Figure 2.

Bmi-1 inhibits c-Myc-induced apoptosis and strongly enhances proliferation in collaboration with myc in an ink4a–ARF-dependent manner. (A) Wild-type MEFs were infected at passage 1 with control (C) or bmi-1 (B) encoding retroviruses, at passage 2 with either control, mycER or mycHA-encoding retroviruses and analyzed for cell viability by trypan blue exclusion. mycER overexpressing cell populations were analyzed for cell death 0, 24, and 48 hr after transfer to 0.1% serum in the presence (circles) or absence (squares) of 125 nm 4-OHT (left). mycHA overexpressing cells were analyzed for cell death 0, 24, and 48 hr after transfer to 0.1% (circles) or 10% (squares) serum (right). Control-infected cultures remained viable for >95% during the entire experiment (not shown). Apoptotic cell death was confirmed by flow-cytrometric analysis of cells with a subdiploid DNA content. (B) Wild-type or ink4a–ARF^−/− MEFs were infected at passage 1 with control (C, black bars) or bmi-1 (B, gray bars)-encoding retroviruses and subsequently at passage 2 with control or mycER retroviruses. After infection, cells were analyzed for subdiploid DNA content 24 hr after transfer to 0.1% serum (left), or for cell viability by trypan blue exclusion 0, 16, and 26 hr after transfer to 0.1% serum in the presence of 125 nm 4-OHT (right). (□ +/+C; █ +/+B; ○ −/−C; ● −/−B.) (C) Growth curves of wild-type (left) or ink4a–ARF^−/− MEFs (right) infected at passage 1 with control (C) or bmi-1 (B) encoding retroviruses and at passage 2 with control or mycHA-encoding retroviruses. Experiments were performed at least three times, yielding highly reproducible results (all standard deviations were within 10% of the means shown) and similar data were obtained with lower levels of Myc by use of the mycER retrovirus in the absence of 4-OHT. (□ Control C; █ Control B; ○ MycHA C; ● MycHA B.)