Extended Data Fig. 7: Disruption of the TOPBP1-CIP2A interaction is lethal in BRCA-deficient cells.
a, DLD1 BRCA2-/- cells transduced with either an empty virus containing only the destabilization domain (DD; EV, left) or a virus encoding B6L (right) were treated with AS1 (1 μM) for the indicated periods or left untreated (UT). Shown are micrographs of mitotic cells stained with antibodies to CIP2A, γH2AX or FLAG (labeling the DD). Quantitation is shown in Fig. 6b. Representative of three independent immunostainings. b, Anti-FLAG immunoblots of whole-cell extracts derived from DLD1 parental (WT) or BRCA2-/- cells untreated or treated with either Shield-1 (S1) or Aqua-Shield-1 (AS1) for 72 h. KAP1 is used as a loading control. Representative of three independent immunostainings. c, Representative images of the clonogenic survival experiment presented in Fig. 6f. Representative of three independent clonogenic experiments. d, Anti-FLAG immunoblots of whole-cell extracts from RPE1-hTERT p53-/- Cas9 parental (WT) or BRCA1-/- cells treated with AS1 for 72 h. Histone H2A is used as a loading control. Representative of two independent immunoblots. e, Clonogenic survival of RPE1-hTERT p53-/- Cas9 parental (WT) or BRCA1-/- cells following expression of B6L for the indicated periods of time prior to replating without AS1. Data presented as mean ± S.D. n = 3 clonogenic assays; analyzed with two-way ANOVA with repeated measures and Šídák’s multiple comparisons. n.s.=0.99907, ∗∗p = 0.0076 (d4) for WT and BRCA1-/- respectively. f, Additional representative micrographs from the experiment shown in Fig. 7c. Left panel shows a normal anaphase cell; right panel shows a cell with a lagging centromere-positive chromosome. All scale bars = 10 µm. Statistical analyses and full blots are found in Source Data ED Fig. 7.
