Fig. 8.
Trx1-mediated transnitrosylation protects HeLa cells from apoptosis. HeLa cells overexpressing WT Trx1, Trx1C32S/C35S, and Trx1C32S/C35S/C73S were treated with 5 μg/ml CHX and 20 ng/ml TNF-α for 6 h. The apoptosis assay by flow cytometry is described under “Experimental Procedures.” All cell types showed an increase in apoptosis when treated with TNF-α and CHX (A and B). Overexpression of WT Trx1 significantly inhibited apoptosis (*, p = 0.005) (B). Trx1C32S/C35S/C73S-overexpressing cells had significantly higher cellular apoptosis compared with Trx1C32S/C35S-overexpressing cells (***, p = 0.002) (A and B) and inversely correlated with protein nitrosylation levels in these cells (Fig. 2C). Values are the mean ± S.E.; n = 4; *, p = 0.005; **, p = 0.04; ***, p = 0.002. Expression of WT Trx1 and mutants was confirmed by Western blotting using an anti-FLAG antibody (C). PE, phycoerythrin; Vec, vector.