TRIM21 enhances the anti-MM activity of bort. (A) MM1R and MM.1S cells were lentivirally transduced with shNC and shTRIM21 (1, 2, 3, 4, and 5). qRT-PCR was performed to verify the transfection efficiency. (B) Western blot assay of MM1R and MM.1S cells lentivirally transduced with shNC and shTRIM21 (1 and 5). The TRIM21 and GAPDH protein levels are shown. (C) CCK-8 assay of MM1R and MM.1S cells lentivirally transduced with shNC or shTRIM21 (1 and 5) and treated with different concentrations of bort. (D) MM1R and MM.1S cells lentivirally transduced with shNC and shTRIM21 (1 and 5) were cultured in the absence or presence of bort (5 nM for both cells; 24 hours). Apoptosis was evaluated using flow cytometry with Annexin-V-APC/proteasome inhibitor (PI)-PE staining. The representative and summarized results show that TRIM21 KD prevents bort-induced apoptosis of MM1R and MM.1S cells. (E) Western blot assay and CCK-8 assay of JJN3 and ARP-1 cells transduced with TRIM21NC and TRIM21 and treated with different concentrations of bort. (F) Flow cytometry assay of JJN3 and ARP-1 cells transduced with TRIM21NC and TRIM21 in the absence or presence of bort (5 nM for both cells; 24 hours). The representative and summarized results show that TRIM21 OE increased bort-induced apoptosis. (G) Stable transfected MM cells were treated with phosphate-buffered saline (PBS) or bort for 24 hours and then lysed and extracted. Western blotting was performed to detect the expression levels of the PARP and Caspase-3 (as reflected by apoptosis). GAPDH was used as a loading control. (H) FALG-TRIM21 and FALG-TRIM21(△RING) were overexpressed in ARP-1 cells, which were then treated with bort (0 and 5 nmol/L) for 24 hours and detected using flow cytometry. (I) The summarized results show the percentage of cells undergoing apoptosis. The values are presented as the mean ± standard deviation (SD) of 3 independent experiments. (∗P < .05; ∗∗P < .01; ∗∗∗P < .001).