Autophagy is required for human primary monocyte differentiation. (A) Morphological changes associated with GM-CSF–induced monocyte/macrophage differentiation from days 1, 3, and 6. Representative phase-contrast images are shown for GM-CSF treatment alone (left panel) and for pretreatment with 3-MA (middle panel) or CQ (right panel). Scale bar indicates 100 μm. (B) Representative FACS plots showing CD14 and CD11b expression in GM-CSF–treated monocytes/macrophages treated or not with 3-MA or CQ. Live Cells were gated and stained with FITC–anti-CD14 Ab and PE–anti-CD11b Ab. (C) Representative FACS plots showing LPS/IFN-γ–stimulated CD86 expression in GM-CSF macrophages pretreated or not with 3-MA or CQ. Gated live cells stained with PE–anti-CD86 Ab are shown. (D) Quantitative real-time PCR analysis of human TNF-α and IL-12p40 mRNA in macrophages pretreated with 3-MA or CQ, presented relative to GAPDH mRNA. P < .005 for comparisons between GM-CSF alone and cotreatment with 3MA or CQ for both graphs. Data are representative of 5 (A) or 3 (B-D) experiments. Histogram shows mean ± SD in panel D.