Figure 4

Lentiviral shRNA vector-mediated knockdown of MCC induced apoptosis in human MM cells. The human MM cell line LP1 cells were transduced with lentiviruses expressing MCC shRNAs (including 1332, 1388, 2284 or 2689) or a scrambled shRNA (Scr). (A) Knockdown of the MCC protein determined by immunoblot analysis. Total cellular proteins were prepared from LP1 cells successfully transduced with a scrambled shRNA (Scr), or MCC shRNA 1332, 1388, 2284 or 2689 on day 5 post-transduction. Proteins were immunoblotted for MCC, followed by actin. Immunoblot of actin was used as a loading control. Results shown are representative of 3 independent experiments, and similar results were obtained in the human MM cell line KMS11 cells. (B) Growth curves of live cells and percentage of live versus dead cells determined by Trypan blue-stained cell counting. On day 4 post-transduction, successfully transduced LP1 cells were cultured in a 6-well plate for growth curve determination. Live and dead cells were counted daily for 4 days using Trypan blue staining and a hemocytometer. The graphs depict the results of 3 independent experiments (mean ± S.D.). (C) Representative FACS profiles of transduced cells analyzed by annexin V and PI staining. On day 6 post-transduction, transduced LP1 cells were stained with annexin V and PI, and then analyzed by a flow cytometer. In the FACS profiles, apoptotic cells were identified as annexin V+ PI-, dead cells were annexin V+ PI+, and live cells were annexin V- PI-. Data shown are representative of 3 independent experiments, and similar results were obtained in the human MM cell line KMS11 cells.