Fig. 3

BID expression drives sensitivity to SAC abrogation in cells with Chr22q11 amplification. A PC9-GR3 cells (AURKBi/TTKi sensitive) were electroporated with a CAS9-gRNA ribonucleoprotein knock-out (KO) CRISPR library targeting genes located in Chr22q11 (Table S11). Resulting clones were treated with AZD2811 and submitted to a functional assay to determine apoptosis and polyploidy, as described in Methods. Values shown are means and range (n=2). B PC9-ER cells (AURKBi/TTKi sensitive) stably expressing Cas9 were lipofected with the same CRISPR library; the resulting clones were treated with AZD2811 and cell viability determined by MTT at 72 h. Viability data were normalized against the viability observed for PC9-ER Cas9 parental cells treated with AZD2811. A value >1 indicates that a lipofected clone is more resistant to the compound than the parental PC9-ER cells. Values shown are means and range (n=2). C Dose-response curves for AZD2811 of PC9-GR3 and PC9-ER cells CRISPR-KO for selected genes. 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). D Micrographs of PC9-GR3 and PC9-ER cells CRISPR-KO for selected genes. AZD2811 was added at 150 nM. E PC9-ER, PC9-GR3, PC9, PC9-GR4 and PC9-GR4AZD1 cells were transfected with lentiviral particles for shRNA-based silencing of BID. After puromycin selection, selected colonies were analyzed by Western blotting. Chr22q11-positive and negative cells are indicated in green and blue, respectively. F Dose-response curves for AZD2811 of PC9-ER, PC9-GR3, PC9, PC9-GR4 and PC9-GR4AZD1 colonies with shRNA-based silencing of BID. 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). G Cell cycle analysis of PC9-GR3 and PC9-ER colonies with shRNA-based silencing. Cells were allowed to attach for 24 h, AZD2811 (150 nM) was added and cultures submitted to PI staining at 72 h. H Percentage of apoptotic/necrotic cells in PC9-GR3 and PC9-ER colonies with shRNA-based silencing. Cells were treated as in (G) and submitted to Annexin V staining. I Acidic beta-galactosidase staining of PC9-ER with shRNA-based silencing after a 72 h treatment with AZD2811