Extended Data Fig. 4: CIP2A and TOPBP1 localize to under-replicated DNA in mitotic cells.
a, Additional micrographs of CIP2A/TOPBP1 structures observed in mitotic cells after treatment with low-dose aphidicolin. Relates to Fig. 4e. Shown are curved (upper panels) and straight (lower panels) filaments. Representative of n = 3 independent immunostainings. b, Micrographs of U2OS parental (WT) and MDC1-/- anaphase cells that were either treated with aphidicolin (400 nM) for 16 h or left untreated. Quantitation of this experiment is shown in Fig. 4f. c, d, Colocalization analysis of TOPBP1 and CIP2A foci in U2OS parental (WT) or MDC1-/- cells using Squassh, size parameter. Representative micrographs (of two independent immunostainings) are shown in (c) quantitation shown in (d). Threshold for colocalization is 0.5 (dashed line). Bar represents the mean ± 95% C.I. n = 24 (WT) or 23 (MDC1-/-). e, Colocalization analysis of RPA and CIP2A in early prophase DLD1 BRCA2-/- cells by Squassh, size parameter. Threshold for colocalization is 0.5 (dashed line). Bar represents the mean ± 95% C.I. n = 15 aggregated from 3 independent immunostainings. Refers to Fig. 4h. f, Quantitation of spontaneous RPA and CIP2A foci in early prophase DLD1 BRCA2-/- cells. Data points are cell-based data, bar represents the mean ± S.D. n = 32 cells aggregated from 3 independent immunostainings. Analyzed with a Mann-Whitney test; U value (normal approximation)=-6.49202, ∗∗∗p = 1.30 × 10−14. g, Representative micrographs of the experiment shown in Fig. 4i with additional MDA-MB-436 cells showing elongation of CIP2A-TOPBP1 filaments during mitosis. h, Immunoblot of lysates from cells used in the experiment shown in Fig. 4j. Lysates were probed for GFP, CIP2A and tubulin (loading control). Representative of n = 2 independent immunoblots. Full blots are provided in Blot Source Data ED Fig. 4. All scale bars = 10 µm. Statistical analyses are found in Source Data ED Fig. 4.
