Suppression of neurosphere formation in vitro in pGBM cells by lentivirus-mediated shRNA specific to BMI1. (A) Representative images showing the suppression of neurosphere formation in two pGBM xenograft models (IC-1206GBM and IC-2305GBM). MTT was added 4 hr prior to the microscopic examination. Dark-colored intracellular crystal was indicative of viable cells. (B) Graphs showing the quantitative analysis of cell proliferation (** P < 0.01) in neurospheres derived from 3 independent PDOX models following the silencing of BMI1 with a panel of five lentivirus-mediated gene-specific shRNAs. O.D. indicates optical density. (C) Western hybridization showing the decreased BMI1 protein expression. CD133+ cells were purified from 3 PDOX models and transduced with Lenti-BMI1-693 (sh693) for 48 hr before being harvested for protein extraction. Tumor cells not treated with lentivirus (Control) and transduced with Lentiviruses containing non-target shRNA (Non-target) were included as references.