Figure 1. PI3K but not Akt is required for engulfment of apoptotic cell by macrophage.

(A) Effect of isoform specific PI3K inhibitors on engulfment of apoptotic cells by macrophages. Macrophages pretreated with 50 nM Wortmannin (pan-), 50 nM Compound 15e (p110α-specific), 50 nM TGX-221 (p110β-specific), or 50 nM AS252424 (p110γ-specific) were incubated with apoptotic thymocytes for 90 min and the engulfment of apoptotic cells was analyzed by HEMA3 staining. Percentage of macrophage containing one or more apoptotic body is counted. (B) Transient generation of PtdIns(3,4,5)P₃ during efferocytosis. RAW264.7 macrophage-like cells were transfected with a PH-Akt-EGFP construct and fed with SNARF1 labeled apoptotic thymocytes for 60 min. Cells were washed, fixed and analyzed by fluorescence microscopy. Top panel represents early phase of apoptotic cell recognition by macrophage and the bottom panel represents a macrophage with an apoptotic cell after completion of engulfment. Bar represents 10 μm. (C) Pharmacological inhibition of Akt by Akti-VIII (3 μM) in peritoneal macrophages did not affect engulfment of apoptotic cells. (D) Overexpression of a constitutively activated Akt (myr-Akt) in RAW264.7 cells did not affect engulfment of apoptotic cells. Inset, Akt activation (Ser473-phosphorylation) upon expression of myr-Akt. Results shown are means ± SD (n>600). *p<0.005.