Caspase-8 involvement in monocyte differentiation into macrophages. (A-B) Human monocytes (Mo) were purified from healthy donor peripheral blood and exposed for indicated times to 100 ng/mL M-CSF to trigger their macrophagic differentiation (Mac) or 100 ng/mL GM-CSF plus 10 ng/mL IL-4 and 50 μM β-mercaptoethanol for inducing their differentiation into dendritic cells (DC) before flow cytometry analysis of (A) cleaved caspase-8 expression and (B) FAM-LETD cleavage activity. (C-E) Monocytes were transfected with either Luciferase (siLuc) or caspase-8 siRNA (siC8). (C) Expression of caspase-8 and cleavage fragments and cleaved caspase-3 were analyzed by immunoblotting. Hsc70 indicates loading control. Molecular weights are indicated in kDa. The asterisk indicates cleavage fragments. Caspase-8 (▪) and cleavage fragments (⊡) were normalized to Hsc70 expression and represented as fold decreases. (D) Expression of CD71 or CD1a was studied by fluorescence-activated cell sorting (FACS) analysis. Results are normalized to values obtained in cells transfected with luciferase siRNA; ▪, siLuc; □, siC8. (E) Percentages of cells with a fibroblastic-like shape, indicating macrophagic differentiation, as observed microscopically (mean ± SD of 3 measurements). (F) U937 cells were treated with 20 nM TPA for indicated times before flow cytometry analysis of FAM-LETD cleavage activity. (G-H) U937 cells were stably transfected with an empty vector or a vector encoding a caspase-8 dominant-negative mutant (C8-DN) before flow cytometry analysis of (G) FAM-LETD, FAM-DEVD, and FAM-LEHD cleavage activities (gray curves, control vector; white curves, C8-DN) and (H) CD11b expression at the cell surface (○, control vector; •, C8-DN). One representative of at least 3 independent experiments is shown or mean ± SD of 3 independent experiments. *P < .05; ***P < .005.