Fig. 5

JNK and AP-1 stress responses promote CHK1i, ATRi and WEE1i apoptosis in ovarian carcinoma. A, Cells were treated with diluent or prexasertib (20 nM) for 48 h and analyzed by RNAseq. Biological replicates were prepared on three separate days. Heatmap comparing prexasertib treatment to vehicle in PEO1 (left) and COV362 (right). Differentially expressed apoptotic genes (|log2FC|> 2 and p < 0.05 by two-sided t-test, n = 3 per group) are noted in purple and orange. B, Aliquots of PEO1 and COV362 cells independent of those in panel A were treated for 48 h with prexasertib (20 nM) and assayed for the indicated mRNAs by qRT-PCR. C, D, PEO1 (C) and COV362 cells (D) treated with the caspase inhibitor Q-VD-OPh (5 µM) and diluent, prexasertib, ceralasertib, or adavosertib at the concentrations indicated for Fig. 4C were blotted for the indicated proteins. E, PEO1 cells were transfected with the indicated pools of siRNAs, treated with prexasertib for 5 days, and assayed for annexin V binding. F, Immunoblots to assess JNK levels in PEO1 cells transfected with the indicated siRNA pools. G, H, Pooled PEO1 cells transduced with empty vector (EV), FADD sgRNA (G) or BID sgRNA (H) were treated for 5 days with prexasertib and assayed for annexin binding. Error bars in B, E, G, and H, mean ± sem of 3 independent experiments. *, *** and ****: p < 0.05, p < 0.001 and p < 0.0001, respectively, for indicated comparison by unpaired t-test corrected for multiple comparisons. FADD^−/− vs PEO1 EV p = 0.1585; PEO1p vs PEO1 EV p = 0.0786; BID^−/− #2 vs PEO1 EV p = 0.1850; BID^–/– #4 vs PEO1 EV p = 0.0286 using two-way ANOVA with Dunnett’s test for multiple comparisons