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. 2021 Aug 16;12:660263. doi: 10.3389/fphys.2021.660263

Figure 3.

Figure 3

Hypoxia inducible factor-1α (Hif-1α), as the downstream of AMPK, participates in the endothelial protective action of Lira against hyperglycemia, in vitro. (A) Cell lysates of HUVECs were used to detect the Hif-1α protein levels by immunoblotting. HUVECs were cultured either in NG, or HG medium alone or with Lira (100 nM) for 72 h, MAN served as the osmotic control for the HG. For signaling pathway analysis, Cpd C inhibitor of AMPK (10 μM) was pretreated for 2 h before Lira administration. (B) The quantitative analysis of each immunoblot. (C) Cell lysates of HUVECs were used to detect the Hif-1α protein levels by immunoblotting. HUVECs were transfected with si-Hif-1α or control siRNA, respectively. After transduction, HUVECs were cultured in HG medium alone for 72 h. (D) Representative immunofluorescence with Hif-1α in HUVECs, which treated as indicated in (A). Scale bars = 5 mm. (E) The quantitative analysis of nuclear/perinuclear Hif1α fluorescence intensity ratio in (D). (F) Nuclear and cytosolic extracts were isolated to detect the Hif-1α protein levels by immunoblotting. HUVECs treated as indicated in (A). (G) The quantitative analysis of each immunoblot. (H,L) Cell lysates of HUVECs were used to detect the Bax, Bcl-2, c-Caspase-3, and HO-1 protein levels by immunoblotting. HUVECs were transfected with si-Hif-1α or control siRNA, respectively. After transduction, HUVECs were cultured either in NG, or HG medium alone or with Lira (100 nM) for 72 h. (I,J,K,M) The quantitative analysis of each immunoblot. All values displayed are means ± SEM of five independent experiments. #p < 0.05 vs. NG or MAN; *p < 0.05 vs. HG; $p < 0.05 vs. HG co-incubated with Lira.