Figure 1. Serum starvation (SS) in EC induces both apoptotic and autophagic features. (A and B) Electron micrographs of normal and apoptotic EC. The nucleus (Nu) of EC exposed to complete growth medium (Normal) displays an elongated aspect whereas that of serum-starved EC (SS) for 4 h appears rounded and condensed, characteristic of apoptosis. Scale bar: 1 µm. (C) Percentages of cells with increased chromatin condensation (apoptosis) and cell membrane permeabilization (necrosis), as evaluated by HO and PI staining, in EC exposed to normal medium (N) or SS for 1–4 h. *p ≤ 0.0007 vs. N, n = 6. (D) Immunoblot for uncleaved and cleaved forms of PARP in EC treated as in (C), representative of 4 experiments. (E) Upper panel: Time-course of LC3 turnover, by immunoblot, in EC treated as in (A–D). Lower panel: Densitometry analysis of LC3-II/LC3-I ratios in EC exposed to N or SS for 1–4 h. *p ≤ 0.01 vs. N. Representative of four experiments. (F) LC3-I and -II immunoblot in EC exposed to N, SS and SS + bafilomycin A₁ 5 nM for 4 h (the immunoblot corresponds to two parts of the same gel). Representative of four experiments. (G–K) Morphological characterization of AV in EC serum starved for 4 h. (G) AV with intact cytoplasmic portions or organelles delimited by multiple membranes with internal electron-lucent material and cytoplasm. (H and I) AV with various stages of degraded cytoplasmic material, characterized by increased electron density within vacuoles surrounded by single or double delimiting membranes. (J) AV displaying a more advanced degradation stage with multilamelar lysosomal bodies (white star). (K) Amphisomes are characterized by the presence of both autophagosomal electron-dense material with a delimiting membrane and MVB nanovesicular content. Scale bar: 0.5 µm. (L) Total cytoplasmic area (µm²) occupied by AV per cell profile in EC exposed for 4 h to N and SS, respectively. Area of AV per cell profile (n = 20) was assessed in relation to the cell cytoplasm (nuclei were not included in the evaluation); *p ≤ 0.001 vs. N.