FIGURE 5:

TRAF2 phosphorylation inhibits oxidative stress–induced cell death. (A) DKO-T2-WT and ‑S11/55A cells were left untreated or treated with ME (12.5 μM) for 30 h, at which point cell death was assessed by microscopy (200×). (B) TRAF2/5 DKO cells stably transduced with empty vector (DKO-C), TRAF2-WT (DKO-T2-WT), ‑S11/55A (DKO-T2-S11/55A), or ‑ΔN (DKO-T2-ΔN) were left untreated or were treated with mTNF-α (20 ng/ml), ME (12.5 μM), tBHP (75 μM), or H₂O₂ (0.075 mM) for 48 h. Total cell death was then assessed via the trypan blue exclusion assay. Data are presented as the average of three experiments performed in triplicate. (C) DKO-T2-WT and ‑S11/55A cells were left untreated or treated with ME (12.5 μM) in the absence or presence of antioxidant NAC (5 mM), the JNK inhibitor SP600125 (SP6; 15 μM), or the p38 inhibitor SB203580 (SB2; 15 μM) for 48 h. Total cell death was then assessed as in (B). (D) DKO-T2-WT and ‑S11/55A cells were transfected with a scrabbled control siRNA-A or an siRNA specific to mouse JNK1 (siRNA-JNK1), using Lipofectamine 2000. At 68 h after transfection, the expression level of JNK1 was assessed by Western blotting. (E) DKO-T2-WT and ‑S11/55A cells transfected with siRNA-A or siRNA-JNK1 were left untreated or treated with ME (12.5 μM), and total cell death was then assessed as in (B). (F) DKO-T2-WT and ‑S11/55A cells were left untreated or treated continuously with ME (10 μM) or tBHP (50 μM) for 14 d or were treated temporarily with etoposide (5 μM) or doxorubicin (5 μM) for 6 h. On day 14, colonies were stained and those containing more than 50 cells were counted. The averages are represented as mean ± SD. *p < 0.05; **p < 0.01.