Figure 5.

Inhibition of TrxR augments polysulfide-mediated suppression of caspase-9 activity and apoptosis. A, apoptotic cell lysates were prepared from HeLa cells treated with STS for 3 h. Then, equal amount of lysates (200 μg) were incubated for 30 min at 37 °C with the indicated concentrations of Na₂S₄ followed by determination of caspase-3 and -9 activities. *, p < 0.05; **, p < 0.01 versus vehicle control. B, HeLa cells were simultaneously treated for 4 h with AUR, STS, and Na₂S₄, as indicated. Thereafter, caspase-9 activity in cell lysates was determined. *, p < 0.05; **, p < 0.01 versus STS alone. C, cells were simultaneously treated with (+) or without (−) AUR (2 μm), STS (1 μm), and Na₂S₄ (250 μm), followed by determination of pro- and cleaved–caspase-9 persulfidation using the BTA. D, cells were treated with (+) or without (−) AUR/STS/Na₂S₄ as in C. Thereafter, the presence of cytochrome c (Cyt c) in cytosolic fractions was determined by immunoblotting and TrxR1 served as loading control. E, cells were treated as in C followed by analysis of DNA fragmentation. F, cells were treated as in C followed by determination of TrxR activity. **, p < 0.01 versus vehicle control unless noted. G, cytosolic extracts from THP-1 cells were treated with Cyt C and dATP in the presence of the indicated concentrations of Na₂S₄, followed by assessment of caspase-9 activity and cleavage. *, p < 0.05; **, p < 0.01 versus Cyt c/dATP alone. Data in the graphs represent mean ± S.D. (n ≥ 3).