Fig. 4

GYY4137 inhibited autophagy in BBB endothelial cells in the early stage after resuscitation; and autophagy mediated the regulation of occludin protein content. (A) Representative immunoblot images and (B) quantitation of LC3B II, ATG5 and p62 in cerebral microvessels of mice in Sham, CA/CPR, CA/CPR + GYY, and CA/CPR + Rapa groups at 24 h after ROSC. β-actin was used as the protein loading control. The images of western blot data derived from triplicate blots conducted as three independent experiments. (B: LC3B II: P < 0.0001, F = 63.68; ATG5: P = 0.0002, F = 52.14; p62: P = 0.0010, F = 26.98). (C) Representative immunoblot images and (D) quantitation of LC3B II, ATG5 and p62 in bEnd.3 cells from Control, OGD/R, and OGD/R + GYY groups. β-actin was used as the protein loading control. The images of western blot data derived from triplicate blots conducted as three independent experiments. (D: LC3B II: P = 0.0001, F = 60.76; ATG5: P < 0.0001, F = 118.6; p62: P < 0.0001, F = 269.2). (E) The bEnd.3 cells were transfected with flag-tagged RFP-mWasabi-LC3 lentiviral vector in Control, OGD/R, and OGD/R + GYY groups. Representative images of fluorescent LC3 dots are shown. (F) Mean number of autophagosomes (dots with both red and green color; i.e., dots with yellow color in merged images) and autolysosomes (dots with only red but not green color; i.e., dots with red color in merged images) per cell. Scale bar indicates 20 μm. (F: red: P < 0.0001, F = 186.4; yellow: P < 0.0001, F = 28.39). The data are the means ± SD. n ≥ 3. ^#P < 0.05 versus OGD/R. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.