Inhibition of USP39 Triggers G2/M Cell Cycle Arrest and Apoptosis in Multiple Myeloma Cells. A OPM2 cells were transfected with control or USP39 siRNAs for 72 h, 96 h or 120 h. In parallel, cells were stimulated with nocodazole (1 µg/ml) for 24 h to block the cells in G2/M phase. Cell cycle distribution was examined by flow cytometry, and percentage of cells in each phase is indicated (top left and right). A representative flow cytometry profile of cells transfected with control (blue area) or USP39 siRNAs (red area) for 96 h (bottom left). B In parallel, OPM2 cells were transfected with either control or single USP39 siRNAs for 72 h. Then, lysates from these cells were subjected to immunoblots using GAPDH, USP39, CDK4 and CyclinB1 antibodies. C OPM2 cells were transfected with either control or single USP39 siRNAs for 96 h or stimulated with BTZ for 48 h. Then, cells were stained by Annexin and PI and analyzed by flow cytometry. % of apoptotic cells (annexin V + /DAPI-) and dead cells (annexin V + /DAPI +) are represented in grey and black respectively. D Lysates from these cells were subjected to immunoblots using USP39, PARP, cleaved caspase 3 and GAPDH antibodies as a loading control. E, F OPM2 cells were transfected with either control or single USP39 siRNAs for 72 h and 96 h, or stimulated with BTZ for 48 h. Then, cells lysates were subjected to caspase 3 (E) and caspase 9 (F) assays
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