Figure 8.

Autophagy mediates the degradation of endocytosed CAV1 under hypoxia. (A and B) CoCl₂-induced hypoxia caused degradation of CAV1 in monolayer bEnd.3 cells. Blocking of autophagy by CQ significantly inhibited the degradation of CAV1 while enhancing of autophagy by Rapa promoted its degradation. n = 5. P value indicates one-way ANOVA with Dunnett’s multiple comparisons test. (C and D) Immunogold-labeled CAV1 (black arrows) was imaged by immuno-electronmicroscope (IEM) and was found in autophagosome-like vesicles of bEnd.3 cells after CoCl₂-induced hypoxia treatment. The right panel is a high magnification scan of the red line-marked region in the left panel. The numbers of gold particles which represent packaged CAV1 in caveolae per image were counted for quantification analyses. n = 6 images analyzed. Scale bars: 200 nm. (E) knock-down of Cav1 itself in bEnd.3 cells showed no effect on the paracellular permeability of cell monolayer under normoxic condition. n = 3. CoCl₂: 200 μmol/L, treated for 12 h. Norm: normoxia; Hyp: hypoxia; sicontrol: monolayer of bEnd.3 was transiently transfected with scrambled negative control siRNA. siCav1: monolayer of bEnd.3 was transiently transfected with Cav1 siRNA. CQ: chloroquine. Rapa: rapamycin. FITC-dextran: Fluorescein-labeled dextran. Papp: apparent permeability coefficient. LC3: microtubule-associated protein 1 light chain 3. Data were presented as mean ± SEM. P value indicates two-tailed unpaired t test. *, P < 0.05, **, P < 0.01. N.S: no significance