Inhibition of autophagy restores apoptosis induced by bort. (A) ShTRIM21 or shNC MM1R, MM.1S, JJN3, and ARP-1 cells were incubated with or without 3-MA (4 mM) and bort (5 nM; 24 hours). Western blot analysis was employed to assess LC3 and P62 expression. GAPDH was used as a loading control. (B) Flow cytometry analysis of apoptosis by Annexin-V-APC/PI staining in a representative experiment. (C) The effect of 3-MA in TRIM21-mediated bort resistance is shown as means ± SD. (D) ShTRIM21 or shNC MM1R and MM.1S cells treated with shATG5 (1, 2, 3) compared with shNC showed a remarkable reduction of ATG5 messenger RNA (mRNA) level at 48 hours using qRT-PCR. (E) Western blot assay of ATG5 protein at 48 hours in shTRIM21 or shNC MM1R and MM.1S cells lentivirally transduced with shNC and shATG5 (1, 2, 3). GAPDH was used as a loading control. (F) Western blot analysis of the effect of shATG5 on LC3 and P62 expression in shNC or shTRIM21 MM1R, MM.1S, JJN3, and ARP-1 cells in the absence or presence of bort (5 nM; 24 hours). (G) Effect of bort (5 nM; 24 hours) on apoptosis in shTRIM21 or shNC MM1R, MM.1S, JJN3, and ARP-1 cells according to a representative flow cytometry experiment after ATG5 shRNA treatment. Notably, ATG5 KD–induced apoptosis. (H) The effect of shATG5 in TRIM21-mediated bort resistance is shown as means ± SD. The values are presented as the mean ± SD of 3 independent experiments. (∗P < .05; ∗∗P < .01; ∗∗∗P < .001).