Figure 3.
Figure 3. Inactivation of UBE2G1 confers resistance to LEN and POM, but not CC-122. (A) Representative western blot analysis confirms efficient CRISPR-induced inactivation of UBE2G1 in BC-3/Cas9 cells by 2 independent UBE2G1-specific sgRNAs (sg1, sg2). sgAAVS1 is a negative control guide. GAPDH served as loading control. (B-C) Growth curve analyses of UBE2G1 inactivated BC-3/Cas9 cells after treatment with 5 µM LEN (B) or 1 µM POM (C). BC-3/Cas9 cells transduced with sgAAVS1 were included as a CM-sensitive negative control, whereas previously described clonal CRBN-inactivated BC-3 cells (CRBN KO) served as a positive control for complete CM resistance. Absolute live cell numbers were normalized to corresponding DMSO vehicle-treated cells at each passage (represented by the dotted line). These experiments were performed in parallel with SENP8-inactivated cells shown in Figure 4B-C, and thus share common negative and positive controls for several or all replicates. (n = 3; error bars represent standard error of the mean [SEM]). (D) Representative western blot analysis (anti-Flag) confirms lentiviral expression of sg2-resistant, Flag-tagged UBE2G1 in UBE2G1-inactivated BC-3/Cas9 cells (UBE2G1 sgRNA sg2). GAPDH served as loading control. (E) Growth curve analysis of UBE2G1-inactivated BC-3/Cas9 cells (UBE2G1 sgRNA sg2) expressing Flag tagged UBE2G1 (sg2-resistant; UBE2G1/sg2R) or control ZsGreen after treatment with 5 µM LEN confirms restoration of CM sensitivity on UBE2G1 reexpression. BC3/Cas9/sg AAVS1 was included as a LEN-sensitive positive control. Live cell counts were normalized to corresponding DMSO vehicle-treated cells, which are represented by the dotted line. These assays were run in parallel with those in Figure 4E, and thus share common controls (n = 3; error bars represent SEM). (F-G) Representative time course western blot analyses of CM neosubstrates IKZF1, IKZF3, and CK1α, and IRF4 in WT or UBE2G1-inactivated BC-3/Cas9 cells upon treatment with 5 µM LEN (F) or 1 µM POM (G). Lysates are matched with growth curves shown in panels B and C, respectively. GAPDH served as loading control. The asterisk marks a nonspecific band in panel F. (H) Growth curve analyses of UBE2G1-inactivated BC-3/Cas9 cells treated with 1 µM CC-122. sgAAVS1 and CRBN KO cells were included as negative and positive controls, respectively. Live cell counts were normalized to corresponding DMSO vehicle-treated cells, represented by the dotted line (n = 3; error bars represent SEM). (I) Representative western blot analysis of IKZF1, IKZF3, CK1α, and IRF4 at indicated points confirms a delay in neosubstrate degradation and IRF4 downregulation in UBE2G1-inactivated BC-3/Cas9 cells compared with control pools as in panel H. GAPDH served as loading control. Statistical analyses throughout this figure were performed by unpaired Student t tests comparing specified conditions to corresponding sgAAVS1 or ZsGreen controls. *P <. 05; **P < .01; ***P < .001; ****P < .0001. n.s., not significant.

Inactivation of UBE2G1 confers resistance to LEN and POM, but not CC-122. (A) Representative western blot analysis confirms efficient CRISPR-induced inactivation of UBE2G1 in BC-3/Cas9 cells by 2 independent UBE2G1-specific sgRNAs (sg1, sg2). sgAAVS1 is a negative control guide. GAPDH served as loading control. (B-C) Growth curve analyses of UBE2G1 inactivated BC-3/Cas9 cells after treatment with 5 µM LEN (B) or 1 µM POM (C). BC-3/Cas9 cells transduced with sgAAVS1 were included as a CM-sensitive negative control, whereas previously described clonal CRBN-inactivated BC-3 cells (CRBN KO) served as a positive control for complete CM resistance. Absolute live cell numbers were normalized to corresponding DMSO vehicle-treated cells at each passage (represented by the dotted line). These experiments were performed in parallel with SENP8-inactivated cells shown in Figure 4B-C, and thus share common negative and positive controls for several or all replicates. (n = 3; error bars represent standard error of the mean [SEM]). (D) Representative western blot analysis (anti-Flag) confirms lentiviral expression of sg2-resistant, Flag-tagged UBE2G1 in UBE2G1-inactivated BC-3/Cas9 cells (UBE2G1 sgRNA sg2). GAPDH served as loading control. (E) Growth curve analysis of UBE2G1-inactivated BC-3/Cas9 cells (UBE2G1 sgRNA sg2) expressing Flag tagged UBE2G1 (sg2-resistant; UBE2G1/sg2R) or control ZsGreen after treatment with 5 µM LEN confirms restoration of CM sensitivity on UBE2G1 reexpression. BC3/Cas9/sg AAVS1 was included as a LEN-sensitive positive control. Live cell counts were normalized to corresponding DMSO vehicle-treated cells, which are represented by the dotted line. These assays were run in parallel with those in Figure 4E, and thus share common controls (n = 3; error bars represent SEM). (F-G) Representative time course western blot analyses of CM neosubstrates IKZF1, IKZF3, and CK1α, and IRF4 in WT or UBE2G1-inactivated BC-3/Cas9 cells upon treatment with 5 µM LEN (F) or 1 µM POM (G). Lysates are matched with growth curves shown in panels B and C, respectively. GAPDH served as loading control. The asterisk marks a nonspecific band in panel F. (H) Growth curve analyses of UBE2G1-inactivated BC-3/Cas9 cells treated with 1 µM CC-122. sgAAVS1 and CRBN KO cells were included as negative and positive controls, respectively. Live cell counts were normalized to corresponding DMSO vehicle-treated cells, represented by the dotted line (n = 3; error bars represent SEM). (I) Representative western blot analysis of IKZF1, IKZF3, CK1α, and IRF4 at indicated points confirms a delay in neosubstrate degradation and IRF4 downregulation in UBE2G1-inactivated BC-3/Cas9 cells compared with control pools as in panel H. GAPDH served as loading control. Statistical analyses throughout this figure were performed by unpaired Student t tests comparing specified conditions to corresponding sgAAVS1 or ZsGreen controls. *P <. 05; **P < .01; ***P < .001; ****P < .0001. n.s., not significant.

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