Fig 2: Loss of CIP2A impairs maintenance of chromosomal stability during mitosis.a Clonogenic survival analysis of IR treated RPE-1 wild type (WT) cells and RPE-1 CIP2A knock-out cells (?CIP2A). Data points represent the mean of 3 independent experiments, error bars represent the SD. Statistical significance was assessed by linear regression (a = 0.05). b Quantification of MNi formation, c quantification of CENPA positive (CENPA + ) and CENPA negative (CENPA-) as well as ?H2AX positive (?H2AX + ) and ?H2AX negative (?H2AX-) MNi, d quantification of residual ?H2AX foci 24 h after irradiation of mitotic cells and e quantification of chromosomal aberrations in metaphase spreads in RPE-1 parental cells (RPE-1), RPE-1 MDC1 knock-out cells (RPE-1 ?MDC1), RPE-1 CIP2A knock-out cells (RPE-1 ?CIP2A) and RPE-1 CIP2A knock-out cells stably transduced with empty vector (+ empty) and Flag-tagged wild type CIP2A ( + Flag-WT). Columns in b represent mean of 3–4 independent experiments (RPE-1 ± IR, RPE-1 ?MDC1 ± IR, + empty -IR, + Flag-WT -IR: n = 3, +empty +IR, + Flag-WT + IR: n = 4), data points represent percentage of cells with MNi in the experiments. Parts of whole are displayed for each cell line in c. The numbers of MNi assessed for each cell line (n) in c are indicated above each column. Each data point in d represents log of total ?H2AX foci intensity per cell (RPE-1: n = 368; RPE-1 ?MDC1: n = 330; +Empty: n = 219; +Flag-WT: n = 224), bars and error bars represent mean and SD. Statistical significance was assessed by one-way ANOVA and Sidak’s multiple comparison test (a = 0.05) ns: not significant. Aberrations in e were counted manually. Each data point represents one metaphase (RPE-1: n = 16, RPE-1 ?MDC1: n = 12, RPE-1 ?CIP2A: n = 19, +Empty: n = 13, +Flag-WT: n = 13), bars represent the median. Statistical significance was assessed by the Kruskal-Wallis test and Dunn’s multiple comparison test (a = 0.05). ns: not significant. f Examples of chromosomal aberrations in metaphase spreads derived from RPE-1 parental (RPE-1) and RPE-1 CIP2A knock-out cells (?CIP2A). Aberrations including single chromatid telomere loss, sister chromatid telomere loss, interstitial telomeres, telomere duplications and dicentric chromosomes are indicated with white arrowheads. For d, e: One representative of two independent experiments is shown. Source data are provided as a Source Data file.

Fig 3: Two conserved sequence segments in TOPBP1 mediate its interaction with CIP2A.a GFP-immunoprecipitation from 293FT cells either Mock transfected (Mock) or co-transfected with a GFP-tagged full-length TOPBP1 wild type (WT), and various deletion mutants as indicated, and Flag-tagged CIP2A. Relative intensities of co-immunoprecipitated Flag-CIP2A bands are indicated. b Confocal micrographs (maximum intensity projections) of Nocodazole-arrested GFP-TOPBP1 (WT and deletion mutants) expressing U2OS cells, treated with 1 Gy of IR. Endogenous TOPBP1 was depleted by 3'-UTR targeting TOPBP1 siRNA. c Quantification of GFP-TOPBP1/CIP2A foci per cell. Each data point represents one mitotic cell (WT: n = 29, ?C: n = 30, ?751–899: n = 31, ?774–798: n = 31, ?813–892: n = 30, ?774–789 + ?813-892: n = 32, pooled from three independent experiments). Statistical significance was assessed with the Kruskal-Wallis test and Dunn’s multiple comparison test (a = 0.05). All scale bars = 10 µm. Source data are provided as a Source Data file.

Fig 4: CIP2A is a TOPBP1 interaction partner.a Schematic showing the layout of conserved domains and regions in TOPBP1. Key amino acids in the AAD (W1145) and BRCT7 (K1317) are indicated. Deletion constructs of TOPBP1 used in b, lacking either the C-terminal portion of the protein (?C), the region between BRCT5 and 6 (?751-899) and BRCT domain 6 (?BRCT6). b Localization of GFP-TOPBP1 wild type and mutants in mitotic cells after 1 Gy of IR and interphase cells after 3 Gy of IR. Displayed are maximum intensity projections of confocal z-stacks. All scalebars = 10 µm. c HA-immunoprecipitation from 293FT cells transfected with a HA-tagged TOPBP1 fragment spanning the entire region between BRCT5 and 6 (amino acids 740-899). d HA-immuno-precipitation of the TOPBP1 fragment in c with the purified GST-ArmRP of CIP2A. e Flag-immunoprecipitation from 293FT transfected with Flag-tagged full-length CIP2A and treated with 3 Gy of IR and Nocodazole as indicated. f Quantification of the experiment in e. Columns represent the mean ratio between TOPBP1 and Flag band intensities in the Flag-IP blots, error bars represent the SEM of four independent experiments. Source data are provided as a Source Data file.

Fig 5: CIP2A-TOPBP1 recruitment to sites of mitotic DSBs is mediated by MDC1.a Confocal micrographs (maximum intensity projections) of Nocodazole-arrested RPE-1 wild type, MDC1 knock-out (?MDC1) and CIP2A knock-out (?CIP2A) cells, irradiated with 1 Gy and stained for TOPBP1 and CIP2A. b Quantification of the experiment in a. CIP2A/TOPBP1 foci were manually counted. Each data point represents one mitotic cell (no IR WT: n = 52, ?MDC1: n = 56, ?CIP2A: n = 51, IR WT: n = 80, ?MDC1: n = 61, ?CIP2A: n = 53, pooled from three independent experiments) and bars represent the median. Statistical significance was assessed with the Kruskal-Wallis test and Dunn’s multiple comparison test (a = 0.05). c Flag-immunoprecipitation from 293FT cells transfected with Flag-tagged full-length CIP2A and HA-tagged MDC1 wild type (WT) and Ser168/Ser196 double mutant (S168A/S196A). d Quantification of CIP2A-MDC1 proximity by in situ PLA in U2OS wild type (WT) and U2OS MDC1 knock-out cells (?MDC1), arrested in mitosis by Nocodazole and mock treated or treated with 1 Gy of IR. Each data point represents one cell (WT -IR: n = 131, WT + IR: n = 114, ?MDC1 -IR: n = 85, ?MDC1 + IR n = 54, pooled from two independent experiments), and bars represent median. Statistical significance was assessed by Kruskal-Wallis test and Dunn’s multiple comparison test (a = 0.05). e Confocal micrographs (maximum intensity projections) of Nocodazole-arrested U2OS ?MDC1 cells stably transfected with GFP-tagged wild type and S168A/S196A mutated MDC1, stained for CIP2A and TOPBP1 1 h treatment with 1 Gy of IR. f Quantitative analysis of GFP-MDC1 and CIP2A co-localization by SQUASSH: Left graph: object size colocalization (area of object overlap divided by total object area). Right graph: object number colocalization (fraction of objects in each channel that overlap = 50%). Data points represent individual mitotic cells (n = 24, except S168A/196 A object number: n = 21, pooled from three independent experiments). Bars and error bars represent mean and SD. Statistical significance was assessed by unpaired t-tests with Welch’s correction (a = 0.05). Source data are provided as a Source Data file.