Autophagy is activated during human monocyte differentiation. (A-B) Immunoblot analysis of lysates from human primary elutriated monocytes incubated with or without GM-CSF (10nM) for the indicated times. (B) Where indicated, cells were pretreated with 3-MA at 10mM or CQ at 50μM. Representative data from at least 3 independent experiments for panels A and B are shown. The ratio of LC3II/LC3I was quantified from 3 experiments and is presented in bar graphs (A-B). (C) Human monocytes treated or not with GM-CSF for 14 hours, stained with anti-LC3 Ab and then Alexa Fluor 568 goat anti–mouse IgG (red), and processed for confocal microscopy. Scale bars indicate 5 μm. (D) Human monocytes were incubated with or without GM-CSF for 14 hours and processed for electron microscopy. Black arrows indicate autophagosomes with double membranes and partially degraded material. Scale bars indicate 500 nm. (E) Human monocytes were incubated with or without GM-CSF (10nM) in the absence or presence of 3-MA (10mM) for 14 hours, fixed, and stained with 4′,6-diamidino-2-phenylindole to visualize the nuclei (blue), and immunolabeled with an anti-LC3 Ab followed by 568 goat anti–mouse IgG (red). Scale bars indicate 20 μm. Representative images from 4 independent experiments are shown in panels B through D and quantitation of the percentage of cells with LC3⁺ punctate from the 4 experiments is shown in panel E (*P < .002 for comparisons between GM-CSF alone and untreated or cotreatment).