Fig. 4: Pharmacologic induction of replication stress sensitizes solid tumor cells to BCL-XL inhibitors.

A Immunoblotting analysis of replication stress and apoptosis markers in LNCaP cells treated with nolatrexed (48 h) followed by navitoclax for 6 h. B Single cell electrophoresis (Comet assay) of LNCaP cells treated for 48 h with DMSO, nolatrexed (1 μM), raltitrexed (1 μM), or doxorubicin (250 nM). Data were analyzed by one-way ANOVA ***p < 0.001 (p = 0.0007). Scale bar is 10 μM. C Immunoblotting analysis of cells treated with nolatrexed or raltitrexed, alone or combined with thymidine rescue for 48 h. D Caspase 3/7 activity in LNCaP cells treated with raltitrexed and navitoclax. Mean and SEM for 3 biological replicates are shown. Data at each raltitrexed concentration were analyzed by unpaired t-test, *p < 0.05. Two-way ANOVA showed effect of raltitrexed was significant, p < 0.001. E, F Caspase 3/7 activity in LNCaP cells treated with BML-277 plus navitoclax (E) or AZD4320 (F). Both were significant by two-way ANOVA, p = 0.0001. G Caspase 3/7 activity in LNCaP cells treated with nolatrexed and AZD4320. Mean and SEM for 3 biological replicates are shown. p < 0.0001 by two-way ANOVA. H Caspase 3/7 activity in indicated cell lines (A375P - melanoma, RKO - poorly differentiated colorectal carcinoma, ZR75 - breast ductal carcinoma, A549 - lung carcinoma) treated with nolatrexed or raltitrexed for 48 h followed by navitoclax for 6 h. Mean and SEM for 3 biological replicates are shown. Two-way ANOVA showed effects of nolatrexed and raltitrexed were significant, p < 0.001. I Immunoblotting of BCL-2 family proteins in LNCaP cells treated with PZ18753B (BCL-XL/BCL2 degrader) for 24 h. J Caspase 3/7 activity in LNCaP cells treated with nolatrexed (48 h) and PZ18753B (16 h). Data are presented as mean values +/− SEM. Data analyzed by two-way ANOVA showed significant effect of nolatrexed, p < 0.001. Source data are provided as a Source Data file.