NSs, which could subsequently impair the RNA processing functions associated with these cellular structures. Regulation of Anti-Apoptotic Gene Expression by Tubercidin in Conditions of Ischemia and Hypoxia This investigation assessed how Tubercidin affects the expression of key anti-apoptotic genes—RFFL, RNF144B, and RIF1—under ischemic and/or hypoxic stress. Given the role of exon skipping in altering the levels of major transcripts within nuclear speckles, we hypothesized that Tubercidin might modulate these transcripts. Using RT-PCR, we quantified the expression of the genes. In FMC84 cells, hypoxia, and serum starvation independently induced RFFL expression, which was then significantly diminished by Tubercidin, pointing to a potential disruption in splicing regulation (Fig. 4 A). In HL-1 cells, however, RFFL expression did not significantly vary with stress or Tubercidin exposure, indicating cell-specific responses to these conditions (Fig. 5 A). RIF1 expression followed a similar pattern, being upregulated by stress, and downregulated by Tubercidin in both cell lines (Figs. 4 B and 5 B). RNF144B expression in FMC84 cells was slightly increased by stress and significantly enhanced by Tubercidin, especially under normoxic conditions and serum starvation (Fig. 4 C). In contrast, RNF144B expression was not detected in HL-1 cells (Fig. 5 C), underscoring the cell type dependency of these effects. (A) After 6 hours of Tubercidin treatment, the relative expression of antiapoptotic gene RFFL in FMC84 decreased. (B) The relative expression of DNA repair and antiapoptotic gene RIF1 is reduced after Tubercidin treatment. (C) The relative expression of proapoptotic gene RNF144B in FMC84 is increased after Tubercidin treatment. These findings suggest that Tubercidin can alter the expression of anti-apoptotic genes, likely through the interference with splicing processes associated with nuclear speckles, with the extent of this effect being dependent on both the cell type and the combined stress of ischemia and hypoxia. (A) HL-1 has no significant change in the relative expression of anti-apoptosis gene RFFL after tubercidin treatment for 6 hours. (B) The relative expression of HL-1 involved in DNA repair and antiapoptotic gene RIF1 decreased after tubercidin treatment. (C) No proapoptotic gene RNF144B expression observed in HL-1 cells. Discussion Tubercidin, a purine ribonucleoside analog, has a broad-spectrum antimicrobial profile, effective against bacteria, fungi, and parasites, including Mycobacterium tuberculosis and Streptococcus faecalis (1). Its antiviral potential has also been recognized, with activity against SARS-CoV-2 [ 15 , 16 ]and influenza virus [ 17 ]. However, its clinical application is constrained by significant nephrotoxicity, leading to the discontinuation of trials for cancer chemotherapy. Consequently, pharmaceutical research has focused on structural modifications to reduce its toxicity [ 18 ]. In addition to its antimetabolic effects, Tubercidin has been identified as an inhibitor of nuclear speckle (NS) formation [ 14 ]. NSs, stress-induced nuclear bodies, are implicated in the assembly of splicing factors, which are crucial in the pathogenesis of ischemic heart disease. Our findings confirm that Tubercidin enhances cardiomyocyte apoptosis under conditions simulating ischemia and hypoxia, as evidenced by increased apoptosis in FMC84 and HL-1 cells (Figs. 1 & 2 ). This suggests that Tubercidin may exacerbate the cardiomyocyte toxicity associated with ischemic heart disease. Tubercidin's incorporation into nucleic acids leads to RNA processing disruptions, with NSs condensation observed upon treatment [ 19 ]. Our study extends these findings by showing that serum starvation and hypoxia increase the size and reduce the number of SC35-labeled NSs in cardiomyocytes, with Tubercidin further promoting NS condensation (Fig. 3 ). Alternative splicing within NSs, particularly exon skipping, is a common mechanism affecting the expression of apoptosis-related genes [ 20 – 22 ]. Our unpublished data indicate that the expression of RFFL, RIF1, and RNF144B, genes associated with apoptosis, is altered by the knockdown of an NS component. RFFL, a ubiquitin-protein ligase, is involved in the degradation of caspases and p53, thereby inhibiting apoptosis [ 23 , 24 ]. RIF1, a regulator of DNA repair, suppresses apoptosis under DNA damage [ 25 – 27 ]. RNF144B, a mitochondrial ubiquitin-protein transferase, negatively regulates apoptosis [ 28 , 29 ]. Our RT-qPCR analysis revealed that serum starvation and hypoxia upregulated RFFL and RIF1, while downregulating RNF144B, indicative of a cardiomyocyte stress response mechanism linked to NSs and RNA splicing. Tubercidin disrupts NSs-mediated expression of apoptotic genes and increases the cardiomyocyte apoptotic toxicity under stresses such as serum starvation and hypoxia through condensing NSs. Conclusion Tubercidin disrupted this stress-induced expression pattern, suggesting that it interferes with NSs-mediated regulation of apoptotic gene expression, potentially increasing cardiomyocyte toxicity under ischemic and hypoxic conditions (Fig. 6 ). These findings underscore the importance of considering Tubercidin's cardiotoxic potential, particularly in patients with ischemic cardiomyopathy, where its clinical use should be approached with caution. Declarations Ethics approval Not applicable. Patient consent for publication Not applicable. Competing interests The authors declare that they no competing interests. Funding This study was financially supported by the Guangxi BaGui Scholars Special Project and the Guilin City Science Research and Technological Development Program (Grant No. 2020011204-3). Author Contribution Guowen Shen contributed to data curation, investigation, validation, and was a major writer of the original draft. Qingni Cheng engaged in investigation and data curation, and played a role in drafting the manuscript. Lunmin Liang participated in investigation, data curation, visualization, and co-wrote the original draft. Yue Fu was involved in investigation and data curation, also contributing to the original draft. Yunzhu Cao assisted with investigation and data curation. Xiaoling Zhang was instrumental in conceptualizing the study, developing methodology, and was a key figure in software utilization, validation, visualization, and drafting the original manuscript. Quanzhong Li was pivotal in conceptualization, formal analysis, securing funding, project administration, and provided critical review and editing of the manuscript. Shengjun Xiao played a significant role in conceptualization, data curation, securing funding, and was a major contributor to the original writing, review, and editing of the manuscript. Acknowledgements Not applicable. Availability of data and materials The data generated in the present study may be requested from the corresponding author. References Swain, S. S., Paidesetty, S. K., & Padhy, R. N. (2017). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5366953","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":376382515,"identity":"b87584df-0ff0-46b2-a95d-545540d584bc","order_by":0,"name":"Guowen Shen","email":"","orcid":"","institution":"The Second Affiliated Hospital of Guilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guowen","middleName":"","lastName":"Shen","suffix":""},{"id":376382516,"identity":"dfc9053c-c8ff-4fba-afaa-3d44533d659b","order_by":1,"name":"Qingni Cheng","email":"","orcid":"","institution":"The Affiliated Hospital of Guilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qingni","middleName":"","lastName":"Cheng","suffix":""},{"id":376382518,"identity":"fb79407e-1249-49db-9122-64dd5b84b17a","order_by":2,"name":"Lunmin Liang","email":"","orcid":"","institution":"The Affiliated Hospital of Guilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lunmin","middleName":"","lastName":"Liang","suffix":""},{"id":376382521,"identity":"7683c444-bbb9-4cde-aa2d-514aa56bae51","order_by":3,"name":"Yaping Qin","email":"","orcid":"","institution":"The Second Affiliated Hospital of Guilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yaping","middleName":"","lastName":"Qin","suffix":""},{"id":376382528,"identity":"e385cfc0-42b5-43d4-a6dd-e511dc439915","order_by":4,"name":"Yunzhu Cao","email":"","orcid":"","institution":"Guilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yunzhu","middleName":"","lastName":"Cao","suffix":""},{"id":376382530,"identity":"ac3be3a8-ce1a-4e2a-94b0-5b0829c0edfb","order_by":5,"name":"Quanzhong Li","email":"","orcid":"","institution":"The Affiliated Hospital of Guilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Quanzhong","middleName":"","lastName":"Li","suffix":""},{"id":376382533,"identity":"d2b9cefa-baf5-459d-9f98-a6a2d4918c13","order_by":6,"name":"Shengjun Xiao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYDACZiBmbACxDjAwfGCwAAtKEK2FcQZUMX4tDHAtQO08xGgxOM788OHPHTZ58o5nzB7b/JKI5m9gPnibh8EuD5cWyWY2Y2PeM2nFhgfOmBvn9knkzjjAlmzNw5BcjEsLPzODmTRj2+HEjQ1nzKRzeyRyNzDwmEnzMBxIbMChhY2Z/Zvkz7b/EC2WYC383/Bq4WfmMZPgbTuQOJ8BqIXhB9gWNrxaJJt5io1525ITNzAcK5PsbQD65TCbseUcg2ScWgzOH9/48GebXeL8GYe3Sfz4Y5Pb39788MabCjucWhB6bxwARlAbAyRyGQwIqQcC+X6QqX+IUDkKRsEoGAUjDgAAccxVe6hmQ6EAAAAASUVORK5CYII=","orcid":"","institution":"The Second Affiliated Hospital of Guilin Medical University","correspondingAuthor":true,"prefix":"","firstName":"Shengjun","middleName":"","lastName":"Xiao","suffix":""}],"badges":[],"createdAt":"2024-10-31 10:53:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5366953/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5366953/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70286401,"identity":"2f9b1de4-0e3a-4879-8b9b-be7c5faefd58","added_by":"auto","created_at":"2024-12-01 16:35:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":247097,"visible":true,"origin":"","legend":"\u003cp\u003eTubercidin treatment increased apoptosis of mouse cardiomyocyte FMC84 under the stress of serum starvation or/and hypoxia.\u003c/p\u003e\n\u003cp\u003e(A) Apoptosis of FMC84 cells after 1 hour of Tubercidin treatment. (B) Apoptosis of FMC84 cells after 3 hours of Tubercidin treatment .(C) Apoptosis of FMC84 cells after 6 hours of Tubercidin treatment. (D) Percentage of apoptotic cells in each group. (*, Compared with Tubercidin (0 μg/ml), P \u0026lt; 0.05; #, Compared with 21% O2 + 10% FBS, P \u0026lt; 0.05; ∆, Compared with 21% O2 + 1% FBS, P \u0026lt; 0.05; $, Compared with 1% O2 + 10% FBS, P \u0026lt; 0.05.)\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5366953/v1/60ec61df297930eb95328ca4.png"},{"id":70286402,"identity":"b6b58139-83d9-4bb2-a7ff-3b4131511ae0","added_by":"auto","created_at":"2024-12-01 16:35:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":259653,"visible":true,"origin":"","legend":"\u003cp\u003eTubercidin treatment increased apoptosis of mouse cardiomyocyte HL-1 under the stress of serum starvation or/and hypoxia.\u003c/p\u003e\n\u003cp\u003e(A) Apoptosis of HL-1 cells after 1 hour of Tubercidin treatment. (B) Apoptosis of HL-1 cells after 3 hours of Tubercidin treatment .(C) Apoptosis of HL-1 cells after 6 hours of Tubercidin treatment. (D) Percentage of apoptotic cells in each group. (*, Compared with Tubercidin (0 μg/ml), \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; #, Compared with 21% O\u003csub\u003e2\u003c/sub\u003e + 10% FBS, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ∆, Compared with 21% O\u003csub\u003e2\u003c/sub\u003e + 1% FBS, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; $, Compared with 1% O\u003csub\u003e2\u003c/sub\u003e + 10% FBS, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.)\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5366953/v1/4f05817773cd9776d57d920b.png"},{"id":70286040,"identity":"7f5fd3c4-26b2-4045-9f3d-ecf6ae4d4341","added_by":"auto","created_at":"2024-12-01 16:27:55","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":194484,"visible":true,"origin":"","legend":"\u003cp\u003eSC35-labeled NSs aggregated in cardiomyocytes in mouse cardiomyocytes after tubercidin treatment for 6 hours. (A) The number SC35-labeled of NSs condensates deacreased and the size increased in FMC84 cells treated with tubercidin for 6 hours. (B) The number SC35-labeled of NSs condensates deacreased and the size increased in HL-1 cells treated with tubercidin for 6 hours. Bar, 10 μm.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5366953/v1/ef4ff5dc73188d409c9e751e.jpeg"},{"id":70286037,"identity":"c7e01517-c988-41f2-9c1e-3cab2071e89f","added_by":"auto","created_at":"2024-12-01 16:27:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":31228,"visible":true,"origin":"","legend":"\u003cp\u003eMajor transcripts of three apoptosis-related gene changes in FMC84 cells treated with Tubercidin for 6 hours.\u003c/p\u003e\n\u003cp\u003e(A) After 6 hours of Tubercidin treatment, the relative expression of antiapoptotic gene RFFL in FMC84 decreased. (B) The relative expression of DNA repair and antiapoptotic gene RIF1 is reduced after Tubercidin treatment. (C) The relative expression of proapoptotic gene RNF144B in FMC84 is increased after Tubercidin treatment.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5366953/v1/d2ba1edb1e201a9bed98f269.png"},{"id":70286035,"identity":"0e1272ed-29a5-41ff-a6e9-153a6a37eb40","added_by":"auto","created_at":"2024-12-01 16:27:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29816,"visible":true,"origin":"","legend":"\u003cp\u003eMajor transcripts of three apoptosis-related gene changes in HL-1 cells treated with Tubercidin for 6 hours.\u003c/p\u003e\n\u003cp\u003e(A) HL-1 has no significant change in the relative expression of anti-apoptosis gene RFFL after tubercidin treatment for 6 hours. (B) The relative expression of HL-1 involved in DNA repair and antiapoptotic gene RIF1 decreased after tubercidin treatment. (C) No proapoptotic gene RNF144B expression observed in HL-1 cells.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5366953/v1/2dd828139543e79f03b66cd1.png"},{"id":70286038,"identity":"f713caeb-d1b9-482c-8736-11eab9b2a7ff","added_by":"auto","created_at":"2024-12-01 16:27:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":33901,"visible":true,"origin":"","legend":"\u003cp\u003eThe proposed mechanism of the cardiomyocyte toxicity of Tubercidin\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5366953/v1/83b74bb3b90a0369599167b5.png"},{"id":71451169,"identity":"7752021d-9ebf-47eb-8cb9-7de3f1a4f1cb","added_by":"auto","created_at":"2024-12-15 17:01:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1554737,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5366953/v1/c306de46-8407-421a-9601-09755f4a4e6b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tubercidin Enhances Apoptosis in Serum-Starved and Hypoxic Mouse Cardiomyocytes by Inducing Nuclear Speckle Condensation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTubercidin, an adenosine analog derived from Streptomyces tubercidicus, possesses a broad spectrum of pharmacological activities, exerting a potent inhibitory influence on diverse microorganisms, including bacteria, fungi, viruses, and protozoa [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Moreover, this compound has demonstrated promise as a chemotherapeutic agent for tumor treatment [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Upon cellular uptake through nucleoside transporters, Tubercidin undergoes intracellular phosphorylation to generate mono-, di-, and triphosphate forms that competitively inhibit adenosine nucleotides, thereby disrupting polymerase activity and subsequently halting DNA replication, RNA transcription, and protein synthesis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Additionally, structural modifications of Tubercidin have been shown to confer inhibitory effects on adenosine kinase [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe clinical deployment of Tubercidin is significantly limited by its considerable hepatotoxicity, nephrotoxicity, and cardiotoxicity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], especially in individuals with cardiac comorbidities such as ischemic cardiomyopathy. Therefore, deciphering the molecular pathways leading to its cardiotoxic effects is essential for devising strategies to alleviate these adverse outcomes. In the context of schistosomiasis, combination therapies have been employed to ameliorate the toxicity profile and reduce patient side effects [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Furthermore, chemical modification of Tubercidin has been investigated as a means to diminish its toxicity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Despite these efforts, a thorough understanding of Tubercidin's toxicity mechanisms remains critical for enhancing its clinical safety profile.\u003c/p\u003e \u003cp\u003eNuclear speckles (NSs) are RNA-binding protein-rich nuclear bodies, numbering approximately 20 to 30 per eukaryotic nucleus [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. They are populated by a multitude of SR (Serine/Arginine-rich) proteins, which are pivotal in pre-mRNA splicing, mRNA transport, RNA stability, and translation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Consequently, NSs serve as key assembly and storage sites for pre-mRNA splicing machinery [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. During eukaryotic RNA splicing, SR proteins operate as essential splicing factors, in conjunction with snRNPs (small nuclear ribonucleoproteins) and other factors, to bind specific RNA sequences, identify splice sites, and facilitate both constitutive and alternative splicing [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The SR proteins are also instrumental in regulating apoptosis through the alternative splicing of genes associated with cell death [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], a process that is dependent on NSs [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To date, over ten SR protein varieties have been identified, with SRSF2 (Serine/Arginine-rich splicing factor 2), also known as SC35, being a critical member of the SR family and a central constituent of NSs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTubercidin has been observed to perturb the assembly of NSs and consequently disrupt mRNA processing. Following Tubercidin exposure, poly (A)\u0026thinsp;+\u0026thinsp;RNAs dispersed throughout the nucleoplasm undergo decay, whereas SC35-labeled NSs persist in a condensed state [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This suggests that Tubercidin specifically impairs mRNA processing within NSs without disassembling these condensed nuclear subcompartments.\u003c/p\u003e \u003cp\u003eNSs are known to form under various stress conditions, including hypoxia and serum starvation, with their RNA processing events presumed to aid in cellular stress survival. Therefore, the disruption of these RNA processing events by Tubercidin, including mRNA splicing, is hypothesized to augment cardiomyocyte toxicity in individuals with ischemic heart diseases. Utilizing murine cardiomyocytes, this study provides evidence that Tubercidin induces the condensation of NSs and exacerbates apoptosis under hypoxic and/or serum-starved conditions. These findings suggest a cautious approach to the clinical use of Tubercidin in patients with ischemic cardiomyopathy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaintenance of Cardiomyocyte Cell Cultures\u003c/h2\u003e \u003cp\u003eThe HL-1 cell line, derived from mouse atrial cardiomyocytes, was sourced from Cellcook (Guangzhou, China) and maintained in a customized Claycomb medium (Sigma-Aldrich). This medium was fortified with 10% fetal bovine serum (FBS; Gibco), 4 mM L-glutamine, 0.1 mM noradrenaline, and an antibiotic-antimycotic mix (100 U/mL penicillin and 100 \u0026micro;g/mL streptomycin) to support cell viability and prevent contamination. The FMC84 cell line, which models mouse ventricular cardiomyocytes, was provided by Jennio Biotech (GuangZhou, China) and cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM; Gibco) supplemented with the same additives as the HL-1 medium. Both cell lines were housed in a humidified incubator set at 37\u0026deg;C with a 5% CO2 atmosphere to maintain homeostatic culture conditions. Cellular experiments were performed during the exponential growth phase to ensure the cells exhibited consistent growth kinetics and biochemical responses.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eQuantification of Apoptosis Induced by Tubercidin\u003c/h3\u003e\n\u003cp\u003eFollowing exposure to Tubercidin for 1, 3, or 6 hours, cells were harvested, including supernatant fractions, and centrifuged at 1000 rpm for 5 minutes to pellet. Cells were washed twice with ice-cold PBS to remove culture medium components. Subsequently, cells were stained with Annexin V-FITC and PI according to the Cell Apoptosis Kit protocol (LIFE iLAB BIO, China), incubated in the dark for 15 minutes at room temperature, and analyzed using a flow cytometer (Invitrogen) to differentiate viable (Annexin V-FITC negative/PI negative), early apoptotic (Annexin V-FITC positive/PI negative), and late apoptotic or necrotic (Annexin V-FITC positive/PI positive) cells, thereby characterizing the apoptotic response over time.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence Assay\u003c/h3\u003e\n\u003cp\u003eCells were cultured on coverslips in 24-well plates and treated with Tubercidin for 6 hours. After PBS rinses and 15-minute fixation with 4% PFA, cells were permeabilized with 0.5% Triton X-100. Following three PBST washes, cells were blocked with 10% FBS for 30 minutes and incubated overnight at 4\u0026deg;C with a 1:200 diluted mouse anti-SC35 primary antibody (Novus Biologicals, USA). The next day, following three PBST washes, cells received a 1-hour incubation with a 1:100 diluted Alexa 488-labeled goat anti-mouse IgG (ZSGD-BIO, China). Nuclei were stained with DAPI for 5 minutes, and coverslips were mounted using ProLong Gold Antifade Reagent (Thermo Fisher Scientific, USA) before imaging under a fluorescence microscope.\u003c/p\u003e\n\u003ch3\u003eGene Expression Analysis by RT-PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated from cells using TRIZOL Reagent (Sigma) and reverse-transcribed into cDNA (1 \u0026micro;g input) with the Reverse Transcription Kit (Biosharp, China). Subsequent RT-PCR employed M5 HiPer plus Taq HiFi PCR mix (Mei5 Biotechnology) for amplification. Specific primer pairs for GAPDH (5'-CGCCTGGAGAAACCTGC-3' and 5'-CGCCTGGAGAAACCTGC-3'), RNF144B (5'-CTCCAAGAGTGCCAGTGTATC-3' and 5'-CCATGTCAGGACAAGTGATAGG-3'), RIF1 (5'-CGTATGACTGGAGAAGAAGG-3' and 5'-GCCTACAATGAAGAAACCAAT-3'), and RFFL (5'-AGTACCTACTGAGGATGAGACC-3' and 5'-CGGTCAGGCCTTCAATGT-3') were used, with GAPDH serving as a normalizer. The PCR conditions were optimized as follows: an initial denaturation at 95\u0026deg;C for 3 minutes, followed by 30 cycles of 95\u0026deg;C for 1 minute, 60\u0026deg;C for 30 seconds, and 72\u0026deg;C for 30 seconds, with a final extension at 72\u0026deg;C for 10 minutes to ensure complete synthesis of the target amplicons.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eExperiments were conducted in triplicate, and data were subjected to statistical analysis using IBM SPSS Statistics 22.0. Differences among groups were assessed via one-way analysis of variance (ANOVA) followed by post hoc tests for pairwise comparisons, where appropriate. For two-group comparisons, an independent samples t-test was applied. A p-value of less than 0.05 was considered to denote statistical significance. Data visualization was performed using GraphPad Prism 8.0 to create informative and publication-quality graphs and charts.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEnhanced Apoptosis in Mouse Cardiomyocytes Following Tubercidin Exposure Under Ischemic and Hypoxic Conditions\u003c/h2\u003e \u003cp\u003eThis investigation revealed that exposure to Tubercidin significantly elevated apoptosis levels in FMC84 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)and HL-1(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) cardiomyocytes under conditions mimicking ischemia and hypoxia (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Under normoxic conditions with standard serum, Tubercidin (5 \u0026micro;g/mL) initiated apoptosis in FMC84 cells by 6 hours and consistently increased apoptosis in HL-1 cells throughout the 1 to 6-hour monitoring period. The apoptotic effect was markedly intensified under conditions of serum deprivation and hypoxia, with both cell lines exhibiting increased apoptosis at each monitored time point (1h, 3h, 6h). The concomitant presence of serum starvation and hypoxia was found to have a synergistic effect, significantly amplifying the cardiotoxic impact of Tubercidin and leading to a pronounced increase in apoptosis. These results underscore the heightened sensitivity of cardiomyocytes to the apoptotic effects of Tubercidin when subjected to combined stressors, providing insights into the mechanisms of drug-induced cardiotoxicity within the context of ischemic heart disease.\u003c/p\u003e \u003cp\u003eS, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003cspan\u003e$\u003c/span\u003e, Compared with 1% O\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;10% FBS, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) Apoptosis of FMC84 cells after 1 hour of Tubercidin treatment. (B) Apoptosis of FMC84 cells after 3 hours of Tubercidin treatment .(C) Apoptosis of FMC84 cells after 6 hours of Tubercidin treatment. (D) Percentage of apoptotic cells in each group. (*, Compared with Tubercidin (0 \u0026micro;g/ml), P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; #, Compared with 21% O2\u0026thinsp;+\u0026thinsp;10% FBS, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ∆, Compared with 21% O2\u0026thinsp;+\u0026thinsp;1% FBS, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003cspan\u003e$\u003c/span\u003e, Compared with 1% O2\u0026thinsp;+\u0026thinsp;10% FBS, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe impact of Tubercidin on cell apoptosis of FMC84 and HL-1 cells under stress of serum starvation or/and hypoxia (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c8\" namest=\"c2\"\u003e \u003cp\u003eApoptotic rate(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eFMC84\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eHL-1\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6h\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e21% O\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u0026thinsp;\u003cb\u003e+\u0026thinsp;10% FBS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTubercidin (0 \u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e6.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e6.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTubercidin (5 \u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e7.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e6.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e8.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e8.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e21% O\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u0026thinsp;\u003cb\u003e+\u0026thinsp;1% FBS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTubercidin (0 \u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e8.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e9.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e6.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e7.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e7.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTubercidin (5 \u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e14.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60*\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e16.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58*\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e19.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52*\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e11.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41*\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e12.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48*\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e12.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39*\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1% O\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u0026thinsp;\u003cb\u003e+\u0026thinsp;10% FBS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTubercidin (0 \u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e6.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e6.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e7.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTubercidin (5 \u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57*\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e13.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46*\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e16.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65*\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e10.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45*\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e11.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43*\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e11.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47*\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1% O\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u0026thinsp;\u003cb\u003e+\u0026thinsp;1% FBS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTubercidin (0 \u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003e#∆$\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e9.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003e#∆$\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e10.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003csup\u003e#∆$\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e8.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003e#∆$\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e8.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003e#∆$\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e9.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003e#∆$\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTubercidin (5 \u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e18.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62*\u003csup\u003e#∆$\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e24.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48*\u003csup\u003e#∆$\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e41.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87*\u003csup\u003e#∆$\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e13.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36*\u003csup\u003e#∆$\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e14.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41*\u003csup\u003e#∆$\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e16.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48*\u003csup\u003e#∆$\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*, Compared with Tubercidin (0 \u0026micro;g/ml), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; #, Compared with 21% O\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;10% FBS, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e∆, Compared with 21% O\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;1% FB\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eApoptosis of HL-1 cells after 1 hour of Tubercidin treatment. (B) Apoptosis of HL-1 cells after 3 hours of Tubercidin treatment .(C) Apoptosis of HL-1 cells after 6 hours of Tubercidin treatment. (D) Percentage of apoptotic cells in each group. (*, Compared with Tubercidin (0 \u0026micro;g/ml), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; #, Compared with 21% O\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;10% FBS, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ∆, Compared with 21% O\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;1% FBS, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003cspan\u003e$\u003c/span\u003e, Compared with 1% O\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;10% FBS, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.)\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTubercidin-Induced Condensation of SC35-Labeled Nuclear Speckles Under Ischemic and Hypoxic Stress\u003c/h3\u003e\n\u003cp\u003eThe influence of Tubercidin on the structural integrity of nuclear speckles (NSs) was assessed through immunofluorescence staining with an anti-SC35 antibody. Our findings indicate that after 6 hours, a timepoint chosen based on the maximal condensation of NSs (19), cells exposed to both serum starvation and hypoxia exhibited a pronounced increase in NS size and a concomitant decrease in their number, suggesting a synergistic impact of these stressors on NSs assembly. Moreover, Tubercidin treatment under conditions of serum starvation or hypoxia, individually or in combination, consistently resulted in enlarged SC35-labeled NSs with a reduced count in FMC84 and HL-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u0026amp; B). These observations imply that Tubercidin may induce condensation and fragmentation of NSs, which could subsequently impair the RNA processing functions associated with these cellular structures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRegulation of Anti-Apoptotic Gene Expression by Tubercidin in Conditions of Ischemia and Hypoxia\u003c/h2\u003e \u003cp\u003eThis investigation assessed how Tubercidin affects the expression of key anti-apoptotic genes\u0026mdash;RFFL, RNF144B, and RIF1\u0026mdash;under ischemic and/or hypoxic stress. Given the role of exon skipping in altering the levels of major transcripts within nuclear speckles, we hypothesized that Tubercidin might modulate these transcripts. Using RT-PCR, we quantified the expression of the genes.\u003c/p\u003e \u003cp\u003eIn FMC84 cells, hypoxia, and serum starvation independently induced RFFL expression, which was then significantly diminished by Tubercidin, pointing to a potential disruption in splicing regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In HL-1 cells, however, RFFL expression did not significantly vary with stress or Tubercidin exposure, indicating cell-specific responses to these conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). RIF1 expression followed a similar pattern, being upregulated by stress, and downregulated by Tubercidin in both cell lines (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). RNF144B expression in FMC84 cells was slightly increased by stress and significantly enhanced by Tubercidin, especially under normoxic conditions and serum starvation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In contrast, RNF144B expression was not detected in HL-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), underscoring the cell type dependency of these effects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) After 6 hours of Tubercidin treatment, the relative expression of antiapoptotic gene RFFL in FMC84 decreased. (B) The relative expression of DNA repair and antiapoptotic gene RIF1 is reduced after Tubercidin treatment. (C) The relative expression of proapoptotic gene RNF144B in FMC84 is increased after Tubercidin treatment.\u003c/p\u003e \u003cp\u003eThese findings suggest that Tubercidin can alter the expression of anti-apoptotic genes, likely through the interference with splicing processes associated with nuclear speckles, with the extent of this effect being dependent on both the cell type and the combined stress of ischemia and hypoxia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) HL-1 has no significant change in the relative expression of anti-apoptosis gene RFFL after tubercidin treatment for 6 hours. (B) The relative expression of HL-1 involved in DNA repair and antiapoptotic gene RIF1 decreased after tubercidin treatment. (C) No proapoptotic gene RNF144B expression observed in HL-1 cells.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTubercidin, a purine ribonucleoside analog, has a broad-spectrum antimicrobial profile, effective against bacteria, fungi, and parasites, including Mycobacterium tuberculosis and Streptococcus faecalis (1). Its antiviral potential has also been recognized, with activity against SARS-CoV-2 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]and influenza virus [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, its clinical application is constrained by significant nephrotoxicity, leading to the discontinuation of trials for cancer chemotherapy. Consequently, pharmaceutical research has focused on structural modifications to reduce its toxicity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to its antimetabolic effects, Tubercidin has been identified as an inhibitor of nuclear speckle (NS) formation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. NSs, stress-induced nuclear bodies, are implicated in the assembly of splicing factors, which are crucial in the pathogenesis of ischemic heart disease. Our findings confirm that Tubercidin enhances cardiomyocyte apoptosis under conditions simulating ischemia and hypoxia, as evidenced by increased apoptosis in FMC84 and HL-1 cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This suggests that Tubercidin may exacerbate the cardiomyocyte toxicity associated with ischemic heart disease.\u003c/p\u003e \u003cp\u003eTubercidin's incorporation into nucleic acids leads to RNA processing disruptions, with NSs condensation observed upon treatment [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our study extends these findings by showing that serum starvation and hypoxia increase the size and reduce the number of SC35-labeled NSs in cardiomyocytes, with Tubercidin further promoting NS condensation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlternative splicing within NSs, particularly exon skipping, is a common mechanism affecting the expression of apoptosis-related genes [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Our unpublished data indicate that the expression of RFFL, RIF1, and RNF144B, genes associated with apoptosis, is altered by the knockdown of an NS component. RFFL, a ubiquitin-protein ligase, is involved in the degradation of caspases and p53, thereby inhibiting apoptosis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. RIF1, a regulator of DNA repair, suppresses apoptosis under DNA damage [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. RNF144B, a mitochondrial ubiquitin-protein transferase, negatively regulates apoptosis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Our RT-qPCR analysis revealed that serum starvation and hypoxia upregulated RFFL and RIF1, while downregulating RNF144B, indicative of a cardiomyocyte stress response mechanism linked to NSs and RNA splicing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTubercidin disrupts NSs-mediated expression of apoptotic genes and increases the cardiomyocyte apoptotic toxicity under stresses such as serum starvation and hypoxia through condensing NSs.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTubercidin disrupted this stress-induced expression pattern, suggesting that it interferes with NSs-mediated regulation of apoptotic gene expression, potentially increasing cardiomyocyte toxicity under ischemic and hypoxic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These findings underscore the importance of considering Tubercidin's cardiotoxic potential, particularly in patients with ischemic cardiomyopathy, where its clinical use should be approached with caution.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003ePatient consent for publication\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was financially supported by the Guangxi BaGui Scholars Special Project and the Guilin City Science Research and Technological Development Program (Grant No. 2020011204-3).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eGuowen Shen contributed to data curation, investigation, validation, and was a major writer of the original draft. Qingni Cheng engaged in investigation and data curation, and played a role in drafting the manuscript. Lunmin Liang participated in investigation, data curation, visualization, and co-wrote the original draft. Yue Fu was involved in investigation and data curation, also contributing to the original draft. Yunzhu Cao assisted with investigation and data curation. Xiaoling Zhang was instrumental in conceptualizing the study, developing methodology, and was a key figure in software utilization, validation, visualization, and drafting the original manuscript. Quanzhong Li was pivotal in conceptualization, formal analysis, securing funding, project administration, and provided critical review and editing of the manuscript. Shengjun Xiao played a significant role in conceptualization, data curation, securing funding, and was a major contributor to the original writing, review, and editing of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eThe data generated in the present study may be requested from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSwain, S. S., Paidesetty, S. K., \u0026amp; Padhy, R. N. (2017). Antibacterial, antifungal and antimycobacterial compounds from cyanobacteria. Biomedicine \u0026amp; pharmacotherapy\u0026thinsp;=\u0026thinsp;Biomedecine \u0026amp; pharmacotherapie, 90, 760\u0026ndash;776.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, J., Barrett, L., Lin, Z., Kendrick, S., Mu, S., Dai, L., \u0026amp; Qin, Z. (2022). 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The EMBO journal, 29(8), 1458\u0026ndash;1471.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Tubercidin, Apoptosis, Hypoxia, Nuclear speckles, Mouse Cardiomyocytes","lastPublishedDoi":"10.21203/rs.3.rs-5366953/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5366953/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTubercidin, known for its antimicrobial, antiparasitic, and anticancer effects, faces clinical limitations due to adverse effects, especially cardiotoxicity risks for those with ischemic cardiomyopathy. This study aims to clarify the molecular pathways of Tubercidin-induced cardiotoxicity, focusing on nuclear speckles (NSs) disruption in cardiomyocytes under serum deprivation and/or hypoxia. To simulate ischemic cardiomyopathy in vitro, we utilized FMC84 and HL-1 murine cardiomyocyte cell lines, exposing them to conditions of serum limitation and/or hypoxia to evaluate the cardiotoxic impact of Tubercidin and the contributing mechanisms. Apoptosis was quantified using flow cytometry, NSs condensation was visualized via immunofluorescence with an anti-SC35 antibody, and the expression levels of key apoptotic transcripts (RFFL, RIF1, and RNF144B) were analyzed by RT-PCR. Our findings revealed that Tubercidin significantly increased apoptosis in both HL-1 and FMC84 cell lines under conditions mimicking serum deprivation (21% O2 with 1% FBS), hypoxia (1% O2 with 10% FBS), or a combination of both. Furthermore, Tubercidin treatment led to a pronounced enlargement of NSs, as detected by immunofluorescence. Concurrently, we documented significant alterations in the expression of critical apoptotic regulatory genes, implying that Tubercidin may modulate the apoptotic pathway in stressed cardiomyocytes. It is hypothesized that Tubercidin induces NSs condensation, affecting alternative splicing of cell death genes, potentially worsening ischemic cardiomyocytes' damage. Therefore, a cautious clinical use of Tubercidin for ischemic cardiomyopathy patients is advised to reduce cardiotoxicity risks.\u003c/p\u003e","manuscriptTitle":"Tubercidin Enhances Apoptosis in Serum-Starved and Hypoxic Mouse Cardiomyocytes by Inducing Nuclear Speckle Condensation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-01 16:27:50","doi":"10.21203/rs.3.rs-5366953/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f20a99fe-06ba-4e21-8ff8-f1cb70bf12d2","owner":[],"postedDate":"December 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-18T11:23:10+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-01 16:27:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5366953","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5366953","identity":"rs-5366953","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}