Figure 7
PHD3 depletion potentiates IL-6–STAT3 signalling and the PERK–ATF4 branch of the UPRER pathway independent of prolyl hydroxylase activity. (A,B) Effects of shRNA-mediated PHD3 knockdown on Tyr705 phosphorylation and STAT3 protein level in mouse hepatocytes without (A) or with (B) exposure to 100 ng/ml LPS for indicated times, as detected by immunoblotting. The lines in (B) indicate the deletion of non-relevant bands from the blots. (C) Time course analysis of IL-6 mRNA expression in mouse hepatocytes with or without PHD3 depletion exposed to 100 ng/ml LPS for indicated times. (D) Effects of ectopic expression of shRNA-resistant PHD3(WT) or PHD3(ΔPH) on Atf4 and Chop gene expression induced by PHD3 depletion in primary mouse hepatocytes, as detected by qRT-PCR. (E) Effect of PHD3 depletion on Thr980 phosphorylation of PERK and total PERK, XBP1s, XBP1u, PHD3, and α-tubulin levels in whole cell lysates, and total amount of nuclear ATF4 and histone H3 in AML12 cells with or without exposure to 5 μg/ml tunicamycin for 24 h, as determined by immunoblotting. Histone H3 and α-tubulin served as loading controls for immunoblot analyses of the nuclear fraction and whole cell lysates, respectively. *Non-specific. Complete immunoblots are presented in Supplementary Fig. S7. Quantitative data are shown as mean ± SEM (n = 3 (C,D)) and are representative of at least two independent experiments. Differences between groups were evaluated by ANOVA followed by Bonferroni’s post hoc test. **P < 0.01 vs. indicated groups. Adenoviral vectors encoding PHD3 shRNA, sh-R PHD3(WT), or sh-R PHD3(ΔPH) were used for experiments.
