Inhibition of ERK- or JNK- pathway blocks zVAD- but not TNF-induced cell death.

<p>(<b>A</b>) Inhibition of ERK- or JNK- pathway blocked zVAD-induced cell death. L929 cells were untreated or treated with ERK inhibitor PD98059 (100 µM), JNK inhibitor SP600125 (10 µM), p38 inhibitor SB203580 (10 µM), and PI3K inhibitor LY294002 (100 µM), respectively. Then the cells were treated with mock or zVAD (20 µM) for 24 h and cell viabilities were measured. (<b>B</b>) Inhibition of MAPKs does not block TNF-induced cell death. L929 cells were untreated or treated with PD98059 (100 µM), SP600125 (10 µM), p38 inhibitor SB203580 (10 µM), and LY294002 (100 µM). Then the cells were treated with mock or TNF (10 ng/ml) for 24 h and cell viabilities were measured. (<b>C</b>) zVAD had little effect on MAPKs’ activation. L929 cells were treated with zVAD or zVAD+LY294002 for 0, 0.5, 1, 2, 4 h, respectively. Cell lysates were subjected to western blot analysis with antibodies against p-ERK, ERK, p-JNK, JNK, p-p38 and p38. (<b>D</b>) Inhibition of zVAD-induced cell death has no effect on MAPKs’ activation. L929 cells were treated with zVAD or zVAD+Nec-1 for 0, 0.5, 1, 2, 4, 6 h, respectively. Cell lysates were subjected to western blot analysis with the indicated antibodies. (<b>E</b>) Inhibitors of MAPKs did not affect zVAD-induced LC3 modification. L929 cells were untreated or treated with PD98059 (100 µM), SP600125 (10 µM), SB203580 (10 µM), or LY294002 (100 µM). Then the cells were treated with mock or zVAD (20 µM) for 12 h and LC3 were analyzed by western blot. (<b>F</b>) Hypothetical pathway network of zVAD-induced cell death. The main pathways of zVAD-induced cell death are autophagy and autocrine of TNF. The induction of TNF by zVAD may depend on the collective effect of a number of pathways such as ERK, JNK, and autophagy. Mechanisms of zVAD-induced L929 cells could be different due to the variation of L929 sublines. **, p<0.01.