Figure 4
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.

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.

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