Fig. 6.

aFGF promoted the combination of HXK2 with mitochondria to protect the endothelial function against HG + PA. (A) Mitochondrial and cytosolic extracts were isolated to detect the HXK2 and Cytochrome c protein levels in HUVECs. 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, cell-permeable form of HXK2VBD (100 μM) was pretreated for 1 h before aFGF administration. (B) OCR was analysed using a Seahorse XF analyser. (C) ATP production in HUVEC. (D) Mitochondrial O₂^•− in HUVEC was measured by mitochondria targeted probe MitoSOX and UPLC after accumulation of O₂^•−-specific product 2-OH-Mito-E⁺. (E) Representative immunofluorescence analysis of HXK2 (red) in HUVECs. The COX IV immunostaining (green) highlights mitochondria, and nuclei were stained with DAPI (blue), scale bars = 5 μm, (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. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)