Fig. 2.

Autophagic flux is blocked under low SS (LSS). (A) LC3 II/LC3 I ratio quantified by Western blot in HUVECs exposed to high SS (HSS) and low SS (n = 5; 6 h of SS including 4 h of bafilomycin A1, 100 nmol/L). (B) Quantification of HUVECs transfected with the tandem mRFP-GFP-LC3 plasmid and exposed for 24 h to high or low SS. Yellow bars represent autophagosomes, and red bars represent autolysosomes. Images are representative of three independent experiments in which more than 100 cells were observed. Autophagosomes are labeled with a yellow signal, whereas autolysosomes are labeled with a red signal. (Scale bar, 10 μm.) (C) Immunofluorescent LAMP2 and LC3 staining on HUVECs exposed to high or low SS for 24 h. (Left) Quantification. (Right) Representative images (red, LC3; green, LAMP2; blue, DAPI). Data are presented as mean ± SEM (*P < 0.05, **P < 0.01, and ***P < 0.001). (D) LC3 en face staining of the aorta of 8-wk-old C57BL/6 male mice injected i.p. with PBS solution or chloroquine (60 mg/kg/d; 48 h, 24 h, and 4 h before euthanasia; n = 7–9 per group; green, CD144; red, LC3; blue, DAPI). (Scale bar, 20 μm.) (Left) Quantification of LC3 area. Data are given as median (horizontal bar) and interquartile range (error bar). (Right) Representative images.