Bort activates prosurvival autophagy in TRIM21 KD MM cell lines. (A) Flow cytometry assay of total ROS expression of TRIM21 KD and TRIM21 OE MM1R, MM.1S, JJN3 and ARP-1 in the absence or presence of bort (5 nM; 24 hours). (B) Western blot analysis of IRE1α, PERK, Ero1-Lα, CHOP, and ATF6 proteins in TRIM21 KD, TRIM21 OE, and negative control MM cells, either untreated or treated with bort (5 nM, 24 hours). GAPDH was used as a loading control. (C) Western blot analysis of p62 and LC3 proteins (as autophagy markers), ATG5, ATG7, and ATG16L1 proteins (ATGs), and mTOR and p-mTOR in TRIM21 KD, TRIM21 OE, and negative control MM cells, either untreated or treated with bort (10 nM, 24 hours). GAPDH was used as a loading control. (D) Immunofluorescence assay of LC3 expression in TRIM21 KD, TRIM21 OE, and negative control MM cells in the absence or presence of bort. LC3-II expression is indicated by the strong red puncta. Original magnification: 600×, scale bar: 50 μm. The values are presented as the mean ± SD from 3 independent experiments. (∗P < .05; ∗∗P < .01; ∗∗∗P < .001).