Extended Data Fig. 4: Loss of CIP2A-TOPBP1, but not MDC1, disperses fragmented chromosomes during mitosis.
From: Mitotic clustering of pulverized chromosomes from micronuclei
a) Quantification of Y chromosome-positive signals in WT and CIP2A KO cells. Data represent median with 5-95 percentiles; ****P ≤ 0.0001 by two-tailed t-test compared to WT controls; WT: n = 214, CIP2A KO sg3: n = 113, CIP2A KO sg4: n = 84 cells pooled from 2 independent experiments. b) Immunoblot of CIP2A, TOPBP1, and MDC1 depletion in WT DLD-1 cells using two or three independent small interfering RNAs. Whole-cell extracts were collected 96 h after transfection. c) Images of fragmented Y chromosomes in mitotic WT DLD-1 cells depleted of CIP2A, TOPBP1, or MDC1 prior to DOX/IAA induction. Scale bar, 5 μm. d) Quantification of fragment clustering and dispersion from (c). Data represent the mean ± SEM of n = 5 (siCtrl) or 3 (all other conditions) independent experiments; *P = 0.0495, **P = 0.001, ****P ≤ 0.0001 by one-way ANOVA with multiple comparisons test compared to siControl sample; siControl = 450, siCIP2A-1 = 145, siCIP2A-2 = 178, siTOPBP1-1 = 138, siTOPBP1-2 = 133, siMDC1-1 = 298, siMDC1-2 = 386, siMDC1-3 = 281 cells. e) Live-cell images of dCas9-SunTag signals from nocodazole-arrested DLD-1 cells showing increased SunTag-positive fragments following CIP2A depletion. Scale bar, 5 μm. f) Quantification of the number of SunTag-positive signals from (e). Individual data points represent a single cell; data pooled from 3 independent experiments; two-tailed unpaired t-test with Welch’s correction. g) Metaphase spreads were collected from DLD-1 cells treated with DOX/IAA and hybridized to Y chromosome FISH probes. Examples of intact and fragmented Y chromosomes are shown along with dispersion index (see Methods for measurements). Scale bar, 10 μm. h) Quantification of metaphase fragment dispersion from (g). Data represent individual metaphase spreads with an intact or fragmented Y chromosome; intact: n = 19, fragmented: n = 79 metaphases from 3 independent experiments. i) Distribution of dispersion indices for intact and fragmented Y chromosomes from data shown in (h); intact: n = 19, fragmented: n = 79 metaphases from 3 independent experiments. j) CIP2A KO cells exhibit increased fragment dispersion on metaphase chromosome spreads. Data represent median with 5-95 percentiles; **P = 0.0042, ***P = 0.0002 by one-way ANOVA with multiple comparisons test compared to WT control sample; WT: n = 83, sg3: n = 54, sg4: n = 44 metaphases from 3 independent experiments. k) CIP2A or TOPBP1 depletion increases fragment dispersion on metaphase chromosome spreads. Data represent median with 5-95 percentiles; *P ≤ 0.05; **P ≤ 0.01 by one-way ANOVA with multiple comparisons test compared to siControl sample; siControl: n = 57, siCIP2A-1: n = 38, siCIP2A-2: n = 52, siTOPBP1-1: n = 63, siTOPBP1-2: n = 49 metaphases from 3 independent experiments.
