GM-CSF induces JNK activation to mediate the disassociation of Beclin1 and Bcl-2, the induction of autophagy, and monocyte survival. (A) Immunoblot analysis with anti-LC3 and actin for monocytes pretreated with JNK inhibitor SP (10μM), MEK inhibitor U0126 (10μM), or p38 inhibitor SB (5μM), and then treated with GM-CSF for 14 hours. (B) Immunoblot analysis with Abs to phosphorylated JNK or JNK of monocytes treated with GM-CSF for the indicated times. (C) Human monocytes were treated with or without GM-CSF for the indicated times and lysates were subjected to immunoblot analysis with Abs to phosphorylated Bcl-2 and actin. (D) Western blots of endogenous Beclin1 co-immunoprecipitated by anti–Bcl-2 Ab. (E) Human monocytes pretreated with the JNK inhibitor SP (10μM) in the presence or absence of GM-CSF for the indicated times and lysates were subjected to immunoblot analysis with Abs to phosphorylated Bcl-2, LC3, and Actin. (F) Cell death assessed by annexin V/PI staining showing apoptosis in monocytes untreated or pretreated with JNK inhibitor SP in the presence or absence of GM-CSF for 48 hours. 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. Error bars indicate± SEM. Data are representative of 3 independent experiments. (G) Lysates were subjected to immunoblotting with Caspase-3 and actin Abs. Western blot data are representative of at least 3 experiments.