Fig 2: The therapeutic effect of SETDB1 knockdown on H/R-treated hepatocytes was reversed upon overexpression of ASK1. (A) The protein levels of total and phosphorylated ASK1, JNK, and p38 were assessed in si-SETDB1-treated and si-NC-treated cell lines infected with adenovirus (ad)-ASK1 or ad-Vector (n = 3 for each group). (B) The mRNA levels of inflammatory factors were measured in the indicated groups (n = 3 for each group). (C) The protein levels of NF-κB signaling pathway components were analyzed in the indicated groups (n = 3 for each group). (D) The mRNA levels of apoptosis-associated genes were determined in the indicated groups (n = 3 for each group). (E) The protein levels of apoptosis-associated markers were examined in the indicated groups (n = 3 for each group). All data are presented as the means ± SD. **P < 0.01 indicates a statistically significant difference.

Fig 3: IRE1α RNase activity regulates the ASK1–JNK pathway and induces apoptotic neuronal damage.A Subcellular localization of JNK in the presence or absence of the IRE1 RNase inhibitor (STF, 30 µM) incubated during GR. Representative immunoblot and quantifications of JNK in the nucleus and cytoplasm. B Chop expression in cortical neurons exposed to GD/GR in the presence or absence of IRE1 RNase inhibitor (STF, 30 µM) incubated during GR. C Quantification and representative micrographs of TUNEL-positive cells exposed to 2 h of GD and 16 h of GR in the presence or absence of IRE1 RNase (STF, 30 µM), ASK1 (MSC, 1 µM), and JNK (SP, 10 µM) inhibitors. The drugs were administered only in the GR phase. Data represent the mean ± SEM of 3 (A, B) and 4 (C) independent experiments and were analyzed by one way ANOVA followed by Fisher’s multiple comparison test, *p < 0.05 vs. control, &p < 0.05 vs. 2 h GD + 16 h GR. Scale bar = 20 µm.

Fig 4: ASK1–JNK contributes to GD/GR-induced neuronal death.A Representative immunoblot and quantification of p-ASK1 at different times of GD and GR. B Subcellular localization of JNK in cortical neurons exposed to GD/GR. Representative immunoblot and quantifications of JNK (46 kDa isoform) in the nucleus and cytoplasm. C Chop mRNA levels in cortical neurons treated with the JNK inhibitor (SP, 10 µM) or ASK1 inhibitor (MSC, 1 µM). D MTT Reduction and E LDH activity in cortical neurons exposed to GD/GR, in the presence or absence of the ASK1 (MSC, 1 µM) or the JNK inhibitors (SP, 10 µM) during GR. Data represent the mean ± SEM of three independent experiments and were analyzed by one way ANOVA followed by Fisher’s multiple comparison test, *p < 0.05 vs. control, &p < 0.05 vs. 2 h GD + 16 h GR (C, E) or vs. 2 h GD + 22 h GR (D).

Fig 5: Downregulation of 14-3-3 θ mRNA by IRE1α RIDD activity stimulates ASK1 activation.A Comparison of human and rat 14-3-3 θ mRNA XBP1 like consensus site (gray). B Predicted secondary structure of the 14-3-3 θ mRNA folded in silico by the RNAfold web server. XBP1 like consensus site (gray area). C Effect of the inhibition of IRE1 RNase activity in the expression of 14-3-3 θ mRNA at different times of GD and GR. STF inhibitor (30 µM) was incubated during GD (2 h) or GR (4, 8, and 16 h). D Representative immunoblot and quantification of 14-3-3 θ /β-Actin at different times of GD and GR. E Representative immunoblot of 14-3-3 θ /β-Actin the presence or the absence of STF (30 µM) incubated during GR. F Analysis of the interaction of 14-3-3 θ with ASK1 by co-immunoprecipitation and detected by immunoblot in neurons exposed to GD and GR. G Representative co-immunoprecipitation of 14-3-3 θ and ASK1 in the presence or the absence of STF (30 µM) incubated during GR. Data represent the mean ± SEM of three independent experiments and were analyzed by unpaired Student’s t test (C) and one-way ANOVA followed by Fisher’s multiple comparison test (D), *p < 0.05 vs. control.