Fig. 4.
Increased autophagy in VEGF-depleted endothelium contributes to cell death phenotype. (A) Autophagic vacuoles (arrowheads) visualized by using the Cyto-ID autophagy detection kit and analyzed by using a confocal microscope. (B) Quantification of fluorescence from cultures analogous to the ones shown in A (n=3). (C) Western blot of autophagic marker LC3A. (D) Quantification of total LC3A (LC3AI and LC3AII) levels normalized to GAPDH (n=8). (E) Autophagic flux measured by quantification of the LC3AI:LC3II ratio in western blots, (n=8). (F) Electron microscopy images. VEGF-ECKO adult animals display an abundance of double-membrane autophagic vacuoles (arrows). (G) Quantification of cell confluence as a measure of viability in siRNA targeting of VEGF or VEGF+Atg7 (n=6). (H) Quantification of cell confluence in KD-HUVECs during autophagy blockade in response to 10 μM chloroquine (n=3). For all graphs, error bars indicate the mean±s.d.; *P<0.05, **P<0.005.