Figure 3.

High glucose suppresses autophagy. (A) HUVECs were treated with indicated concentrations of glucose or mannitol for 24 h. CAV1, CAVIN1 and LC3B levels were analyzed by immunoblot, with ACTB as a loading control. G: glucose; M: mannitol. Representative western blotting analysis of the indicated proteins (upper panel) and summary bar graph showing the expression of the indicated proteins (lower panel). * p < 0.05 versus 5.5 mM glucose. (B) HUVECs were treated with indicated concentrations of glucose for 24 h, followed by electron transmission microscopy analysis. Representative images are shown. Asterisk, caveolae or caveolae cluster, arrow, autophagosomes or autolysosomes, Scale bar: 1 μm. (C-D) HUVECs were treated with indicated concentrations of glucose for 24 h, followed by treatment with bafilomycin A₁ (100 nM, 2 h). LC3B and SQSTM1 levels were analyzed by immunoblot, with ACTB as a loading control. Representative western blotting analysis of the indicated proteins (C) and summary bar graph showing the expression of the indicated proteins (D). * p < 0.05 versus 5.5 mM glucose, † p < 0.05 versus 5.5 mM glucose +bafilomycin A₁, (n = 4). (E-F) HUVECs were transfected with GFP-LC3B plasmids for 24 h, followed by 5.5 mM or 30 mM glucose treatment for 24 h in the presence or absence of bafilomycin A₁ (100 nM, 2 h). GFP-LC3B puncta was visualized by confocal microscopy. (E) Representative fluorescent images are shown. Scale bars: 15 μm. (F) The number of GFP- LC3B puncta in each cell was quantified, and at least 50 cells were included per group. * p < 0.05 versus 5.5 mM glucose; † p < 0.05, versus 5.5 mM glucose+bafilomycin A₁ (n = 3). (G-I) HUVECs were treated with indicated concentrations of glucose for 24 h. Whole-cell lysates from HUVECs immunoprecipitated with CAV1 antibody (H; IP: CAV1), CAVIN1 antibody (I; IP: CAVIN1), or LC3B antibody (J; IP: LC3B). Cellular ACTB from the same samples for IP served as a loading control (n = 3).