Figure 3.

EA-mediated enhancement of ATP-induced cell death is dependent on the ASK1-p38 pathway downstream of the P2X₇ receptor. A, RAW264.7 cells were pretreated with 200 μm EA for 12 h in the presence of various inhibitors, including the P2X₇ receptor inhibitor CBB (1 mm), the antioxidant PG (20 μm), the p38 inhibitor SB203580 (SB, 20 μm), and the JNK inhibitor SP600125 (SP, 20 μm), stimulated with 0.5 mm ATP for 4 h, and then subjected to a cell viability assay. The data are represented as the percentage of viability of the cells treated with 200 μm EA without ATP stimulation and shown as mean ± S.D. Significant differences were determined by one-way ANOVA followed by Tukey-Kramer test. ***, p < 0.001 (versus control without inhibitors). B, RAW264.7 cells were pretreated with 200 μm EA for 12 h in the presence of 0.5 mm NAC or 20 μm PG and then stimulated with 0.5 mm ATP for the indicated periods. Cell lysates were subjected to immunoblotting with the indicated antibodies. Arrowheads indicate bands corresponding to the indicated proteins. C and D, DNA sequences around the gRNA target sites of P2rx7 (C) and ASK1 (D) genes in WT and KO RAW264.7 cells generated by using the CRISPR/Cas9 system. In P2rx7 KO cells, all P2rx7 loci are deleted at the same position (gray letters), in which the start codon is included. The underline indicates exon 1 (C). In ASK1 KO cells, frameshift deletions (dashes) are introduced in exon 5 of ASK1 loci (D). E, WT, P2rx7 KO, and ASK1 KO cells were pretreated with 200 μm EA, and stimulation and immunoblot analysis were performed as for Fig. 2, A and B. F and G, P2rx7 WT and KO cells (F) and ASK1 WT and KO cells (G) were pretreated with or without 200 μm EA for 12 h, stimulated with the indicated concentration of ATP for 6 h, and then subjected to a cell viability assay. The data shown are the mean ± S.D. Significant differences were determined by one-way ANOVA followed by Tukey-Kramer test. ***, p < 0.001.