Fig. 4

MDM2 arrests Osimertinib-induced cancer cell apoptosis through manipulating MCL-1. (A) PC-9 NC and PC-9 MDM2 cells were treated with increasing concentrations of Osimertinib (0, 0.1, 0.5, 1 µmol/L) for 48 h. Cell apoptosis indicators (PARP and Cleaved caspase-3) and BCL-2 family proteins involved in cell apoptosis program (MCL-1, BCL-2, BCL-xL, BAX, BIM) were analyzed by immunoblotting. GAPDH was used as an equal loading control. (B) PC-9 cells were treated with DMSO or 1 µmol/L Osimertinib for 48 h. To block proteasome-mediated protein degradation, MG132 at a final concentration of 20 µmol/L was added into the cell culture medium 8 h before cell harvest. MCL-1 protein level was determined by Western blot. (C) PC-9 MDM2 cells and PC-9 NC cells were split into 6 cm cell culture dishes and treated with 25 µg/mL CHX for indicated time intervals. Proteins of interest were separated by electrophoresis, transferred to nitrocellulose membrane and probed with indicated antibodies. (D) PC-9 OR cells were transfected with siRNA targeting MDM2 or NC. The half-life of candidate proteins was assessed by CHX chase assay. (E) Representative images of colony formation assay in PC-9 OR cells treated with DMSO, Osimertinib, AZD5991 or their combination. (F) Western blot analysis of PARP and caspase-3 fragmentation after 48 h of indicated treatment. (G) Evaluation of PC-9 OR cell apoptosis by TUNEL assay. The magnitude of apoptosis was calculated by the percentage of TUNEL-positive cells. Scale bar = 200 μm. (H-I) Cytotoxicity assessment of PC-9 MDM2 cells treated with DMSO, Osimertinib, AZD5991 or their combination. Scale bar = 200 μm. (J) Transient expression of the degradation resistant MCL-1 S159A mutant and its effect on cancer cell apoptosis induction in PC-9 OR cells treated with the Osimertinib + AZD5991 combinational strategy. **P < 0.01. ***P < 0.001