HeLa Cells Override the Mitotic Entry Checkpoint by Highjacking the Survivin-Cdc25B-Cdk1 Pathway

HeLa Cells Override the Mitotic Entry Checkpoint by Highjacking the Survivin-Cdc25B-Cdk1 Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article HeLa Cells Override the Mitotic Entry Checkpoint by Highjacking the Survivin-Cdc25B-Cdk1 Pathway Pedro M. Cánovas This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3949429/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract The Survivin protein has roles in repairing incorrect microtubule-kinetochore attachments at prometaphase and the faithful execution of cytokinesis, as part of the C hromosomal P assenger C omplex (CPC). In its absence, mitotic errors often ensue that lead to chromosome missegregation, a cause of aneuploidy, polyploidy and ultimately cancer. Adding to these well-known roles of Survivin, this paper now shows for the first time that this protein is required for cancer cells to enter mitosis, and that, in its absence, HeLa cells accumulate at G2/early prophase, or prior to prometaphase, as claimed before. This early mitotic blockage is demonstrated by the inability of the Survivin-depleted cells to disassemble their nuclear lamina and their low Cdk1 activity. Not surprisingly, the Survivin-ablation arrest can be reproduced by a Survivin double mutant SUR D70A/D71A, which cannot bind to Cdk1. Also, escape from this mutant’s blockage leads to multiple mitotic defects, or mitotic catastrophe , and cell death. Mechanistically, Cdk1 does not localize at the centrosome in the absence of Survivin, pointing at this latter protein contributing to the activation of the mitotic kinase via Cdc25B. Furthermore, absence of Survivin leads to an inactive cytosolic Cdc25B-Cdk1-Cyclin B1 complex, which seems to indicate a role for Survivin in bridging this complex and its centrosomal activator/s. Interestingly, the drop in Cdk1 activity caused by interference with the Survivin function could be rescued when Survivin-depleted HeLa cell lysates were incubated with the recombinant Survivin protein. Also, a role for Survivin in the Cdc25B-mediated activation of Cdk1, and concomitant prophase to prometaphase transition could be confirmed by expression of a gain-of-function Cdc25B mutant, which overrode the G2/prophase blockage caused by Survivin depletion. Biological sciences/Cancer Biological sciences/Cell biology Survivin Cdk1 Cdc25B mitosis kinase phosphatase G2/M-phase checkpoint mitotic catastrophe cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Full Text Additional Declarations The authors declare no competing interests. Supplementary Files Suppl.Figure1.jpg Figure S1. Loss of Survivin causes an impairment in the prophase to prometaphase transition in HeLa cells. A , siRNA treatment of cells. B , Cell cycle phase of Cyclin B1-positive siRNA-treated HeLa cells. HeLa cells transfected with control (VIII) or Survivin (S4) siRNA were analyzed by fluorescence microscopy using an antibody to Cyclin Β1. DNA was stained with DAPI. Cells in prophase (P), prometaphase (PM), metaphase (M), anaphase (A), telophase (T) or multinucleated (MN) are indicated. C , Nuclear lamina integrity of siRNA-treated cells. Cells transfected with control (VIII) or Survivin (S4) siRNA were analyzed by fluorescence microscopy using an antibody to Cyclin Β1 and Lamin B. DNA was stained with DAPI. Cells in S-phase (S), prophase (P), prometaphase (PM) or metaphase (M), and intact lamina (IL) or spindle-like structures (SLS) are indicated. Suppl.Figure2.jpg Figure S2. Characterization of the Survivin-Cdk1 interaction in vitro. A , Survivin directly interacts with Cdk1 in vitro : Cdk1-binding Survivin region. GST, GST-Survivin or the indicated GST-Survivin deletion mutant (numbers correspond to amino acid residues in the Survivin sequence) were mixed with His-Cdk1, and bound proteins were analyzed by Western blotting. Membranes were stained with Coomassie Blue to check the amount of the GST-Survivin fragments (*). B , Survivin-binding Cdk1 region. GST-Survivin ( left ) or biotinylated SUR A55-D70 peptide ( right ) were mixed with His-tagged full-length or the indicated Cdk1 fragment (numbers correspond to amino acid residues in the Cdk1 sequence) followed by Western blotting. Suppl.Figure3.jpg Figure S3. Regions involved in the Survivin-Cdk1 complex. A , Cdk1-binding Survivin region. B , Survivin-binding Cdk1 region. C , SUR A55-D70 peptide superimposed on the N-terminal Survivin region. D , Survivin-binding Cdk1 region superimposed on N-terminal Cdk1 region. All protein sequence and functional information used to generate Survivin’s and Cdk1’s graphs and cartoons was extracted from UniProt. Suppl.Figure4.jpg Figure S4. Characterization of the SUR A55-D70 peptide. A , SUR A55-D70 pull down of endogenous Cdk1. HeLa cell lysates were used to pull down Cdk1 using a biotin-conjugated scrambled or Survivin A55-D70 (SUR A55-D70) peptide. Protein complexes were analyzed by Western blotting. B , SUR A55-D70-mediated disassembly and loss of kinase activity of a Survivin-Cdk1 mitotic complex. Lysates were prepared from nocodazole-treated HeLa cells, and incubated with the scrambled or SUR A55-D70 peptide. Cdk1 was IP, and immune complexes were analyzed in a Histone H1 phosphorylation assay or by Western blotting. C , Peptide delivery into HeLa cell cytosol. Cells were incubated with biotinylated, HIV tat-scrambled or SUR A55-D70 peptide, and analyzed by fluorescence microscopy using Texas Red-streptavidin. DNA was stained with DAPI. Suppl.Figure5.jpg Figure S5 . The Survivin Asp70Ala/Asp71Ala (SUR D70A/D71A) double mutant does not bind to Cdk1. Alanine Scanning Mutagenesis of the SUR A55-D70 peptide. The Survivin region spanning the SUR A55-D70 peptide was subjected to alanine mutagenesis, and single alanine GST-Survivin mutants or GST alone were analyzed for their binding to His-Cdk1 by Western blotting ( top ) (*: Two gels had to be run in order to analyze all the mutants). Binding of SUR D70A/D71A to His-Cdk1 was compared to mutant SUR D71A ( bottom ). An unrelated SUR F58A mutant was used as control. Membranes were stained with Coomassie Blue to check the amount of the GST-Survivin mutants. Suppl.Figure6.jpg Figure S6. Detection of a Hsp90-Survivin-Cdk1 complex at mitosis in HeLa cells. Synchronized cells were lysed, IP with an antibody to Hsp90, and analyzed by Western blotting. Suppl.Figure7A.jpg Figure S7. A: Original Figure 8B - Centrosomal Cdk1 levels in the presence and absence of Survivin in HeLa cells . Asynchronous cells were transfected with the control (VIII) or Survivin (S4) siRNA. Centrosomes were isolated and analyzed by Western blotting. Initially, the levels of γ-Tubulin, Cdk1 and Survivin were checked. Then, the same membrane was reprobed with antibodies against RCC1, Ran, Caspase 3 and p21. B: Original Figure 8C - Cdk1 and Survivin panels. Centrosomal Cdk1 and Survivin levels following siRNA treatment of HeLa cells . Synchronized cells treated with VIII ( top ) or S4 ( bottom ) siRNA were released, and samples were taken from 12 to 18 h. Centrosomes were isolated, and Cdk1 and Survivin levels were analyzed. Samples taken at 16 h after both treatments were loaded in reverse fashion, and omitted from Figure 8C. C: Original Figure 8C - P32-Histone H1 panel. Centrosomal Cdk1 activity following siRNA treatment of HeLa cells . Synchronized cells treated with VIII ( top ) or S4 ( bottom ) siRNA were released, and samples taken from 12 to 18 h. Centrosomes were isolated, and Cdk1 activity measured. Samples taken at 16 h after both treatments were loaded in reverse fashion, and omitted from Figure 8C. Suppl.Figure7BC.jpg Figure S7. A: Original Figure 8B - Centrosomal Cdk1 levels in the presence and absence of Survivin in HeLa cells . Asynchronous cells were transfected with the control (VIII) or Survivin (S4) siRNA. Centrosomes were isolated and analyzed by Western blotting. Initially, the levels of γ-Tubulin, Cdk1 and Survivin were checked. Then, the same membrane was reprobed with antibodies against RCC1, Ran, Caspase 3 and p21. B: Original Figure 8C - Cdk1 and Survivin panels. Centrosomal Cdk1 and Survivin levels following siRNA treatment of HeLa cells . Synchronized cells treated with VIII ( top ) or S4 ( bottom ) siRNA were released, and samples were taken from 12 to 18 h. Centrosomes were isolated, and Cdk1 and Survivin levels were analyzed. Samples taken at 16 h after both treatments were loaded in reverse fashion, and omitted from Figure 8C. C: Original Figure 8C - P32-Histone H1 panel. Centrosomal Cdk1 activity following siRNA treatment of HeLa cells . Synchronized cells treated with VIII ( top ) or S4 ( bottom ) siRNA were released, and samples taken from 12 to 18 h. Centrosomes were isolated, and Cdk1 activity measured. Samples taken at 16 h after both treatments were loaded in reverse fashion, and omitted from Figure 8C. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3949429","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":275344343,"identity":"c929316e-0f7b-492f-8d30-d6ee1892dbae","order_by":0,"name":"Pedro M. Cánovas","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYHACAwTzYwOIZGw8QLQWxpkNDBJAqoF4Lcy8YC0MDHi1mLM3b/vwocYuj18i+fFn2x02dbrth4G21NhE49Ji2XOseOaMY8nFkjPSzKRzz6RJmJ1JBGo5lpbbgMtVN3KMge5hTtxw5oAZc27bYQmzA0AtjA2HcWu5/wakpR6o5fjnz5YgLecfEtBygwek5XDihuM9BtKMIC03CNhi2ZNWzDjj2PHEme09ZZK9bWmS224AbUnA4xdz9sObGT7UVCf2M7Nv/vCzzYbf7Hz6wwcfamxwOwy7cAIO5Xi0jIJRMApGwShAAgBsIWKkZ7zaQQAAAABJRU5ErkJggg==","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Pedro","middleName":"M.","lastName":"Cánovas","suffix":""}],"badges":[],"createdAt":"2024-02-11 21:47:12","currentVersionCode":2,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-3949429/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-3949429/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90693625,"identity":"4f646c7b-ac88-44d9-8dab-1fd8a6847c44","added_by":"auto","created_at":"2025-09-05 19:11:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2314782,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003esiRNA-mediated loss of Survivin induces an early prophase blockage in asynchronous HeLa cell cultures. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eA\u003c/em\u003e, Survivin knockdown. Cells transfected with control (VIII) or Survivin (S4) siRNA were analyzed by Western blotting (\u003cem\u003eleft\u003c/em\u003e) or FACS analysis (\u003cem\u003eright\u003c/em\u003e). \u003cem\u003eB\u003c/em\u003e, Survivin knockdown cells. Cells transfected with the indicated siRNA were analyzed by Western blotting (\u003cem\u003eleft\u003c/em\u003e) or fluorescence microscopy using an antibody to α-Tubulin (\u003cem\u003eright\u003c/em\u003e). DNA was stained with DAPI. \u003cem\u003eC\u003c/em\u003e, Absence of spindle in knockdown cells. Cells transfected with the indicated siRNA were analyzed by fluorescence microscopy (\u003cem\u003eleft\u003c/em\u003e). Cells were incubated with antibodies against α-Tubulin (\u003cem\u003etop\u003c/em\u003e) and Survivin (\u003cem\u003emiddle\u003c/em\u003e). DNA was stained with DAPI. \u003cem\u003eRight\u003c/em\u003e, One cell transfected control (VIII) or Survivin (S4) siRNA at higher magnification. \u003cem\u003eD\u003c/em\u003e, Bar graph shows the percentage of mitotic cells in siRNA transfected cultures (n=4).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/50aff12ce384d2cac09be202.jpg"},{"id":90692782,"identity":"7fe2d012-fd72-4b1d-8fd4-e201d609714c","added_by":"auto","created_at":"2025-09-05 18:47:01","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2583878,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eNuclear lamina stays assembled in Survivin-depleted HeLa cells.\u003c/strong\u003e\u003c/em\u003e \u003cem\u003eA\u003c/em\u003e, Nuclear lamina integrity in siRNA-transfected cells. Cells transfected with the indicated siRNA were analyzed by fluorescence microscopy using an antibody to α-Tubulin (\u003cem\u003eleft\u003c/em\u003e), and Survivin (\u003cem\u003etop middle\u003c/em\u003e) or Lamin B (\u003cem\u003ebottom middle\u003c/em\u003e). DNA was stained with DAPI. Several cells are labeled with arrow heads (S: Interphase, P: Prophase, PM: Prometaphase, M: Metaphase, T: Telophase, MN: Multinucleated, IL: Intact lamina and SLS: Spindle-like structure). \u003cem\u003eB\u003c/em\u003e, Nuclear lamina in interphase or mitotic cells. Cells were transfected with the indicated siRNA, stained with an antibody to Lamin B and α-Tubulin, and analyzed by fluorescence microscopy. DNA was stained with DAPI. Cells are shown at the same scale. Arrows show intact nuclear lamina, and arrow head points out at Lamin B colocalization with the mitotic spindle. \u003cem\u003eC\u003c/em\u003e, Percentage of rounded mitotic cells with intact or disassembled nuclear lamina. Rounded mitotic cells transfected and stained as in \u003cem\u003eA\u003c/em\u003e were tallied for the presence or absence of an intact lamina (n=3). \u003cem\u003eD\u003c/em\u003e, Purvalanol A treatment of cells. Cells were treated with the Cdk1 inhibitor purvalanol A (20 μM), stained with an antibody to Lamin B and α-Tubulin, and analyzed by fluorescence microscopy. DNA was stained with DAPI.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/dbc6f09dabc8609c35ee2aa2.jpg"},{"id":90693207,"identity":"3c5ff033-b080-436e-9095-ca7bde74ec26","added_by":"auto","created_at":"2025-09-05 18:55:01","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":518852,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCdk1 activity is lower in Survivin-depleted HeLa cells.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e A\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eCdk1 kinase assay. siRNA-transfected asynchronous cell lysates were used to immunoprecipitate (IP) Cdk1, and the kinase activity was analyzed by a Histone H1 phosphorylation assay (\u003cem\u003eright\u003c/em\u003e). Amounts of Cyclin B1, Cdk1 and Survivin were analyzed by Western blotting as a control (\u003cem\u003eleft\u003c/em\u003e). \u003cem\u003eB\u003c/em\u003e, Survivin ablation vs. prometaphase blockage. Cells were treated with 10 μM nocodazole, and their Cdk1 activity was analyzed by an IP and a Histone H1 phosphorylation assay. Survivin siRNA-transfected HeLa cells were used as a control.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/1580889855473dc70299bfca.jpg"},{"id":90693214,"identity":"8f37f824-c3d4-4b65-b449-bd267570d886","added_by":"auto","created_at":"2025-09-05 18:55:01","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1482289,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSurvivin abrogation in synchronous HeLa cells causes a sustained G2/M-phase blockage and diminished Cdk1 activity. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eA\u003c/em\u003e, Cell cycle analysis of untreated synchronous cells. Cells were synchronized with 2 mM thymidine for 48 h, released into fresh medium, and harvested at the indicated time intervals. Collected samples were subjected to an IP using an antibody to Survivin, and analyzed by Western blotting (\u003cem\u003eleft\u003c/em\u003e) or FACS analysis (\u003cem\u003eright\u003c/em\u003e). \u003cem\u003eB\u003c/em\u003e, Cell cycle analysis of synchronous cells depleted of Survivin. Cells transfected with control (VIII) or Survivin (S4) siRNA were synchronized as in \u003cem\u003eA\u003c/em\u003e, released into fresh medium, and harvested at the indicated time intervals. Collected samples were subjected to Western blotting using a Survivin or Cdk1 antibody (\u003cem\u003etop\u003c/em\u003e), or FACS analysis (\u003cem\u003ebottom\u003c/em\u003e). \u003cem\u003eC\u003c/em\u003e, Cdk1 activity during mitotic transition. Synchronized cells, previously transfected with the indicated siRNA, were harvested when they transitioned through mitosis, and analyzed by FACS (\u003cem\u003eleft\u003c/em\u003e). Lysates were prepared and used to IP Cdk1, and the immune complexes were analyzed in a Histone H1 phosphorylation assay (\u003cem\u003etop right\u003c/em\u003e). The amount of Survivin in the \u0026nbsp;lysates is shown as a control (\u003cem\u003ebottom right\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/79a2e7579657647490083eb6.jpg"},{"id":90693212,"identity":"280b00bc-111e-4f80-9cd2-abbb1bf4f48d","added_by":"auto","created_at":"2025-09-05 18:55:01","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":865946,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eTreatment of HeLa cells with the SUR A55-D70 peptide causes spindle abnormalities. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eA\u003c/em\u003e,\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003eSpindle abnormalities caused by the SUR A55-D70 peptide. Asynchronous cell cultures were transfected with 50 μM scrambled or SUR A55-D70 peptide for 6 h, and cells were stained with an antibody to α-Tubulin. DNA was stained with DAPI. \u003cem\u003eB\u003c/em\u003e, Mitotic phenotypes were quantified (n=2).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/69b817c55f08aa173f3623fd.jpg"},{"id":90693216,"identity":"eca42205-296d-4c2b-b101-e34fe6d11733","added_by":"auto","created_at":"2025-09-05 18:55:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":11427177,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eProlonged incubation of HeLa cells with the SUR A55-D70 peptide causes apoptosis. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eA\u003c/em\u003e, SUR A55-D70 peptide-induced cell death. Asynchronous cell cultures were treated with 50 μM scrambled or SUR A55-D70 peptides for 24 h, and cells were analyzed by FACS (\u003cem\u003etop\u003c/em\u003e), or phase-contrast microscopy (\u003cem\u003ebottom\u003c/em\u003e). Images represent one of several experiments (n=4). The percentage of apoptotic (\u0026lt;\u003cem\u003e2N\u003c/em\u003e), G1 (\u003cem\u003e2N\u003c/em\u003e) and G2/M-phase (\u003cem\u003e4N\u003c/em\u003e) cells is indicated at \u003cem\u003etop\u003c/em\u003e. Dead cells showing some kind of dark material inside are indicated by arrows. \u003cem\u003eB\u003c/em\u003e, SUR A55-D70 peptide dose response of transfected HeLa cells. Asynchronous cell cultures were treated with 200 μM scrambled or SUR A55-D70 peptides for 24 h, and cells were analyzed by FACS (\u003cem\u003eleft\u003c/em\u003e), phase-contrast microscopy (\u003cem\u003emiddle\u003c/em\u003e) or Trypan Blue staining (\u003cem\u003eright\u003c/em\u003e). Images represent one of several experiments (n=4). The percentage of apoptotic (\u0026lt;\u003cem\u003e2N\u003c/em\u003e), G1 (\u003cem\u003e2N\u003c/em\u003e) and G2/M-phase (\u003cem\u003e4N\u003c/em\u003e) cells is indicated on \u003cem\u003eleft\u003c/em\u003e. Dead cells showing some kind of dark material inside are indicated by arrows, and dead cells looking like bubbles are indicated by arrow heads. \u003cem\u003eC\u003c/em\u003e, SUR A55-D70 peptide-induced apoptosis. Asynchronous cell cultures were transfected with 200 μM scrambled or SUR A55-D70 peptides, and cells were analyzed by phase-contrast microscopy (\u003cem\u003eleft\u003c/em\u003e) and FAM-DEVD-FMK fluorescence microscopy (\u003cem\u003eright\u003c/em\u003e) after 24 h. Images represent one of several experiments (n=3). \u003cem\u003eD\u003c/em\u003e, SUR A55-D70 peptide activation of caspase 3. Cells treated as in C for 24-48 h were incubated with an antibody to the caspase 3 proform. \u003cem\u003eE\u003c/em\u003e, SUR A55-D70 peptide-induced apoptosis during G2/M-phase. G2/M-phase synchronized cells were transfected with 200 μM scrambled or SUR A55-D70 peptides for 12 h, and analyzed by FACS (\u003cem\u003etop\u003c/em\u003e), or phase-contrast microscopy (\u003cem\u003ebottom\u003c/em\u003e). The percentage of apoptotic (\u0026lt;\u003cem\u003e2N)\u003c/em\u003e, G1 (\u003cem\u003e2N\u003c/em\u003e) and G2/M-phase (\u003cem\u003e4N\u003c/em\u003e) cells is indicated.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/95a4760f8f2b4e6076a0d00a.png"},{"id":90692796,"identity":"13671236-d537-416a-ada4-bf2063f5fb18","added_by":"auto","created_at":"2025-09-05 18:47:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":8687333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eThe Survivin Asp70Ala/Asp71Ala double mutant (SUR D70A/D71A) causes G2/M-phase arrest, mitotic abnormalities and cell death in HeLa cells. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eA\u003c/em\u003e, SUR D70A/D71A causes mitotic abnormalities. Asynchronous cell cultures were transfected with constructs expressing HA-tagged wild type Survivin (HA-SUR WT) or SUR D70A/D71A (HA-SUR D70A/D71A), and subjected to Western blotting using antibodies to HA-Tag, Survivin or β-Actin (left), or analyzed by fluorescence microscopy using antibodies to HA-Tag or α-Tubulin (\u003cem\u003eright\u003c/em\u003e). DNA was stained with DAPI. Cells are labeled according to their mitotic phase or chromatin fragmentation. \u003cem\u003eB\u003c/em\u003e, Phenotypes observed in SUR D70A/D71A-expressing cells. Graph shows the averages of two independent experiments. \u003cem\u003eC\u003c/em\u003e, Cdk1 activity in SUR D70A/D71A-expressing cells. HA-SUR WT- or HA-SUR D70A/D71A-transfected cell lysates were used to IP Cdk1, and pellets were analyzed in a Histone H1 phosphorylation assay (\u003cem\u003etop\u003c/em\u003e), or by Western blotting (\u003cem\u003ebottom\u003c/em\u003e). \u003cem\u003eD\u003c/em\u003e, Detection of phosphorylated inactive Cdk1 form in cells expressing SUR D70A/D71A. Parallel samples as in \u003cem\u003eC \u003c/em\u003ewere used to determine the level of inactive Cdk1 by Western blotting. \u003cem\u003eE\u003c/em\u003e, \u003cem\u003eF\u003c/em\u003e, Time-lapse video microscopy of SUR D70A/D71A-expressing cells. Synchronous cell cultures were transfected with GFP-SUR WT or GFP-SUR D70A/D71A, released into fresh medium, and imaged every 10 min continuously for 24 h. Images were taken at the indicated time points. \u003cem\u003eE\u003c/em\u003e, Individual cells (bars), transfected with GFP-SUR WT (\u003cem\u003etop\u003c/em\u003e) or GFP-SUR D70/D71A mutant (\u003cem\u003ebottom\u003c/em\u003e) were quantified for time spent at each indicated mitotic transition and assigned specific phenotypes (see legend). \u003cem\u003eBottom\u003c/em\u003e \u003cem\u003eF\u003c/em\u003e, Selected cells undergoing sustained mitotic arrest (magenta and light blue arrows), or cell death after mitosis re-entry (yellow arrow) are shown. \u003cem\u003eTop F\u003c/em\u003e, White arrow shows a control-transfected cell that progressed normally through mitosis.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/a752913bfbf7a39a1c21d23f.png"},{"id":90692801,"identity":"be09ac83-e401-491d-a12f-8e769e68ecec","added_by":"auto","created_at":"2025-09-05 18:47:01","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":6612974,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSurvivin is needed to recruit Cdk1 to Hela cell centrosomes. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eA\u003c/em\u003e, Fast-migrating centrosomal Survivin-Cdk1 complex at mitosis. Centrosomes were isolated from lysates of untreated synchronous HeLa cell cultures, subjected to Survivin IP, and analyzed by Western blotting using antibodies to Cdk1 or Survivin (\u003cem\u003eleft\u003c/em\u003e). \u003cem\u003eB\u003c/em\u003e, Centrosomal Cdk1 levels in cells depleted of Survivin. Centrosomes were isolated from asynchronous HeLa cells transfected with control (VIII) or Survivin (S4) siRNA, and analyzed by Western blotting using several antibodies (see original data in Fig. S7A). \u003cem\u003eC\u003c/em\u003e, Centrosomal Cdk1 levels and activity in siRNA-treated synchronous HeLa cultures. Centrosomal preparations from synchronized HeLa cells transfected with the indicated siRNA that were collected after release into fresh media at the mentioned times, were analyzed by FACS (\u003cem\u003ebottom\u003c/em\u003e) or IP with an antibody to Cdk1, and pellets were analyzed by Western blotting or a Histone H1 phosphorylation assay (*: 16 h time point was omitted in both treatments due to a loading error (see original data in Fig. S7B). The right panel shows the levels of Cyclin B1, Cdk1 and Survivin in the lysate as a control. \u003cem\u003eD\u003c/em\u003e, Centrosomal Cdk1 signal in Survivin-depleted cells. siRNA-treated cells were analyzed by fluorescence microscopy using an antibody to Cdk1 (\u003cem\u003etop\u003c/em\u003e). DNA was stained with DAPI. Control cells (VIII) were either in early prophase (a, b), prometaphase (c, d) or metaphase (e, f), and Survivin-depleted cells (S4) were in S-phase/G2 (g, h) or early prophase (i-l). Percentage of splitted Cdk1 signal was scored (\u003cem\u003ebottom\u003c/em\u003e) (n=3).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/b2bbe401d1714e1797c5ab27.png"},{"id":90693215,"identity":"9f41143f-320c-4ff1-b054-0f3a43ea443d","added_by":"auto","created_at":"2025-09-05 18:55:01","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1637137,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCdc25 activity is induced by recombinant Survivin in vitro. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eA\u003c/em\u003e, Phosphorylated inactive Cdk1 isoform in Survivin-depleted Hela cells. Shaken-off cells transfected with control (VIII) or Survivin (S4) siRNA were analyzed by Western blotting using several antibodies (\u003cem\u003eleft\u003c/em\u003e) or a Histone H1 phosphorylation assay (\u003cem\u003eright\u003c/em\u003e). \u003cem\u003eB\u003c/em\u003e, Impaired Cdc25 activity in Survivin-knocked down mitotic HeLa cells. Synchronized mitotic cells (14 h) transfected with the indicated siRNA were collected, and analyzed by Western blotting. \u003cem\u003eC\u003c/em\u003e, \u003cem\u003eIn vitro\u003c/em\u003e induction of Cdc25 activity by recombinant Survivin. G2/M-phase HeLa cell lysates were immunodepleted of Survivin (\u003cem\u003eleft\u003c/em\u003e), supplemented with an ATP-regenerating system, incubated with GST or GST-Survivin, used to IP Cdk1, and analyzed by Western blotting (\u003cem\u003etop\u003c/em\u003e \u003cem\u003eright\u003c/em\u003e). Binding of the recombinant GST-Survivin protein (*) to the immunoprecipitated Cdk1 was checked by staining the IP membrane with Coomassie Blue. U indicates a nonspecific protein band. \u003cem\u003eD\u003c/em\u003e, \u003cem\u003eE\u003c/em\u003e, Cdk1 activation by recombinant Survivin in interphase. \u003cem\u003eD\u003c/em\u003e, Interphase HeLa cell lysates (\u003cem\u003etop left\u003c/em\u003e), depleted of Survivin (\u003cem\u003ebottom left\u003c/em\u003e), and supplemented with an ATP-regenerating system, were incubated with different concentrations of GST or GST-Survivin, and analyzed by Western blotting (\u003cem\u003etop right\u003c/em\u003e), or a Histone H1 phosphorylation assay (\u003cem\u003eE\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/3bea0320619e699d45f714e8.jpg"},{"id":90692803,"identity":"8c7ec69d-934b-4699-bb30-75b162ecb6f9","added_by":"auto","created_at":"2025-09-05 18:47:01","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1839159,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSurvivin regulation of Cdc25B phosphatase activity in HeLa cells. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eA\u003c/em\u003e, Survivin binds directly to Cdc25B. GST or GST-Survivin were mixed with His-Cdc25B, and analyzed by Western blotting. \u003cem\u003eB\u003c/em\u003e, Accumulation of a cytosolic Cdc25B-Cdk1-Cyclin B1 complex in the absence of Survivin. Asynchronized cells transfected with control (VIII) or Survivin (S4) siRNA were collected, lysates were used to IP Cyclin B1, and pellets were analyzed by Western blotting. \u003cem\u003eC\u003c/em\u003e, Cdc25B accumulation in Survivin-depleted cell cultures. Asynchronous (\u003cem\u003etop\u003c/em\u003e) or synchronous (\u003cem\u003ebottom\u003c/em\u003e) cells treated with the indicated siRNA were analyzed by Western blotting. \u003cem\u003eD\u003c/em\u003e, Cdc25B activity at mitosis onset in siRNA-treated cells. Synchronized HeLa cells transfected with the indicated siRNA were collected at G2/M-phase (\u003cem\u003etop left\u003c/em\u003e), analyzed for Survivin expression (\u003cem\u003ebottom left\u003c/em\u003e), and used to IP Cdc25B (\u003cem\u003ebottom right\u003c/em\u003e). Cdc25B phosphatase activity in the immune complexes was measured by the rate of OMFP hydrolysis (\u003cem\u003etop right\u003c/em\u003e). \u003cem\u003eE\u003c/em\u003e, Quantification of several experiments as in D (n=4) is shown. \u003cem\u003eF\u003c/em\u003e, Cdc25B activity during the cell cycle in siRNA-treated cells. Synchronized cells transfected with the indicated siRNA were collected at the indicated times following thymidine release, analyzed by FACS (\u003cem\u003ebottom\u003c/em\u003e), and used to IP Cyclin B1. Immune complexes were analyzed by Western blotting (\u003cem\u003etop\u003c/em\u003e \u003cem\u003eleft\u003c/em\u003e). Cdc25B was IP from the same samples and its activity measured by the rate of OMFP hydrolysis (\u003cem\u003eright\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/71eb4f6e1322847034b5f797.jpg"},{"id":90693213,"identity":"54da1959-6df8-430b-b047-8f0491dd0157","added_by":"auto","created_at":"2025-09-05 18:55:01","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":2134910,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eA gain-of-function Cdc25B mutant can override the blockage induced by Survivin abrogation in HeLa cells. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eA\u003c/em\u003e, Cdc25B-mediated mitotic entry. Cells transfected with control (VIII) or Survivin (S4) siRNA were synchronized for 40 h. Cells were then transfected with a pcDNA3 or pCdc25B plasmid, and placed back in thymidine for another 8 h. Upon release, cells were collected at 16 h, as they transited through G2/M-phase. Cells were analyzed by Western blotting (\u003cem\u003eleft\u003c/em\u003e) and FACS analysis (\u003cem\u003eright\u003c/em\u003e). The percentage of cells in apoptosis (\u0026lt;\u003cem\u003e2N\u003c/em\u003e), G1 (\u003cem\u003e2N\u003c/em\u003e), G2/M-phase (\u003cem\u003e4N\u003c/em\u003e) and polyploid (\u003cem\u003e\u0026gt;4N\u003c/em\u003e) is indicated. Arrows and percentages indicate changes in cell populations. \u003cem\u003eB\u003c/em\u003e, Gain-of-function ∆NCdc25B mutant overrides blockage induced by Survivin abrogation. siRNA-treated synchronized cells were transfected with a pGFP (control), pCdc25B (wild type Cdc25B) or p∆NCdc25B (constitutively active Cdc25B mutant) plasmid (the last 2 were also transfected with the GFP monitoring plasmid), released into fresh medium, and analyzed by Western blotting (\u003cem\u003eleft\u003c/em\u003e), or fluorescence and phase-contrast microscopy (\u003cem\u003eright\u003c/em\u003e) after 12 h (cells entering mitosis). Averages of 3 independent experiments are shown in \u003cem\u003ebottom left\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/421763a965ee4ef82dcafb74.jpg"},{"id":91148495,"identity":"0b0fe7f9-31fd-41c8-8bd4-c0dc35a36e83","added_by":"auto","created_at":"2025-09-12 06:44:36","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17633175,"visible":true,"origin":"","legend":"","description":"","filename":"ResearchSquareManuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2_covered_d8119444-269b-4cce-83db-c773bb6872a6.pdf"},{"id":90692789,"identity":"84a2f308-194d-4023-b312-9e80332409fb","added_by":"auto","created_at":"2025-09-05 18:47:01","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1957838,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S1. \u003cem\u003e\u003cstrong\u003eLoss of Survivin causes an impairment in the prophase to prometaphase transition in HeLa cells.\u003c/strong\u003e\u003c/em\u003e \u003cem\u003eA\u003c/em\u003e, siRNA treatment of cells. \u003cem\u003eB\u003c/em\u003e, Cell cycle phase of Cyclin B1-positive siRNA-treated HeLa cells. HeLa cells transfected with control (VIII) or Survivin (S4) siRNA were analyzed by fluorescence microscopy using an antibody to Cyclin Β1. DNA was stained with DAPI. Cells in prophase (P), prometaphase (PM), metaphase (M), anaphase (A), telophase (T) or multinucleated (MN) are indicated. \u003cem\u003eC\u003c/em\u003e, Nuclear lamina integrity of siRNA-treated cells. Cells transfected with control (VIII) or Survivin (S4) siRNA were analyzed by fluorescence microscopy using an antibody to Cyclin Β1 and Lamin B. DNA was stained with DAPI. Cells in S-phase (S), prophase (P), prometaphase (PM) or metaphase (M), and intact lamina (IL) or spindle-like structures (SLS) are indicated.\u003c/p\u003e","description":"","filename":"Suppl.Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/a32b91e40cb4ef3be0ba584e.jpg"},{"id":90693274,"identity":"2dbe1b9d-2e59-4855-bd5f-37a91f80c013","added_by":"auto","created_at":"2025-09-05 19:03:01","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1044180,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S2. \u003cem\u003e\u003cstrong\u003eCharacterization of the Survivin-Cdk1 interaction in vitro.\u003c/strong\u003e\u003c/em\u003e \u003cem\u003eA\u003c/em\u003e, Survivin directly interacts with Cdk1 \u003cem\u003ein vitro\u003c/em\u003e: \u003cem\u003eCdk1-binding Survivin region.\u003c/em\u003e GST, GST-Survivin or the indicated GST-Survivin deletion mutant (numbers correspond to amino acid residues in the Survivin sequence) were mixed with His-Cdk1, and bound proteins were analyzed by Western blotting. Membranes were stained with Coomassie Blue to check the amount of the GST-Survivin fragments (*). \u003cem\u003eB\u003c/em\u003e, Survivin-binding Cdk1 region. GST-Survivin (\u003cem\u003eleft\u003c/em\u003e) or biotinylated SUR A55-D70 peptide (\u003cem\u003eright\u003c/em\u003e) were mixed with His-tagged full-length or the indicated Cdk1 fragment (numbers correspond to amino acid residues in the Cdk1 sequence) followed by Western blotting.\u003c/p\u003e","description":"","filename":"Suppl.Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/8346111feedf298096df88dc.jpg"},{"id":90693211,"identity":"dd381d82-d784-4b86-a7fe-77584bd51f3f","added_by":"auto","created_at":"2025-09-05 18:55:01","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1696572,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S3. \u003cem\u003e\u003cstrong\u003eRegions involved in the Survivin-Cdk1 complex.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eA\u003c/em\u003e,\u003cstrong\u003e \u003c/strong\u003eCdk1-binding Survivin region. \u003cem\u003eB\u003c/em\u003e, Survivin-binding Cdk1 region. \u003cem\u003eC\u003c/em\u003e, SUR A55-D70 peptide superimposed on the N-terminal Survivin region. \u003cem\u003eD\u003c/em\u003e, Survivin-binding Cdk1 region superimposed on N-terminal Cdk1 region. All protein sequence and functional information used to generate Survivin’s and Cdk1’s graphs and cartoons was extracted from UniProt.\u003c/p\u003e","description":"","filename":"Suppl.Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/7f503ec9e9fb7d1d8121d054.jpg"},{"id":90692802,"identity":"ae3c8a03-6f4a-40de-b9f6-e17ca0fea9ca","added_by":"auto","created_at":"2025-09-05 18:47:01","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":710075,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S4. \u003cem\u003e\u003cstrong\u003eCharacterization of the SUR A55-D70 peptide.\u003c/strong\u003e\u003c/em\u003e \u003cem\u003eA\u003c/em\u003e, SUR A55-D70 pull down of endogenous Cdk1. HeLa cell lysates were used to pull down Cdk1 using a biotin-conjugated scrambled or Survivin A55-D70 (SUR A55-D70) peptide. Protein complexes were analyzed by Western blotting. \u003cem\u003eB\u003c/em\u003e, SUR A55-D70-mediated disassembly and loss of kinase activity of a Survivin-Cdk1 mitotic complex. Lysates were prepared from nocodazole-treated HeLa cells, and incubated with the scrambled or SUR A55-D70 peptide. Cdk1 was IP, and immune complexes were analyzed in a Histone H1 phosphorylation assay or by Western blotting. \u003cem\u003eC\u003c/em\u003e, Peptide delivery into HeLa cell cytosol. Cells were incubated with biotinylated, HIV tat-scrambled or SUR A55-D70 peptide, and analyzed by fluorescence microscopy using Texas Red-streptavidin. DNA was stained with DAPI.\u003c/p\u003e","description":"","filename":"Suppl.Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/279f872a32a71e6eed162a15.jpg"},{"id":90692793,"identity":"be63f9a0-d5a9-490d-9d77-54b6fc728745","added_by":"auto","created_at":"2025-09-05 18:47:01","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1007194,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S5\u003cem\u003e.\u003c/em\u003e \u003cem\u003e\u003cstrong\u003eThe\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eSurvivin Asp70Ala/Asp71Ala (SUR D70A/D71A) double mutant does not bind to Cdk1.\u003c/strong\u003e\u003c/em\u003e Alanine Scanning Mutagenesis of the SUR A55-D70 peptide. The Survivin region spanning the SUR A55-D70 peptide was subjected to alanine mutagenesis, and single alanine GST-Survivin mutants or GST alone were analyzed for their binding to His-Cdk1 by Western blotting (\u003cem\u003etop\u003c/em\u003e) (*: Two gels had to be run in order to analyze all the mutants). Binding of SUR D70A/D71A to His-Cdk1 was compared to mutant SUR D71A (\u003cem\u003ebottom\u003c/em\u003e). An unrelated SUR F58A mutant was used as control. Membranes were stained with Coomassie Blue to check the amount of the GST-Survivin mutants.\u003c/p\u003e","description":"","filename":"Suppl.Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/9ee1ab81a8a1de9ad14b8bcc.jpg"},{"id":90692804,"identity":"b5a247a8-3172-48b1-bb88-25d36985a40c","added_by":"auto","created_at":"2025-09-05 18:47:01","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":554390,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S6. \u003cem\u003e\u003cstrong\u003eDetection of a Hsp90-Survivin-Cdk1 complex at mitosis in HeLa cells.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eSynchronized cells were lysed, IP with an antibody to Hsp90, and analyzed by Western blotting.\u003c/p\u003e","description":"","filename":"Suppl.Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/5f90e61ceb6856b4473adfbe.jpg"},{"id":90692794,"identity":"3cdfbc6a-1734-4977-b7e0-6beeaad0712e","added_by":"auto","created_at":"2025-09-05 18:47:01","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":882948,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S7. \u003cem\u003e\u003cstrong\u003eA: Original Figure 8B - Centrosomal Cdk1 levels in the presence and absence of Survivin in HeLa cells\u003c/strong\u003e\u003c/em\u003e. Asynchronous cells were transfected with the control (VIII) or Survivin (S4) siRNA. Centrosomes were isolated and analyzed by Western blotting. Initially, the levels of γ-Tubulin, Cdk1 and Survivin were checked. Then, the same membrane was reprobed with antibodies against RCC1, Ran, Caspase 3 and p21. \u003cem\u003e\u003cstrong\u003eB: Original Figure 8C - Cdk1 and Survivin panels.\u003c/strong\u003e\u003c/em\u003e \u003cem\u003e\u003cstrong\u003eCentrosomal Cdk1 and Survivin levels following siRNA treatment of HeLa cells\u003c/strong\u003e\u003c/em\u003e. Synchronized cells treated with VIII (\u003cem\u003etop\u003c/em\u003e) or S4 (\u003cem\u003ebottom\u003c/em\u003e) siRNA were released, and samples were taken from 12 to 18 h. Centrosomes were isolated, and Cdk1 and Survivin levels were analyzed. Samples taken at 16 h after both treatments were loaded in reverse fashion, and omitted from Figure 8C. \u003cem\u003e\u003cstrong\u003eC: Original Figure 8C - P32-Histone H1 panel.\u003c/strong\u003e\u003c/em\u003e \u003cem\u003e\u003cstrong\u003eCentrosomal Cdk1 activity following siRNA treatment of HeLa cells\u003c/strong\u003e\u003c/em\u003e. Synchronized cells treated with VIII (\u003cem\u003etop\u003c/em\u003e) or S4 (\u003cem\u003ebottom\u003c/em\u003e) siRNA were released, and samples taken from 12 to 18 h. Centrosomes were isolated, and Cdk1 activity measured. Samples taken at 16 h after both treatments were loaded in reverse fashion, and omitted from Figure 8C.\u003c/p\u003e","description":"","filename":"Suppl.Figure7A.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/7060ca2a95da19ad27691189.jpg"},{"id":90692799,"identity":"d2992c90-f08a-4612-8a6c-04d81cbe830b","added_by":"auto","created_at":"2025-09-05 18:47:01","extension":"jpg","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":1375861,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S7. \u003cem\u003e\u003cstrong\u003eA: Original Figure 8B - Centrosomal Cdk1 levels in the presence and absence of Survivin in HeLa cells\u003c/strong\u003e\u003c/em\u003e. Asynchronous cells were transfected with the control (VIII) or Survivin (S4) siRNA. Centrosomes were isolated and analyzed by Western blotting. Initially, the levels of γ-Tubulin, Cdk1 and Survivin were checked. Then, the same membrane was reprobed with antibodies against RCC1, Ran, Caspase 3 and p21. \u003cem\u003e\u003cstrong\u003eB: Original Figure 8C - Cdk1 and Survivin panels.\u003c/strong\u003e\u003c/em\u003e \u003cem\u003e\u003cstrong\u003eCentrosomal Cdk1 and Survivin levels following siRNA treatment of HeLa cells\u003c/strong\u003e\u003c/em\u003e. Synchronized cells treated with VIII (\u003cem\u003etop\u003c/em\u003e) or S4 (\u003cem\u003ebottom\u003c/em\u003e) siRNA were released, and samples were taken from 12 to 18 h. Centrosomes were isolated, and Cdk1 and Survivin levels were analyzed. Samples taken at 16 h after both treatments were loaded in reverse fashion, and omitted from Figure 8C. \u003cem\u003e\u003cstrong\u003eC: Original Figure 8C - P32-Histone H1 panel.\u003c/strong\u003e\u003c/em\u003e \u003cem\u003e\u003cstrong\u003eCentrosomal Cdk1 activity following siRNA treatment of HeLa cells\u003c/strong\u003e\u003c/em\u003e. Synchronized cells treated with VIII (\u003cem\u003etop\u003c/em\u003e) or S4 (\u003cem\u003ebottom\u003c/em\u003e) siRNA were released, and samples taken from 12 to 18 h. Centrosomes were isolated, and Cdk1 activity measured. Samples taken at 16 h after both treatments were loaded in reverse fashion, and omitted from Figure 8C.\u003c/p\u003e","description":"","filename":"Suppl.Figure7BC.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3949429/v2/59b22280a28d0d400a154143.jpg"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eHeLa Cells Override the Mitotic Entry Checkpoint by Highjacking the Survivin-Cdc25B-Cdk1 Pathway\u003c/strong\u003e\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"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":"Survivin, Cdk1, Cdc25B, mitosis, kinase, phosphatase, G2/M-phase checkpoint, mitotic catastrophe, cancer","lastPublishedDoi":"10.21203/rs.3.rs-3949429/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3949429/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Survivin protein has roles in repairing incorrect microtubule-kinetochore attachments at prometaphase and the faithful execution of cytokinesis, as part of the \u003cu\u003e\u003cem\u003eC\u003c/em\u003e\u003c/u\u003e\u003cem\u003ehromosomal \u003c/em\u003e\u003cu\u003e\u003cem\u003eP\u003c/em\u003e\u003c/u\u003e\u003cem\u003eassenger \u003c/em\u003e\u003cu\u003e\u003cem\u003eC\u003c/em\u003e\u003c/u\u003e\u003cem\u003eomplex \u003c/em\u003e(CPC). In its absence, mitotic errors often ensue that lead to chromosome missegregation, a cause of aneuploidy, polyploidy and ultimately cancer. Adding to these well-known roles of Survivin, this paper now shows for the first time that this protein is required for cancer cells to enter mitosis, and that, in its absence, HeLa cells accumulate at G2/early prophase, or prior to prometaphase, as claimed before. This early mitotic blockage is demonstrated by the inability of the Survivin-depleted cells to disassemble their nuclear lamina and their low Cdk1 activity. Not surprisingly, the Survivin-ablation arrest can be reproduced by a Survivin double mutant SUR D70A/D71A, which cannot bind to Cdk1. Also, escape from this mutant’s blockage leads to multiple mitotic defects, or \u003cem\u003emitotic catastrophe\u003c/em\u003e, and cell death. Mechanistically, Cdk1 does not localize at the centrosome in the absence of Survivin, pointing at this latter protein contributing to the activation of the mitotic kinase \u003cem\u003evia\u003c/em\u003e Cdc25B. Furthermore, absence of Survivin leads to an inactive cytosolic Cdc25B-Cdk1-Cyclin B1 complex, which seems to indicate a role for Survivin in bridging this complex and its centrosomal activator/s. Interestingly, the drop in Cdk1 activity caused by interference with the Survivin function could be rescued when Survivin-depleted HeLa cell lysates were incubated with the recombinant Survivin protein. Also, a role for Survivin in the Cdc25B-mediated activation of Cdk1, and concomitant prophase to prometaphase transition could be confirmed by expression of a gain-of-function Cdc25B mutant, which overrode the G2/prophase blockage caused by Survivin depletion.\u003c/p\u003e","manuscriptTitle":"HeLa Cells Override the Mitotic Entry Checkpoint by Highjacking the Survivin-Cdc25B-Cdk1 Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2025-09-05 18:46:56","doi":"10.21203/rs.3.rs-3949429/v2","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}},{"code":1,"date":"2024-02-28 08:59:01","doi":"10.21203/rs.3.rs-3949429/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":"031097ed-2d28-4fd1-9c14-3667213796a8","owner":[],"postedDate":"September 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54156389,"name":"Biological sciences/Cancer"},{"id":54156390,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2025-01-08T22:26:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-05 18:46:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-3949429","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3949429","identity":"rs-3949429","version":["v2"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}