Fig. 3

Effects of checkpoint inhibitors MK-1775 (Wee1i) and AZD7762 (Chki) on parental and resistant melanoma cells. a Dose-response curves and IC₅₀ values (in nM) of MK-1775 and AZD7762 in A375, IGR37 and 501Mel melanoma cells. Response to 3-fold serial dilutions of each kinase inhibitor was assessed 72 h after treatment. The IC₅₀ values (nM) were calculated as indicated in Methods. Values represent the mean of at least three biological replicates. SD: standard deviation; XP: cells resistant to Vemurafenib, GP: cells resistant to Dabrafenib. b The combination of MK-1775 and AZD7762 efficiently induces apoptosis in parental and BRAFi-resistant A375 cells. Cells were treated for 72 h with the indicated concentrations of MK-1775 (Wee1i) or AZD7762 (Chki) or a combination thereof. Etoposide (Eto) treatment was used as positive apoptosis control. Resulting caspase-3 activity was normalized to the untreated control. Error bars represent the standard deviation of four biological replicates. Statistical significance was determined with one-way repeated measures ANOVA followed by Dunnett’s post-test. *p > 0.05, **p > 0.01, ***p > 0.001. c Western blot analysis of A375, A375-XP and A375-GP cells after treatment for 3 or 24 h with indicated amounts of drugs. P-cdc2 (CDK1), cdc2 (CDK1), p-Chk1 and Chk1 were detected after 3 h drug treatment, while PARP cleavage was detected after 24 h treatment. Vinculin and α-tubulin were used as loading controls. AZD: AZD7762, MK: MK-1775