Causal relationship between degradation of STK33 and killing of mutant KRAS-dependent cancer cells by HSP90 inhibitors. (a) Immunoblots of mutant KRAS-dependent breast cancer (MDA-MB-231) and lung cancer (A549) cell lines incubated with 1 µM PU-H71 for the indicated times. (b) Immunoblot of MDA-MB-231, A549, and Calu-1 cells stably transduced with empty vector (EV) or STK33 and incubated with different concentrations of PU-H71 for 24 h. Cleaved PARP bands were quantified by densitometric analysis using the ImageJ program. Results of two to three independent experiments are shown. RU, relative unit. (c) Caspase 9 activity in KRAS mutant breast cancer (MDA-MB-231) and lung cancer (Calu-1) cell lines transduced with empty vector (EV) or STK33 and incubated with 0.5 µM PU-H71 for 24 h. Experiments were done in triplicate. Error bars represent mean ± SEM. (d) Immunoblot of KRAS WT MDA-MB-453 breast cancer (ERBB2-amplified) and MOLM-14 AML (FLT3-mutated) cells stably transduced with empty vector (EV) or STK33 and incubated with different concentrations of PU-H71 for 24 h. (e) Immunoblot of MDA-MB-231 cells that evaded apoptosis induced by shRNA-mediated STK33 knockdown after incubation with increasing concentrations of PU-H71 for 24 h. All results were confirmed in at least one additional independent experiment.