Fig 2: Activation of SAC in Kv10.1-depleted HeLa cells decreases the occurrence of lagging chromosomes. HeLa cells were transiently transfected with either Scr- or KCNH1-siRNA and synchronized with double thymidine block at the G1/S border (A–I) or with consecutive thymidine and nocodazole treatments at metaphase and anaphase (J–M). (A–G) Samples were collected at the indicated time-points after cell cycle re-initiation and immunoblotted. Representative immunoblots and densitometry analysis results are given. The time interval corresponding to Kv10.1 expression is highlighted in pink (n = 3 independent synchronization experiments; mean ± SEM, two-way ANOVA, repeated measures, Bonferroni post hoc test, the given p-value shows the significance of the global difference between the two groups. (H,I) PLK1 enzymatic activity was measured in whole-cell lysates from HeLa (left grey panel) and hTERT RPE1 (right blue panel) cells synchronized at G2/M (H) and G1/S (I) (n = 4 independent synchronization experiments; two-way ANOVA, repeated measures, mean ± SEM is shown). (J–M) The mitotic spindles in synchronized HeLa cells were visualized with α-tubulin (green), and the chromosomes—with CENP-B (kinetochores, red) and Hoechst 33342 (DNA, blue) (scale bars represent, 10 µm and apply for all panels). (J) Metaphase is considered defective when chromosomes (overlap of CENP-B and Hoechst stains) are not properly aligned (indicated with arrows) or damaged DNA (only Hoechst positive) is present. Example images of normal and defective metaphases are shown. (K) The percentage of defective metaphases from the total number of metaphases is plotted (600 metaphases per experiment, n = 4 independent synchronization experiments; Mann–Whitney U-test, min to max range with the median indicated as a line are shown). (L) Lagging chromosomes in anaphase are detected as CENP-B- and Hoechst-positive signal located in the middle part of the spindle (indicated with arrows). (M) The percentage of anaphases with lagging chromosomes from the total number of anaphases is plotted (400 anaphases per experiment, n = 4 independent synchronization experiments; Mann–Whitney U-test, min to max range with the median indicated as a line are shown). (SAC—spindle assembly checkpoint; CDC—cell division cycle protein; MAD2—mitotic arrest deficient 2; pVHL—von Hippel-Lindau protein; AURKB—Aurora kinase B; PLK1—Polo-like kinase 1; CENP-B—centromere protein B).

Fig 3: Induction of CIN/aneuploidy is not tolerated in the developing cerebellum and does not provoke medulloblastoma. (A) Schematic overview of the transgenic mouse model and experimental setup. Five experimental groups of P4 pups containing the tdTomato reporter were generated: CreNegative (white), Mad2f/f (pink), p53f/f (green); Mad2+/f, p53f/f (yellow), and Mad2f/f; p53f/f (blue). P4 pups were treated once with Tamoxifen by oral gavaging. Mice were monitored for tumor formation. Additionally, fluorescent CGNPs were isolated at different time points for further analyses. (B) Stereoscopic fluorescent images showing tdTomato expression in cerebellum between postnatal day P7 and P21. (C) IVIS images demonstrating tdTomato expression in the cerebellum of a 6.5-month-old transgenic and control mouse. (D) Kaplan–Meier survival curve showing overall survival of the five experimental groups described at (A). (E) Conventional genomic PCR assessing the switching efficiency of the floxed Mad2l1 alleles in Mad2f/f cerebella of postnatal day P7 (n = 1), P15 (n = 1), and P22 (n = 1) mice. Upper bands: floxed (unswitched) allele; middle bands: switched Mad2l1 allele; lower bands: wild type Trp53. (F) Bar charts showing the switching efficiency of the Mad2l1 floxed allele in Mad2f/f (n = 1), Mad2f/f; p53f/f (n = 1), and Mad2+/f; p53f/f (n = 1) sorted CGNPs or titrated control at P7, P15, and P21 at the genomic level, as assessed by quantitative genomic PCR. (G) Bar charts showing the switching efficiency of the Mad2l1 and Trp53 floxed alleles at the mRNA level in P7 CGNPs, as assessed by quantitative RT-PCR. (H) Western blot showing the switching efficiency of the Mad2l1 floxed allele at the protein level in P7 CGNPs. (I) scWGS data of sorted P8 and P13 Mad2f/f; p53f/f CGNPs. Each colored line represents the karyotype of a single cell (P8, n = 19; P13, n = 19 cells). Chromosomes are indicated on the X-axis. Colors indicate the ploidy state of their corresponding region, as stated in the legend.

Fig 4: Elimination of ATM increased phosphorylation of Mad2. (A) Immunoblotting analysis of control or ATM shRNA cells in absence or presence of nocodazole. (B) Immunoblotting analysis in simian virus 40-transformed fibroblast cell lines GM9607 and GM0637. (C) Immunoblotting analysis of cells treated with mock or nocodazole in presence or absence of λPPase. (D) Immunoblotting analysis of HeLa cells transfected with vector, WT, S214A, or S214D mutant form of Mad1 of in presence or absence of nocodazole.

Fig 5: Phosphorylation of Mad2 Serine 195 regulated Spindle Checkpoint. (A) FACS analysis of HeLa cells transfected with vector, WT, S195A, or S195D mutant form of Mad2 and treated with nocodazole followed by flow cytometric anti-phospho-H3 staining. (B) Mean mitotic percentages (at least triplicate samples) and error bars represent variations around averages. (C) Co-immunoprecipitation assay of Mad2 complex in 293FT cells transfected with GFP-tagged S195A or S195D mutants of Mad2.