Proteomic analysis of Rpn10 iKO. After being cultured with Dox for 4 days, AMO1 Rpn10-iKO cells were subjected to proteomic analysis by multiplexed proteomics with tandem mass spectrometry. (A) Volcano plot of differentially expressed genes in AMO1 Rpn10-iKO cells compared with those in control cells. Blue and red dots represent the genes (P < .05) that were downregulated or upregulated, respectively. (B) Heatmap showing significantly (false discovery rate [FDR] < .05) altered genes in Rpn10 iKO cells. (C) Gene set enrichment analysis (GSEA) and gene ontology biological process (GOBP) significantly enriched after Rpn10 iKO. For all pathways shown, FDR < 10%. (D) GSEA-derived enrichment plots for the lysosomal transport pathway (left). AMO1 Rpn10-iKO cells were cultured with Dox as described for the indicated time points (middle) and when cultured with Dox, 25 nM BafA1 was added to the culture of Rpn10 iKO cells for the last 16 hours of day 4 (right). Cells were then lysed and subjected to immunoblot using antibodies against p62, LC3, LAMP2, or β-actin. (E) GSEA-derived enrichment plots for antigen processing and presentation of peptide antigen (left). AMO1 Rpn10-iKO cells were cultured with Dox for 4 days. Cells were subjected to flow cytometry after staining with anti–HLA-DR or anti–HLA-DQ Abs and 7AAD (middle). 7AAD^– cells were gated out, and the surface expression of HLA-DR or DQ expression was quantified. Rpn10-iKO cells, from which Dox was washed out, were added to total BM-derived mononuclear cells of patients with MM (n = 4) (right). Degranulation marker CD107a was measured on CD3⁺CD4⁺ T cells or CD3^–/CD56⁺ natural killer (NK) cells. FC, fold change; MFI, mean fluorescence intensity.