Journal: bioRxiv

Article Title: Mitotic slippage causes nuclear instability in polyploid cells

doi: 10.1101/2025.07.21.665898

Figure Lengend Snippet: (A) Stills of time lapse movies showing RPE-1 cells stably expressing mCherry-H2B (in magenta) performing MS and treated with DMSO (upper panel) or 2µM Methylstat (lower panel). Microtubules were labelled using SPY650-Tubulin (in cyan). The white arrows indicate the microtubules transiently accumulating at the nuclear envelope. (B) Schematic workflow presenting the method used to treat cells with Methylstat before or after MS. (C) Super resolution representative images showing diploid and MS-generated tetraploid RPE-1 cells treated with DMSO or with 4µM Methylstat before and after MS. Lamin A/C in yellow hot, DNA in magenta. (D-E) Graph showing nuclear circularity and solidity in diploid and in MS-generated tetraploid cells (in grey and blue, respectively) treated with DMSO or with 4µM Methylstat. (F-G) Graph showing nuclear circularity and solidity in diploid and in MS-generated tetraploid cells (in grey and blue, respectively) treated with DMSO or with (F) GSK-J4 or (G) JIB. Mean□±□SEM, >70 G1 cells analysed per condition, three independent experiments. (H) Western blots of total protein extracts obtained from RPE-1 cells transfected with siCtrl or siPHF8 (upper panel) or overexpressing HA-G9a (lower panel). (I-J) Graphs showing nuclear circularity and solidity in diploid and in MS-generated tetraploid cells (in grey and blue, respectively) (I) transfected with siCtrl or siPHF8 or (J) overexpressing HA-G9a. Mean□±□SEM, >140 G1 cells analysed per condition, three independent experiments. (K) Schematic workflow presenting the method used to extend mitotic duration using Monastrol treatment. (L) Left panel – Super resolution representative images showing RPE-1 cells in prometaphase treated with DMSO or with 50µM Monastrol for 16hrs. H3S10 in green, DNA in magenta. Right panel – graph showing H3S10 mean intensity in early mitosis in RPE-1 diploid cells treated with DMSO or with 50µM Monastrol for 16hrs. Mean□±□SEM, >100 mitotic cells analysed, three independent experiments. (M) Graph showing nuclear circularity and solidity in diploid and in MS-generated tetraploid cells (in grey and blue, respectively) treated with DMSO or with 50µM Monastrol. Mean□±□SEM, >100 G1 cells analysed per condition, three independent experiments. (N) Representative images showing RPE-1 diploid cells treated with DMSO or with 5nM Calyculin. H3S10 and H3K9me2 in green and cyan hot, respectively, DNA in magenta. (O) Graph presenting nuclear circularity and solidity in RPE-1 diploid cells treated with DMSO or with 5nM Calyculin. Mean□±□SEM, >230 G1 cells analysed per condition, three independent experiments. Scale bars: 10µm. D=diploid; T=tetraploid; MS=mitotic slippage; ON=overnight. ( D,E,F,G,I,J,M ) Anova-test (one-sided). ( L,O,P ) t-test (two-sided). ns=not significant. *=P ≤ 0.05. **= P ≤ 0.01. ***= P ≤ 0.001. ****= P ≤ 0.0001.

Article Snippet: The drugs were used at the following concentrations: Auxin (I5148 from Sigma), 500μM; Doxycycline (D3447 from Sigma), 2μg/ml; Asunaprevir (S4935 from Selleckchem), 3μM; Monastrol (S8439 from Selleckchem), 50μM; MPI-0479605 (S7488 from Selleckchem), 1μM; Blebbistatin (B0560 from Sigma), 20μM; Palbociclib (S1579 from Selleckchem), 1μM; Methylstat (HY-15221 from Medchem), 2 or 4μM; GSK-J4 (HY-15648B from Medchem), 30μM, JIB (HY-13953 from MedChemExpress), 2,5 or 5μM, Nocodazole (HY-13520 from Medchem), 2μM; Taxol (T1912 from Sigma), 1nM; SP600125 (HY-12041 from Medchem), 20μM; RO3306 (HY-12529 from Medchem), 10μM; Calyculin A (HY-18983 from Medchem), 5nM; Barasertib (HY-10127 from Medchem), 0.1 and 1μM; C646 (HY-13823 from Medchem), 30μM.

Techniques: Stable Transfection, Expressing, Generated, Western Blot, Transfection

Journal: bioRxiv

Article Title: Mitotic slippage causes nuclear instability in polyploid cells

doi: 10.1101/2025.07.21.665898

Figure Lengend Snippet: (A) Representative images of microtubules (in cyan) in megakaryocytes, DNA in magenta. (B) Super resolution representative images of HSC and megakaryocyte. H3K9me2 in cyan hot, DNA in magenta. (C-D) Graph showing H3K9me2 (C) mean intensity and (D) coefficient of variation (define as the ratio of the standard deviation to the mean) in HSCs (in grey) and in MKs (in blue). Mean□±□SEM, >150 interphase cells analysed per condition, four independent experiments. (E) Super resolution representative images showing HSC and megakaryocytes. Lamin A/C and Lamin B1 in yellow hot and red hot, respectively, DNA in magenta. (F-G) Graph showing (F) Lamin A/C and (G) Lamin B1 mean intensity at the NE in HSCs and in MKs. Mean□±□SEM, >35 cells analysed per condition, three independent experiments. (H) Representative images showing MKs in prometaphase treated with DMSO or 2µM Methylstat. H3S10 in green, DNA in magenta. (I) Graph showing H3S10 mean intensity in early mitosis in megakaryocytes treated with DMSO or 2µM Methylstat. Mean□±□SEM, >55 mitotic cells analysed per condition, three independent experiments. (J) Representative images showing MKs treated with DMSO or Methylstat. H3K9me2 in cyan hot, DNA in magenta. (K) Graph showing nuclear circularity (left panel) and solidity (right panel) in HSCs and in megakaryocytes treated with DMSO, 2µM Methylstat or 2,5µM JIB. Mean□±□SEM, >120 G1 cells analysed per condition, three independent experiments. (L) Working model showing the consequences of MS on nuclear stiffness and nuclear organisation. Upon MS, but not CF or EnR, nuclear instability at the population level can be identified. This has immediate consequences in chromatin organization, such as increasing the interactions between large and small chromosomes, leading to differential gene expression detected in a single cell cycle. Scale bars: 10µm. HSC=hematopoietic stem cell; MK=megakaryocyte; NE=nuclear envelope. ( C,D,I ) t-test (two-sided). ( F,G,K ) ANOVA test (one-sided). ns=not significant. *=P ≤ 0.05. **= P ≤ 0.01. ***= P ≤ 0.001. ****= P ≤ 0.0001.

Article Snippet: The drugs were used at the following concentrations: Auxin (I5148 from Sigma), 500μM; Doxycycline (D3447 from Sigma), 2μg/ml; Asunaprevir (S4935 from Selleckchem), 3μM; Monastrol (S8439 from Selleckchem), 50μM; MPI-0479605 (S7488 from Selleckchem), 1μM; Blebbistatin (B0560 from Sigma), 20μM; Palbociclib (S1579 from Selleckchem), 1μM; Methylstat (HY-15221 from Medchem), 2 or 4μM; GSK-J4 (HY-15648B from Medchem), 30μM, JIB (HY-13953 from MedChemExpress), 2,5 or 5μM, Nocodazole (HY-13520 from Medchem), 2μM; Taxol (T1912 from Sigma), 1nM; SP600125 (HY-12041 from Medchem), 20μM; RO3306 (HY-12529 from Medchem), 10μM; Calyculin A (HY-18983 from Medchem), 5nM; Barasertib (HY-10127 from Medchem), 0.1 and 1μM; C646 (HY-13823 from Medchem), 30μM.

Techniques: Standard Deviation, Gene Expression