PI3K and AKT down-regulation prevents CSF-1–mediated caspase activation. (A) Monocytes were transfected with siRNA targeting Luciferase (SC) or AKT (AKT1 or AKT2) or the p85 subunit of PI3K (PI3K1 or PI3K2) 2 days before real-time qPCR analysis of the corresponding mRNAs (mean ± SD of 3 independent experiments). (B) Monocytes were exposed for 2 days to CSF-1 after indicated siRNA transfection. Macrophage differentiation was examined morphologically (fibroblastic-like shape) and by 2-color flow cytometric analysis. Percentages indicate cells that express both CD71 and CD163 or both CD14 and CD16. Similar results were obtained with the 2 siRNAs targeting indicated genes. (C) FAM-DEVD cleavage activity was studied by flow cytometry in monocytes treated for 2 days with CSF-1 in the absence or presence of 10 μmol/L LY added 30 minutes before CSF-1 or in cells transfected at day 0 with siRNA targeting AKT (AKT1 and AKT2) or p85 PI3K subunit (PI3K1 and PI3K2). Numbers: percentages of cells with DEVD activity. One representative of at least 3 independent experiments is shown. (D-E) Expression of p85 PI3K subunit, NPM, and its cleavage fragment (NPM*) in cells transfected with indicated siRNA and treated for 2 days with CSF-1 as above. HSC70 indicates loading control. Molecular weight (MW) is in kilodaltons. In panel D, vertical line indicates repositioned gel lanes caused by the removal of an inefficient control.