Knockdown of MCC induced apoptosis and inhibited proliferation in human MM cells. (A) Cell cycle distribution analyzed by PI staining and flow cytometry. The human MM cell line LP1 cells were transduced with lentiviruses expressing MCC shRNAs (1332 or 2689) or a scrambled shRNA (Scr). Successfully transduced (GFP+) cells were sorted by a FACS sorter on day 4 post-transduction. Top panel shows the FACS histograms of cells right after sorting (Day 0), and bottom panel shows the FACS histograms of sorted cells after cultured for 24 hours (Day 1). Gated populations in the FACS histograms indicate apoptotic cells (DNA content < 2n) or proliferating cells (2n < DNA content ≤ 4n). Results shown are representative of 3 independent experiments, and similar results were also obtained in the human MM cell line KMS11 cells. (B) Cell proliferation analyzed by dilution of the proliferation dye (eFluor 670)-labeling and flow cytometry. On day 4 post transduction, LP1 cells transduced with MCC shRNA 1332 (1332) or a scrambled shRNA (Scr) were labeled with a proliferation dye eFluor 670, which binds to any cellular protein containing primary amines. As cells divide, the dye is distributed equally between daughter cells that can be measured as successive halving or dilution of the fluorescence intensity of the dye. Day 0 FACS profiles and overlay histograms show the eFluor 670 signals of freshly labeled cells, while those of Day 2 and Day 4 show diluted eFluor 670 signals of cells after cultured for 2 and 4 days, respectively. Inhibited proliferation of MCC shRNA 1332-transduced cells was demonstrated by the hampered dilution of the proliferation dye as compared to those observed in both the untransduced (GFP-) cells and the scrambled shRNA (Scr)-transduced cells. Results shown are representative of 3 independent experiments with duplicate samples in each experiment.