Figure 2.

A targeted TSG siRNA screen identifies loss of C8 as a mechanism of resistance to BOLD-100. A, Primary siRNA screen (left): HCT116 colorectal cancer cells were reverse transfected with siRNAs targeting 177 TSGs and 24 hours later treated with either DMSO control or 50 μmol/L BOLD-100. Cell viability was recorded 48 hours later using the CTG assay. Scatter plot showing distribution of rZ-scores for TSG siRNA screen. Positive scores indicate potential mediators of resistance to BOLD-100, whereas negative scores indicate potential mediators of sensitivity to BOLD-100. Secondary siRNA screen (right): HCT116 cells were reverse transfected with two additional independent siRNAs targeting 46 TSGs and positive hits from the primary screen. Waterfall plot showing distribution of rZ-scores. Each siRNA targeting C8 is highlighted in black. B,BRAFMT HT-29 (left) and VACO432 (right) cells were treated with BOLD-100 and preincubated with DMSO or the pan-caspase inhibitor (20 μmol/L). Apoptosis was determined by PARP and caspase 3/7 activity assay. C, HT-29 cells were transfected with 10 nmol/L C8 or/and C9 siRNA for 24 hours and thereafter treated with BOLD-100 for 24 hours. Apoptosis was assessed by WB analysis for PARP (left) and caspase-3/7 activity (right). Expression of pro-caspase-8, pro-caspase-9, GRP78 are also shown. D, Paired CRISPR HCT116C8WT and HCT116C8null cells were treated with increasing concentrations of BOLD-100 for 48 hours. Apoptosis was determined by WB for PARP (left) and caspase-3/7 activity levels (right). Expression of pro-caspase-8, GRP78, and CHOP are also shown.