Figure 5.

Combined WEE1 and ATR inhibition further sensitizes TNBC cancer cells with defective HRR to cisplatin.A, MDA-231 and Hs578t cells were treated for 72 h by the indicated concentrations of cisplatin with 100 nM of AZD1775, or 500 nM of AZD6738 or in combination. Cell viability was detected by CCK8 assay (n = 3, mean ± SD). B, Expression of RAD51, γH2AX and pHH3 of MDA-231 and Hs578t exposed to cisplatin alone, or in combination with AZD1775 or AZD6738, or both, were determined by immunoblot. C, An immunofluorescence assay was used to detect γH2AX and RAD51 expression in the nucleus in Hs578t following exposure to 3 μM of cisplatin with AZD1775 or AZD6738, or both. Scale bar: 2 μm. D, Percentage of cells with γH2AX foci (five or more foci per cell), pan-nuclear γH2AX signal or RAD51 foci (five or more foci per cell) after the indicated treatments in MDA-231 and Hs578t cells (n = 3, mean ± SD). E, Pearson’s coefficient shown as the quantification of RAD51 and γH2AX co-localization of three separate experiments in Figure 5C calculated by Image-Pro Plus software (n = 3, mean ± SD). F, After 48 h of treatment with vehicle or BRCA1 siRNA, MDA-231 cells were treated with the indicated concentrations of AZD1775 or AZD67398 for 72 h. Cell viability was analyzed by the CCK8 assay (n = 3, mean ± SD). G, Two days after the MDA-231 cells were transfected with vehicle or BRCA1 siRNA, cells were exposed to the indicated concentrations of cisplatin with or without the combination of 100 nM of AZD1775 and 500 nM of AZD6738 for 72 h, and cell viability was determined by CCK8 assay (n = 3, mean ± SD).