Characterization of ubiquitin receptor Rpn10 in MM cells. (A) Dox-inducible Cas9 AMO1 cells were made by infecting AMO1 cells with lentivirus-packaged inducible Cas9 plasmid followed by G418 selection. These cells then were infected with either control single-guide RNA (sgRNA) or Rpn13- or Rpn10-targeted sgRNA and selected with puromycin to generate stable iKO cells. Cell growth was evaluated by a CellTiter-Glo (CTG) assay. Immunoblot showing Rpn13 and Rpn10 expression, respectively, in corresponding KO cells to test the KO efficiency. Data are shown as the mean ± standard deviation (SD) of triplicates. (B) Gene expression data were collected using Affymetrix Human Genome U133A array platform. Rpn10 expression in the different stages of MM development, including normal CD138⁺ cells (n = 15), monoclonal gammopathy of undetermined significance (MGUS) (n = 22), smoldering (n = 24), newly diagnosed MM (n = 69), and relapsed MM (n = 28) (data accession number GSE6477). (C) Kaplan-Meier plots of Rpn10 expression vs overall survival of patients with MM. Analysis was performed using samples from 170 newly diagnosed patients with MM (data accession number GSE39754; P = .0064). (D) Total cellular RNA from purified CD138⁺ cells from BM of patients with MM or normal healthy donor PBMCs was subjected to reverse transcription polymerase chain reaction analysis, followed by real-time polymerase chain reaction using primers designed to recognize sequences internal to Rpn10 (mean ± SD, n = 4). (E) Immunohistochemistry analysis of BM biopsies from normal donors and MM patients showing Rpn10 expression (scale bar, 5 μm). (F) Protein lysates from a panel of MM cell lines, normal PCs, or primary cells from patients with MM were subjected to immunoblotting (IB) using Rpn10 and β-actin antibodies. Ab, antibody; CT, control; ns, not significant.