Inhibition of autophagy suppresses GM-CSF–induced human primary monocyte survival. (A) Cell death of human monocytes with different treatments (GM-CSF, 10nM; 3-MA, 10mM; or CQ, 50 mM) for 48 hours was assessed using annexin V/PI staining and flow cytometry. Percentage 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 (A,C). Error bars indicate ± SEM. Data are representative of at least 3 independent experiments. (B) Immunoblot analysis with Abs to LC3, Caspase-3, and actin for GM-CSF–treated primary human monocytes. Representative data are shown for GM-CSF alone and pretreatment with 3-MA (10mM) or CQ (50μM). (C) Cell death assessed by flow cytometry of annexin V/PI–stained cells with Beclin1 siRNA or control siRNA in treated or not with GM-CSF. The percentage of annexin V/PI double-negative cells (percentage survival) is shown. Error bare indicate ± SEM. Data are representative of at least 3 independent experiments. *P < .03. (D) Immunoblot analysis of lysates of monocytes transfected with human Beclin1 siRNA or control siRNA probed with anti–Caspase-3, anti-Beclin1, and anti-actin Abs. Data are representative of at least 3 experiments.