Fig. 8.

Bcl-2 overexpression attenuates cytokine-induced caspase-3 cleavage. A: RINm5F cells (2.0 × 10⁶ cells) were transiently transfected with 2 μg of pRC-CMV (vector) or pRC-CMV.bcl2 (Bcl-2) and cotransfected with pEGFP to determine transfection efficiency (>70%). Forty-eight hours later, 10 U/ml IL-1 was added, and the cells were incubated for 36 h with IL-1 or 36 h with IL-1 followed by the addition of l-NMMA and continued culture for 8 h (IL-1 + l-NMMA 8h). Following these incubations the cells were harvested, and caspase-3 activity was determined. Cells treated with tunicamycin (2 μg/ml) for 36 h are shown as a positive control for caspase activation, and the inhibitory actions of l-NMMA, when coincubated with IL-1, on caspase activity following a 36-h treatment are shown. B: INS 832/13 cells were treated for the indicated times with 10 U/ml IL-1 in the presence or absence of l-NMMA, the cells were isolated, and PUMA mRNA accumulation was determined by real-time PCR. mRNA levels were normalized to the levels of GAPDH. PUMA induction following 24-h incubation with camptothecin is shown as a positive control. Results are the average ± SE for 3 independent experiments. Statistically significant inhibition of caspase-3 activity by Bcl-2 and induction of PUMA vs. control is indicated (*P < 0.05).