Journal: Blood
Article Title: Proteasome subunit PSMD1 is a key therapeutic target in multiple myeloma ∗ †
doi: 10.1182/blood.2025029358
Figure Lengend Snippet: Clinical impact and functional significance of PSMD1 in MM. (A) Gene expression data were obtained using the GPL570 platform. PSMD1 expression was analyzed in normal (n = 22) and MM samples (n = 414) (data accession numbers GSE5900 and GSE4581 ). (B) Kaplan-Meier plots of PSMD1 expression vs overall survival of patients with MM. These data were generated as part of the Multiple Myeloma Research Foundation Personalized Medicine Initiatives. (C) Immunohistochemical analysis of BM biopsies from normal donors and patients with MM, demonstrating PSMD1 expression (scale bar, 20 μm). Arrows point to plasma cells. (D) Protein lysates from a panel of MM cell lines, normal plasma cells, and primary MM patient tumor cells were analyzed by immunoblotting for PSMD1 and β-actin expression. The right membrane was blotted with PSMD1 antibody after being previously blotted with PSMD4 and β-actin antibodies (as published in Du et al ). The β-actin blot result was reused here. (E) MM cell lines were transfected with scrambled siRNA (CT) or PSMD1-siRNA (KD), and cell viability was assessed 48 hours/72 hours post-transfection using CellTiter-Glo (CTG) assay (mean ± standard deviation [SD]; n = 3; P < .01). Bar graph: percentage of cell viability normalized to CT. Immunoblot analysis confirms the expression of PSMD1 in cells at 72 hours post-transfection except for H929 cells at 48 hours. (F) Dox-inducible-Cas9 AMO1 cells were generated by infecting AMO1 cells with lentivirus-packaged–inducible Cas9 plasmid. These cells were then infected with either control sgRNA or PSMD1-targeted sgRNA, followed by puromycin selection to establish a stable iKO cell line. Cells were treated with 0.5 μg/mL Dox every 2 days, and cell growth was assessed using the CTG assay. Immunoblot was performed to confirm PSMD1 knockout efficiency. Data are presented as the mean ± SD of triplicates. The blots share the same PSMD1 and β-actin bands with A left panel. (G) Human AMO1-inducible PSMD1-shRNA knockdown (PSMD1-shKD) cells or control-shRNA cells (shRNA-CT) were subcutaneously inoculated into NSG mice (n = 5 mice per group). Mice were then placed on a diet containing irradiated 0.0625% Dox (delivering 1-6 mg of Dox per mouse per day) starting when tumors became visible (average size of control was 37.27 mm 3 and KD was 28.10 mm 3 ). Upper panel: tumor volume (mm 3 ) over time is illustrated as mean ± SD. Internal blot: immunoblot revealing PSMD1 expression in tumor lysates. Lower panel: Kaplan-Meier survival curves for the mice are displayed. (H) Human AMO1 PSMD1-iKO cells and control sgRNA-CT cells were subcutaneously injected into each flank of NSG mice. Five days after inoculation, mice were treated with a diet containing irradiated 0.0625% Dox (n = 8). Upper panel: tumor volume (mm 3 ) over time is plotted. Lower panel: immunoblot analysis of PSMD1 expression in tumor lysates from the corresponding mice illustrated in the middle panel.
Article Snippet: For the inducible knockout (iKO) experiment, 6-week-old female NSG mice (Jackson Laboratory) were inoculated with 2 × 10 6 AMO1 iKO or nontargeting control (CT) cells, followed by a doxycycline (Dox) diet to induce PSMD1 knockout.
Techniques: Functional Assay, Gene Expression, Expressing, Generated, Immunohistochemical staining, Clinical Proteomics, Western Blot, Membrane, Transfection, CTG Assay, Standard Deviation, Plasmid Preparation, Infection, Control, Selection, Knock-Out, shRNA, Knockdown, Irradiation, Injection
![Clinical impact and functional significance of PSMD1 in MM. (A) Gene expression data were obtained using the GPL570 platform. PSMD1 expression was analyzed in normal (n = 22) and MM samples (n = 414) (data accession numbers GSE5900 and GSE4581 ). (B) Kaplan-Meier plots of PSMD1 expression vs overall survival of patients with MM. These data were generated as part of the Multiple Myeloma Research Foundation Personalized Medicine Initiatives. (C) Immunohistochemical analysis of BM biopsies from normal donors and patients with MM, demonstrating PSMD1 expression (scale bar, 20 μm). Arrows point to plasma cells. (D) Protein lysates from a panel of MM cell lines, normal plasma cells, and primary MM patient tumor cells were analyzed by immunoblotting for PSMD1 and β-actin expression. The right membrane was blotted with PSMD1 antibody after being previously blotted with PSMD4 and β-actin antibodies (as published in Du et al ). The β-actin blot result was reused here. (E) MM cell lines were transfected with scrambled siRNA (CT) or PSMD1-siRNA (KD), and cell viability was assessed 48 hours/72 hours post-transfection using CellTiter-Glo (CTG) assay (mean ± standard deviation [SD]; n = 3; P < .01). Bar graph: percentage of cell viability normalized to CT. Immunoblot analysis confirms the expression of PSMD1 in cells at 72 hours post-transfection except for H929 cells at 48 hours. (F) <t>Dox-inducible-Cas9</t> AMO1 cells were generated by infecting AMO1 cells with lentivirus-packaged–inducible Cas9 plasmid. These cells were then infected with either control sgRNA or PSMD1-targeted sgRNA, followed by puromycin selection to establish a stable <t>iKO</t> cell line. Cells were treated with 0.5 μg/mL Dox every 2 days, and cell growth was assessed using the CTG assay. Immunoblot was performed to confirm PSMD1 <t>knockout</t> efficiency. Data are presented as the mean ± SD of triplicates. The blots share the same PSMD1 and β-actin bands with A left panel. (G) Human AMO1-inducible PSMD1-shRNA knockdown (PSMD1-shKD) cells or control-shRNA cells (shRNA-CT) were subcutaneously inoculated into NSG mice (n = 5 mice per group). Mice were then placed on a diet containing irradiated 0.0625% Dox (delivering 1-6 mg of Dox per mouse per day) starting when tumors became visible (average size of control was 37.27 mm 3 and KD was 28.10 mm 3 ). Upper panel: tumor volume (mm 3 ) over time is illustrated as mean ± SD. Internal blot: immunoblot revealing PSMD1 expression in tumor lysates. Lower panel: Kaplan-Meier survival curves for the mice are displayed. (H) Human AMO1 PSMD1-iKO cells and control sgRNA-CT cells were subcutaneously injected into each flank of NSG mice. Five days after inoculation, mice were treated with a diet containing irradiated 0.0625% Dox (n = 8). Upper panel: tumor volume (mm 3 ) over time is plotted. Lower panel: immunoblot analysis of PSMD1 expression in tumor lysates from the corresponding mice illustrated in the middle panel.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_7978/pmc13197978/pmc13197978__BLOOD_BLD-2025-029358-gr1.jpg)
