Fig. 4
ENO1 promotes PASMC proliferation via the activation of the Akt-GSK3β pathway. a We treated shENO1 and shCTRL PASMC with hypoxia (1% O2) for 8 h, and measured the levels of p-Akt, pan-Akt, p-PRAS40, PRAS40, p-GSK3β, and GSK3β by western blotting. b Normal PASMCs were treated with 10 μM ENOblock and hypoxia for 8 h, and the levels of p-Akt, pan-Akt, p-PRAS40, PRAS40, p-GSK3β, and GSK3β were measured by western blotting. c PASMCs were transfected with pCMV3-ENO1-GFP, and afterwards we measured the level of p-Akt, pan-Akt, p-PRAS40, PRAS40, p-GSK3β, and GSK3β using western blotting. d PASMCs were transfected with pCMV3-ENO1-GFP and immunostained with antibody against p-GSK3β. The green fluorescence indicates the GFP-tagged ENO1 and the red fluorescence indicates p-GSK3β (Scale bars, 50 μm). e PASMC were transfected with pCMV3-ENO1-GFP and treated with 1 μM GSK690693 or MK2206 for 12 h. The level of p-Akt, pan-Akt, and PCNA were measured by western blotting in the cell lysate. We also measured the f cell proliferation (using BrdU assay) and g cell viability in ENO1-overexpressing PASMC after the treatment of two Akt inhibitors: GSK690693 or MK2206 (n = 5 per group, *P < 0.05, n.s. = non-significance). 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
