Fig. 5
ENO1 activates Akt-GSK3β Pathway via the phosphorylation of AMPKα1. a We treated shENO1 and shCTRL PASMC with hypoxia (1% O2) for 8 h, and the levels of p-AMPKα, AMPKα, p-ACC, and ACC were measured by western blotting. b Normalized quantification of p-AMPKα and AMPKα in shCTRL and shENO1–PASMC under normoxic and hypoxic conditions (n = 4, *P < 0.05). c Normal PASMC were treated with 10 μM ENOblock and hypoxia for 8 h, and the levels of p-AMPKα, AMPKα, p-ACC, and ACC were measured by western blotting in cell lysates. d–e PASMC were transfected with pCMV3-ENO1-GFP, and we measured the levels of p-AMPKα, AMPKα, p-ACC, and ACC using western blotting. Actin was used as the loading control. The representative blots were shown in d and the quantification in e. n = 3, *P < 0.05. f PASMC were treated with 20 μM Compound C (CC, AMPK inhibitor) for 12 h or 500 μM AICAR (AMPK activator) for 2 h, and key proteins in the AMPK-Akt-GSK3β cascade and their phosphorylation were measured by western blotting in the cell lysates. g Wild-type (WT), AMPKα1-null, and AMPKα2-null MEFs were treated with 500 μM AICAR or DMSO for 2 h, and the key proteins in the AMPK-Akt cascade were measured by western blotting. h PASMC were overexpressed with ENO1-GFP and then transfected with siRNAs against AMPKα1 and/or α2 (si α1, si α2, or si α1 + 2), afterwards the key proteins in the AMPK-Akt cascade were measured by western blotting. Data represent the mean ± SEM. Student t test and one-way ANOVA were used to compare two and multiple groups. Bonferroni post-tests were carried out after ANOVA
