GM-CSF blocks the cleavage of apoptosis-related truncated Atg5 by inhibiting calpain activity. (A) Immunoblot analysis with Abs to Atg5 or truncated Atg5 in lysates of human primary monocytes treated or not with GM-CSF. (B) Immunoblot analysis of subcellular fractionations (cytoplasm and mitochondria) of human monocytes that were or were not treated with GM-CSF for 14 hours, probed with anti-truncated Atg5, anti-VADC, and anti–β-Tubulin. (C) Immunoblot analysis with Abs to truncated Atg5 in human primary monocytes treated or not treated with GM-CSF. Where indicated, cells were pretreated with 3-MA (10μM) or CQ (50μM) for the indicated times. (D) Calpain activity assay of lysates from monocytes treated or not with GM-CSF. Error bars indicate ± SEM. Data are representative of 5 independent experiments, *P < .02; **P < .002. (E) Immunoblot analysis with Abs to Atg5 or truncated Atg5 in lysates from human primary monocytes treated or untreated with the calpain inhibitor ZVP (10μM). (F) Immunoblot analysis with Abs to Calpain1, calpastatin, and Actin in lysates of GM-CSF–incubated monocytes. (G) Immunoblot analysis with Abs to LC3, truncated Atg5, Caspase-3, and actin in human primary monocytes lysates treated with GM-CSF and ZVP (10μM). (H) Histogram of cell death assessed by flow cytometry of annexin V/PI–stained cells treated or not with GM-CSF and ZVP for 24 hours. The percentages of annexin V/PI double-negative cells (percentage survival) are shown. Dead cells indicate all cells positive for annexin V, including annexin V⁺/PI⁻ early apoptosis and annexin V⁺/PI⁺ later apoptosis. Error bars indicate ± SEM. Data are representative of at least 3 independent experiments. #P < .02. Western blot data are representative of at least 3 (A-C,E) or 5 (F-G) experiments. Asterisk indicates a nonspecial band of truncated Atg5 Ab (A,C,E,G).