Figure 5. Caspase-3-dependent release of AV components in serum-starved murine EC. (A) Percentages of cells with chromatin condensation and cell membrane permeabilization (as evaluated by HO and PI staining) in aortic EC isolated from controls (WT) and CASP3-deficient (C3^−/−) mice exposed to normal medium 4 h or SS for 2–4 h. *p ≤ 0.02 vs. WT, n = 6. (B) Upper panel: Immunoblot for LC3-I/-II in EC treated as described above. Lower panel: densitometry analysis of LC3-II/LC3-I ratios for WT and C3^−/− murine EC serum starved for 4 h. Representative of 16 independent WT mice and 6 C3^−/− mice. (C–E) Electron micrographs of control murine EC (WT) exposed to SS for 4 h showing AV near and/or interacting with the cell membrane [islet in (E)]. Scale bars: (C and D) 2 µm; (E): 0.5 µm. (F–H) Electron micrographs of murine C3^−/− EC exposed to SS for 4 h showing enhanced autophagic vacuolization located away from the cell membrane (islet in H) as compared with WT (E). Scale bars: (F and G) 2 µm; (H) 0.5 µm. (I) Upper panel: Immunoblot analysis for LC3-I and LC3-II in 10 ml of serum-free media conditioned by controls (SSC WT) or CASP3-deficient (SSC C3^−/−) murine EC. Lower panel: Densitometry analysis of LC3-I and LC3-II; n = 3.