Rpn10 blockade induces cell death. (A) Polyubiquitylated proteins were detected in AMO1 Rpn10-iKO cells, MM.1S Rpn10-shKD cells, and ANBL6 Rpn10-shKD cells by western blot analysis. (B) Protein lysates from indicated cell lines were subjected to immunoblotting using antibodies against Rpn10, p-eIF2α, PERK, BiP, p53, p21, PLK1, or β-actin. (C) AMO1 Rpn10-iKO cells were cultured with Dox for indicated time points and fixed in 70% ethanol. After washing with phosphate-buffered saline, cells were stained with propidium iodide, and the DNA content of cells was then analyzed using fluorescence-activated cell sorter. Bar graph shows percentage of cell populations in the G1, G2/M, or S phases of the cell cycle. (D) GSEA-derived enrichment plots for mitotic cell cycle pathway and cell cycle G2-M phase transition pathway (left) and heatmap showing proteins related to G2-M phase transition enriched in AMO1-CT cells (right) have been shown. MM.1S shRNA KD cells, ANBL6-BR shRNA KD cells, and AMO1 Rpn10-iKO cells were cultured with Dox for indicated time points and then analyzed for apoptosis with annexin V/4′,6-diamidino-2-phenylindole double staining assay (mean ± SD, n = 3) (E) or protein lysates were then subjected to immunoblotting using antibodies against caspase-3, caspase-9, PARP, or β-actin (F). (G) AMO1 sgRNA-CT cells transfected with lentivirus-packaged pEV, referred to as CT; AMO1 Rpn10-iKO cells transfected with lentivirus-packaged pEV vector, referred to as KO; and AMO1 Rpn10-iKO transfected with lentivirus-packaged V5-tagged Rpn10 plasmid, referred to as RES. Protein lysates from CT, KO, or RES cells were then subjected to immunoblotting using antibodies against Rpn10 or K48 polyubiquitin, p62, LC3, BiP, PERK, caspase-3, caspase-9, p21, or β-actin.