Figure 2. Caspase inhibition does not prevent development of autophagy in serum-starved EC. (A) Percentages of cells with increased chromatin condensation (apoptosis) and cell membrane permeabilization (necrosis), as evaluated by HO and PI staining, in EC exposed to SS in presence of the pan-caspase inhibitor ZVAD-FMK (SS+ZVAD) 50 µM or SS + vehicle DMSO (SS) for 1–4 h. *p ≤ 0.002 vs. Z, n = 6. (B) Immunoblot for uncleaved and cleaved PARP in EC treated as described above. Representative of 4 experiments. (C and D) Electron micrographs of EC serum starved for 4 h with vehicle DMSO [SS, (C)] or with ZVAD-FMK 50 µM [SS+ZVAD, (D)]. Scale bar: 1 µm. (E) Mean cytoplasmic area of the cell profiles. (F) Percentage of total cytoplasmic area (µm²) occupied by AV per cell profile in EC treated as in (A–D). *p = 0.01 vs. SS; n = 30 cell profiles. (G) Mean number of AV profiles per cell profile in EC serum starved for 4 h in presence of the pan-caspase inhibitor ZVAD-FMK 50 µM (SS+ZVAD) or vehicle DMSO (SS). *p ≤ 0.0005 and **p ≤ 0.005 vs. SS; n = 30 cell profiles. (H) Time course of LC3 turnover by immunoblot in EC treated as in A (the immunoblot corresponds to two parts of the same gel). Representative of three experiments. (I–K) Native LC3 immunostaining evaluated by confocal microscopy in EC exposed either to (I) normal medium (N) or serum-starved and treated with (J) vehicle DMSO (SS) or (K) ZVAD-FMK 50 µM (SS+ZVAD). (L) Mean number of LC3 puncta per cell profile evaluated by confocal microscopy immunostaining in EC treated as above. LC3 puncta were counted in approximatly 50 cells/representative section of the sample in three different trials; p ≤ 0.0001. (M) Upper panel: LC3-I and -II immunoblot in EC exposed to vehicule (SS), SS+ZVAD (50 µM), SS + bafilomycin A₁ (5 nM) or SS + ZVAD + bafilomycin A₁ for 4 h. Lower panel: Densitometry analysis of the LC3-II/LC3-I ratio. *p ≤ 0.02 vs. SS, **p ≤ 0.0002 vs SS+ZVAD. Representative of four experiments.