Figure 5.

EC autophagy machinery regulates cell-surface PECAM-1 intracellular trafficking and degradation

(A–C) Schematic illustrating cell-surface biotinylation method. Control and ATG5 siRNA-silenced HUVECs were stimulated with LPS (A) and (B) immunoblotted for total and biotinylated PECAM-1, ATG5, and β-actin at the indicated times postbiotin incubation and (C) analyzed for fold change in cell surface-derived PECAM-1 (n = 7).

(D–G) GFP-LC3 transfected HUVECs were stimulated with LPS before antibody feeding using a nonblocking anti-PECAM-1 mAb.

(D) Number of GFP-LC3⁺/ PECAM-1⁺ vesicles at the indicated times after incubation with anti-PECAM-1 mAb (n = 3–5; 40–100 cells analyzed per condition).

(E) Time-lapse confocal images (Video S6) showing the formation of GFP-LC3⁺/PECAM-1⁺ vesicles (scale bars, 10 μm).

(F and G) Representative (n = 4) confocal images of GFP-LC3 transfected HUVECs immunostained for PECAM-1 and WIPI2 (scale bars, 10 μm and enlargements, 3 μm) (F) and (G) quantification of the number of GFP-LC3⁺/ PECAM-1⁺ vesicles WIPI2^-/+ (n = 4; >100 cells analyzed per condition).

Means ± SEMs. Statistically significant difference from controls is shown by ^∗p < 0.05 and ^∗∗p < 0.01.

See also Figure S5.