Figure 4. Caspase-dependent release of AV components during serum starvation (A) Immunoblot analysis for LAMP2, LC3-I, LC3-II and ATG16L1 in 25 ml of media conditioned by EC serum staved with vehicle DMSO (SSC) or with ZVAD-FMK 50 µM (SSC ZVAD); n = 3 for ATG16L1, LC3I and LC3-II; n = 2 for LAMP2. (B–E) Electron micrographs of EC serum starved with vehicle for 4 h showing AV near and/or interacting with the cell membrane. Scale bar: (B and C) 0.5 µm; (D) 1 µm. (E) Islet of the autophagic network in (D) located near the cell membrane; contact sites (white arrowheads). Scale bar: 0.5 μm. (F) AV in EC serum starved incubated with ZVAD-FMK 50 µM (SS + ZVAD) are located farther away from the cell membrane as opposed to AV seen in EC serum starved with vehicle (SS) (D and E). Scale bar: 1 µm. (G) Islet of the AV in (F). Scale bar: 0.5 µm. (H) Distance (µm) of AV from the cell membrane in serum-starved EC treated as in (A–G). Each AV distance was measured perpendicularly to the cell membrane in electron micrographs using ImageJ software; n = 30 cell profiles for each condition *p < 0.001 vs. SS.