Fig. 4

Ectopic expression of BID sensitizes Chr22q11 negative cells to SAC abrogation in a dose-dependent manner. A PC9, PC9-GR4 and PC9-GR3 BID KO cells (all of them resistant to AURKBi/TTKi) were transfected with a pTRIPZ lentiviral vector with the BID gene under the control of a doxycycline-inducible promoter. Two transfected colonies per cell type (SE1 and SE2) were cultured in presence of doxycycline (1 µg/mL), Western blotting was used to confirm ectopic expression of the BID protein. B Dose-response curves to AZD2811 of PC9, PC9-GR4 and PC9-GR3 BID KO cells transfected with the pTRIPZ BID vector (SE1 and SE2 colonies). Ectopic expression of BID was induced using 1 µg/mL doxycycline. Cells numbers were determined by MTT at 72 h. Values shown are means ± SD of ≥2 independent experiments. In each experiment, every concentration of drug was tested in six wells (n = 6). C Micrographs of PC9-GR3 BID KO parental and pTRIPZ BID-transfected cells (clone SE1) treated with AZD2811 (150 nM) in the presence and absence of 1 µg/mL doxycycline. D Cell cycle analysis of PC9-SE1 cells treated with AZD2811 (150 nM) for 72 h, in the absence and presence of 1 µg/mL doxocyclin. E Percentage of apoptotic/necrotic cells by Annexin V analysis in PC9-SE1 cells treated with AZD2811 (150 nM) for 72 h, in the absence and presence of 1 µg/mL doxycycline. F Effect of increasing concentrations of doxycycline on the ectopic expression of BID mRNA by PC9-SE1 and SE2 clones, as determined by RT-Q-PCR. G Correlation between the levels of BID protein and BID mRNA ectopic expression in PC9-SE1 and SE2 colonies represented in (F) and (H). H Effect of increasing concentrations of doxycycline on the ectopic expression of BID protein by PC9-SE1 and SE2 colonies, as determined by Western blotting. I Dose-response curves for AZD2811 of PC9-SE1 (left panel) and PC9-SE2 (right panel) clones, ectopic expression of BID protein was induced with increasing concentrations of doxycycline as shown. Cells numbers were determined by MTT at 72 h. Values shown are means ± SD of ≥2 independent experiments. In each experiment, every concentration of drug was tested in six wells (n = 6). J Plot of BID mRNA levels vs. cell growth inhibition at 100 nM AZD2811 (left panel) and 500 nM AZD2811 (right panel) in PC9-SE1 and SE2 cells. K Plot of BID protein levels vs. cell growth inhibition at 500 nM AZD2811 in PC9-SE1 and SE2 cells