Fig 2: CIP2A controls TOPBP1 recruitment to sites of mitotic DSBs independently of PP2A.a Western blots of total cell extract of RPE-1 wild type cells (WT) and RPE-1 CIP2A knock-out cells cells (?CIP2A) either Mock treated (-) or treated with the PP2A inhibitor LB-100. b GFP pull-down from 293FT cells transfected with GFP-tagged full-length MDC1 and either control siRNA (siCtrl) or siRNA against CIP2A (siCIP2A). All scale bars = 10 µm. Source data are provided as a Source Data file.

Fig 3: CRM1 is dispensible for TOPBP1 recruitment in interphase.a Confocal micrographs of unsynchronized of parental RPE-1 cells (RPE-1), RPE-1 CIP2A knock-out cells (RPE-1 ?CIP2A) and RPE-1 ?CIP2A cells stably transduced with empty vector (+ Empty), Flag-tagged wild type CIP2A ( + Flag-WT), treated with 3 Gy of IR and stained for TOPBP1. b Quantification of TOPBP1 foci mean intensity and number of TOPBP1 foci per nucleus in interphase parental RPE-1 cells, RPE-1 CIP2A knock-out cells (?CIP2A) and ?CIP2A cells stably transduced with empty vector (+Empty) or Flag-tagged full-length CIP2A ( + Flag-WT). Each data point represents one cell (RPE-1: n = 200; ?CIP2A: n = 144; +Empty: n = 128; +Flag-WT: n = 125; pooled from two independent experiments) bars and error bars left graph represent mean and SD, bars right graph represent median. Statistical significance in the left graph (mean intensities) was assessed by one-way ANOVA and Dunnett’s multiple comparison test. Statistical significance in the right graph (foci number) was assessed by 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: TOPBP1 accumulation at sites of mitotic DSBs is dependent on CIP2A and vice versa.a Western blots of total cell extract of parental RPE-1 cells, RPE-1 ?CIP2A cells and stably transduced RPE-1 ?CIP2A cells with empty vector (+empty) and vector containing Flag-tagged wild type CIP2A cDNA (+Flag-WT). b Confocal micrographs (maximum intensity projections) of Nocodazole-arrested empty vector (+Empty) and Flag-tagged CIP2A wild type (+Flag-WT) complemented RPE-1 ?CIP2A cells, treated with 1 Gy of IR and stained for TOPBP1 and CIP2A. c Quantification of the experiment in b. Number of CIP2A/TOPBP1 foci per cell was assessed (+Empty: n = 57, +Flag-WT: n = 62, pooled from three independent experiments). Statistical significance was assessed with the two-sided Mann-Whitney test (a = 0.05) d Western blots of total cell extracts of CIP2A knock-out RPE-1 cells stably transduced with Flag-tagged wild type CIP2A cDNA (RPE-1 ?CIP2A + Flag-WT) and transiently transfected with control siRNA (siCtrl) and two different siRNAs against TOPBP1 (siTOPBP1 #1 and siTOPBP1 #2). e Confocal micrographs (maximum intensity projection) of CIP2A knock-out RPE-1 cells stably transduced with Flag-tagged wild type CIP2A cDNA (RPE-1 ?CIP2A + Flag-WT) and transiently transfected with control siRNA (siCtrl) and two different siRNAs against TOPBP1 (siTOPBP1 #1 and siTOPBP1 #2). Cells were arrested in pro-metaphase with Nocodazole, fixed 1 h after 1 Gy of IR and stained for CIP2A and TOPBP1. f Quantification of the experiment in e. Number of CIP2A foci per cell was assessed (siCtrl: n = 62, siTOPBP1 #1: n = 46, siTOPBP1 #2: n = 44, pooled from three independent experiments). Bars and error bars represent mean and SD. 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 5: 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.