Fig. 5: Understanding the molecular mechanism of navitoclax plus AZD8055 combination in vitro in MPM.

Analysis of drug-induced priming and anti-apoptotic dependencies after treatment with navitoclax, AZD8055 and a BCL-xL specific antagonist, A-1331852 in MPM cell lines (H2052, JMN, JMN1B and MSTO-211H). Overall priming is measured by BIM or PUMA, whereas HRK, MS1, FSI and venetoclax are specific for BCL-xL, MCL-1, BFL-1, and BCL-2 dependency respectively (a, b). Drug treatment with navitoclax, AZD8055 and navitoclax plus AZD8055 combination in MPM cell lines (c–e). a, b Cells treated with 1 µM navitoclax, A-1331852 or AZD8055 and then DBP carried out (n = 3). Cytochrome c positive cells % (cytochrome c released = 100 − cytochrome c positive cells %) was measured using immunofluorescence microscopy, on permeabilized cells after 1 h incubation with indicated BH3 peptide concentration. Data are presented as bar graphs as mean values with error bars (±standard deviation). We calculated significance using a two-way ANOVA multiple comparisons test to DMSO-control (n = 3). c Cells treated with indicated concentration of drug/s for 3 days. Fourteen days from initial drug treatment cells were fixed and stained with crystal violet and area confluency and growth rate was calculated. Data presented as violin plots with median as solid line and quartiles as dashed line. We calculated significance using ANOVA multiple comparisons test to DMSO-control (n = 3). a–c P values are shown on the graph. d Representative Immunoblots (n = 3) of MPM cell line lysates after 24-h treatment with indicated drug concentrations for, phosphoSer473-AKT (pAKT), AKT, MCL-1, BCL-xL, BCL-2, BAK, BAX, BIM, PUMA, and β-Actin (loading control). e 24 h after treatment with indicated drug concentration, BCL-xL and MCL-1 were immunoprecipitated in H2052 cells (n = 2) and BIM complexes were determined by western blotting analysis (Input total cell lysate; IP, immunoprecipitated fraction; IgG1 (immunoglobulin isotype 1; MCL-1 isotype) and IgG3 (immunoglobulin isotype 3; BCL-xL isotype) control; S, supernatant).