Autophagy involvement in monocyte differentiation into macrophages. (A) Monocytes were transfected with siRNA targeting Luciferase (Luc), Beclin-1 (BEC), ATG7, or ATG5 and exposed 2 days to CSF-1. Macrophage differentiation was examined morphologically (fibroblastic shape) and by 2-color flow cytometric analysis. Percentages indicate cells that express both CD71 and CD163. (B) Monocytes were transfected with siRNA targeting Luciferase (Luc), Beclin-1 (BEC), ATG7, or ATG5 and exposed 2 days to CSF-1. The expression of Beclin-1, ATG7, ATG5, and LC3 in cells transfected with the indicated siRNA and treated for 2 days with CSF-1 was analyzed by immunoblot. Actin is used as a loading control. Molecular weights (MW) are in kDa. (C) Functional assay of monocytes transfected with Luciferase, Beclin-1, ATG7, or ATG5 siRNA and treated for 2 days with CSF-1. Results are expressed as fold induction compared with untreated monocyte and represent the mean ± SD of 4 independent experiments performed in triplicate. ***P < .001 (vs d2 LUC) according to a student paired t test. (D) Enriched bone marrow mouse monocytes were exposed for the indicated times to 100 ng/mL CSF-1. Differentiation was studied by morphologic examination (fibroblastic shape) and by 2-color flow cytometric analysis at indicated day. Percentages indicate cells that express both high CD11b and F4/80 staining. One representative of 5 independent experiments is shown. (E) Immunoblot analysis of NPM and LC3 in mouse monocytes exposed for the indicated times to CSF-1. Actin is used as a loading control. *Cleavage fragments. (F) Immunoblot analysis of ATG7 in monocytes obtained from WT or vav-Atg7^−/− mice (n = 2). Actin is used as a loading control. (G) Monocytes obtained from WT or vav-Atg7^−/− mice were exposed for the indicated times to 100 ng/mL CSF-1. Differentiation was studied by morphologic examination (fibroblastic shape) and by 2-color flow cytometric analysis at day 4. Percentages indicate cells that express both high CD11b and F4/80 staining.