(A) Schematic depicting the CUL5 ubiquitin ligase scaffold and its interacting partners. (B) Western blots confirming effective knockdown of CUL5, RNF7 and UBE2F by stable lentiviral expression of dCas9-KRAB and corresponding sgRNAs. Asterisk indicates neddylated CUL5. GAPDH serves as loading control. (C) Quantification of western blots as in B. Error bars show standard deviations from three independent biological replicates. (D) Induction of apoptosis when cells depleted of CUL5, RNF7 or UBE2F as in (B) are treated with CDK9i (3 μM for 6 hr), MCL1i (1 μM for 12 hr) or DMSO. Knockdown of Bcl-xL serves as a positive control for induction of apoptosis. Percentage of apoptosis determined by flow cytometry detection of the fluorogenic apoptotic detection reagent, Cell Event. Error bars are standard deviations of three independent biological replicates. (E) Western blotting for cleaved PARP (c-PARP) serves as orthogonal readout for induction of apoptosis following knockdown of target genes and treatment with 3 μM CDK9i for 6 hr or 1 μM MCL1i for 12 hr. Knockdown of Bcl-xL is included as a positive control, however due to extreme toxicity of the combination treatment, cells were harvested 3 hr after treatment with 3 μM CDK9i or 0.5 hr after treatment with 1 μM MCL1i. GAPDH, loading control. (F) Dose response curves of caspase induction showing resensitization of CUL5 knockout (CUL5-KO c3) and UBE2F knockout (UBE2F-KO c1) lines as compared to parental LK2 cells when treated with CDK9i (top) and MCL1i (bottom). Caspase activation was measured at 10 hr post drug treatment at the indicated concentrations by CaspaseGlo 3/7 and normalized to a positive control containing inhibitors of MCL1, BCL2 and Bcl-xL. (G) Colony forming potential is decreased in CUL5-KO and UBE2F-KO as compared to parental LK2 cells after 1 µM treatment with CDK9i or MC1i for 8 hr. Representative images shown of colonies stained 2 weeks after drug treatment with 0.5% crystal violet in 6% glutaraldehyde. Experiments performed in triplicate. (H) Quantification of colonies as in (G), error bars represent standard deviations of three independent experiments.