Autophagy is induced during macrophagic differentiation of human monocytes. Human peripheral blood monocytes from healthy donors were exposed for the indicated times to 100 ng/mL CSF-1. (A) Electron microscopy images showing ultrastructural features of a representative monocyte (d0) and morphologic features of autophagy in monocytes treated for 3 days (d3) with CSF-1. P indicates phagophore; A, autophagosome; and N, nuclei. (B) Immunoblot analysis of caspase-3, NPM, LC3, Lamp2, CTSB, and SQSTM1 in monocytes exposed for the indicated times to CSF-1. Actin is used as a loading control. *Cleavage fragments. Molecular weights (MW) are in kDa. (C) Measurement of CTSB and B + L activities using Z-RR-AMC or Z-FR-AMC as substrates, respectively, in monocytes treated with CSF-1. Results, expressed as arbitrary units (A.U.) per minute and per milligram of protein, are the mean ± SD of 4 independent experiments performed in quadruplicate. (D) Monocytes were exposed for 3 days to 100 ng/mL alone or in association with bafilomycin A1 (10nM) added 48 hours after CSF-1 treatment, and protein expression was analyzed by immunoblot. Actin is used as a loading control. (E) Monocytes were exposed for 2 days to CSF-1 before collecting cytoplasmic (F1) and microsomal (F2) extracts that were analyzed by immunoblot. Lamp2 and γ-tubulin are used as a control for microsomal and cytoplasmic fractions, respectively. (F) Immunoblot analysis of phospho-ULK1 (Ser555) and ULK1 in monocytes exposed for the indicated times to CSF-1. (G) Monocytes were transfected with siRNA targeting Luciferase (Luc) or ULK1 and exposed 2 days to CSF-1. The expression of ULK1 in transfected cells was analyzed by immunoblot. (F-G) Actin is used as a loading control. (H) Monocytes were transfected with siRNA targeting Luciferase (Luc) or ULK1 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.