Extended Data Fig. 1: Data supporting CIP2A as a protein recognizing mitotic DNA lesions.
a, Immunoblotting of whole-cell extracts of RPE1-hTERT p53-/- Cas9 cells, parental (WT) or BRCA1-/-, expressing the indicated sgRNAs and either a virus expressing an sgRNA-resistant FLAG-CIP2A (CIP2A*) or an empty virus (EV). Lysates were probed for FLAG, CIP2A and tubulin (loading control). Representative of two immunoblots. b, Immunoblot examining CIP2A levels in isogenic and BRCA-deficient cells in the DLD1 and RPE1-hTERT p53-/- Cas9 (RPE1-hTERT) backgrounds. Tubulin was used as a loading control. Immunoblotting was performed once. c, Clonogenic survival assay of RPE1-hTERT p53-/- Cas9 cells of the indicated genotype following treatment with camptothecin (CPT). Data presented as the mean ± S.D. n = 4 independent clonogenic assays apart from the 15 nM dose where n = 3; analyzed with extra sum-of-squares F test and compared to WT. ∗∗∗p = 1.27 × 10−7 (#1), 1.68 × 10−6 (#6). d, e, Analysis of sister chromatid exchanges (SCEs) in cells of the indicated genotype. Quantitation is shown in (d) as cell-based data; bars represent the mean ± S.D. n = 31, 30, 31, 30, 30 aggregated from 3 independent metaphase spreads; analyzed with Kruskall-Wallis non-parametric test, followed by Dunn’s multiple comparisons, compared to WT. n.s.= 0.4398, 0.6574, ∗∗∗p = 1.97 × 10−13, 1.18 × 10−13. Representative micrographs of metaphase spreads are shown in (e). Arrowheads indicate an SCE event. f, Immunoblotting for CIP2A in DLD1 WT and CIP2A knockout clones. Tubulin was used as a loading control. Representative of 3 independent immunoblots. g, Analysis of RAD51 foci in EdU+ cells in RPE1-hTERT (WT) and indicated CIP2A-/- clones. Left, aggregate cell-based values. Right, mean of each independent experiment. Bars represent the mean ± S.D. n = 60 aggregated from 3 independent immunostainings; analyzed with Kruskall-Wallis non-parametric test, followed by Dunn’s multiple comparisons, compared to WT; n.s.= 0.5542, 1.0000, 1.0000. All scale bars = 10 μm. Statistical analyses and full blots are found in Source Data ED Fig. 1.
