Fig. 3.
aFGF restored HG + PA-reduced Wnt/β-catenin signaling pathway activity, and the endothelial protective action of aFGF against HG + PA is Wnt/β-catenin signaling pathway dependent, in vitro. (A) Representative immunofluorescence staining with β-catenin in HUVECs, scale bars = 5 μm, (B) Nuclear and cytosolic extracts from HUVECs were isolated to detect β-catenin protein level by immunoblotting. Lamin B1 and β-Actin were served as loading controls for nuclear and cytosolic fractions, respectively, HUVECs were cultured either in NG or HG + PA medium alone or with aFGF (100 ng/mL) for 72 h, MAN was served as the osmotic control for the HG + PA. For manipulation of Wnt/β-catenin pathway, IWR (5 μM) was pretreated for 2 h before aFGF administration. (C) OCR was analysed using a Seahorse XF analyser. (D) ATP production in HUVEC. (E) Mitochondrial O2•− in HUVEC was measured by mitochondria targeted probe MitoSOX and UPLC after accumulation of O2•−-specific product 2-OH-Mito-E+. (F) mtROS of HUVECs was detected by MitoSOX staining assay, scale bars = 1000 μm, (G) Mitochondrial membrane potential was detected by TMRM fluorescence staining, scale bars = 5 μm, (H) TUNEL assay of HUVECs, scale bars = 100 μm, (I) Capillary-like tube formation of HUVECs, scale bars = 300 μm. All values displayed are means ± SEM of 6 independent experiments. #p < 0.05 vs. NG or MAN; *p < 0.05 vs. HG + PA; % p < 0.05 vs. HG + PA co-incubated with aFGF.