Fig. 4.

Apoptosis was reduced by inhibition of Sp3 under normoxia and hypoxia/reoxygenation. A: after overnight incubation in FBS-free medium with 2 mM (control) or 10 mM glutamine, untransfected or siRNA transfected cells were treated with 20 ng/ml of TNF and 25 μg/ml of cycloheximide (CHX) for 4 h. DNA fragmentation was determined using a Cell Death Detection ELISA kit. B: after overnight incubation in FBS-free medium with 2 mM (control) or 10 mM glutamine, untransfected or siRNA-transfected cells were incubated in a hypoxic chamber with 1% O₂-5% CO₂-94.5% N₂ for 4 h, and then cultured under normoxic conditions in presence of 20 ng/ml of TNF and 25 μg/ml of CHX for an additional 4 h. DNA fragmentation in cells was determined using a Cell Death Detection ELISA. Cells treated with neither siRNA nor inducers of apoptosis and grown in basal conditions served as untreated control. Statistical significance was evaluated by one-way ANOVA with post hoc Tukey's test.