Overexpression of CRBN restores LEN sensitivity in SENP8-inactivated cells. (A) Growth curve analyses of SENP8-inactivated BC-3/Cas9 cells (using SENP8-specific sg2) lentivirally transduced to overexpress Flag-tagged CRBN or ZsGreen and treated with 5 µM LEN. sgAAVS1-transduced cells serve as a control for LEN-sensitive BC-3 and CRBN-inactivated BC-3 cells (CRBN KO) were used as a control for LEN-resistant cells. Live cell counts were normalized to DMSO vehicle-treated cells, represented by the dotted line (n = 3; error bars represent SEM). (B) Representative western blot analysis of IKZF1, IKZF3, CK1α, IRF4, CRBN, and SENP8 at indicated points into the experiment shown in panel A. GAPDH served as loading control. Asterisks mark nonspecific bands. (C) Representative western blot analysis of BC-3 cells transduced with lentiviral vectors constitutively expressing WT CUL4A, K705R mutant CUL4A, or ZsGreen. Membranes were probed for CUL4A, CRBN, IRF4, or the loading control GAPDH. Neddylated (N8) and unneddylated forms of CUL4A are indicated by arrows. (D-E) Growth curve analyses of BC-3 cells from panel C and treated with 5 µM LEN (D) or 1 µM POM (E). CRBN-inactivated BC-3 cells (CRBN KO) were used as a control for LEN-resistant cells. Live cell counts were normalized to DMSO-treated control cells, as represented by the dotted line (n = 3; error bars represent SEM). For western blot analysis of this experiment, see supplemental Figure 8. Statistical analyses throughout this figure were performed by unpaired Student t tests comparing specified conditions to corresponding ZsGreen controls. *P < .05; **P < .01; ***P < .001; ****P < .0001.