PI3K inhibition and TNF secretion. (A) RAW264.7 macrophages were stimulated with LPS in the presence of vehicle DMSO or either 15 nM wortmannin or 50 µM LY294002, and supernatants were sampled at the times indicated to measure TNF secretion by ELISA. (B) TNF secretion 2 h after LPS activation and treatment with p110-selective inhibitors (YM024 [p110α], TGX221 [p110β], IC87114 [p110δ], or AS252524 [p110γ]) at the concentrations indicated. The following indicate statistical significance at the lowest effective concentrations: ** (IC87114), P = 0.0025; ** (TGX221), P = 0.0073; * (YM024), P = 0.116. (C) Epifluorescence of RAW264.7 cells stimulated with LPS for 1 h in the presence of a TACE inhibitor, TAPI, to retain surface TNF. Cells treated with DMSO alone or with 50 µM LY294002. Immunostaining of surface TNF (red) on unpermeabilized (unperm) cells was followed by staining of intracellular TNF (green) after permeabilization (perm). DAPI (blue) labeling depicts nuclei. Bars, 10 µm. (D) Flow cytometric analysis of intracellular TNF (Alexa Fluor 488) and surface TNF (PE) staining in activated cells treated with DMSO, 15 nM wortmannin, 25 µM LY294002, or 5 µM IC87114 for 2 h. Mean fluorescence intensity of TNF staining in Alexa Fluor 488 or PE channel expressed as mean ratio ± SEM relative to DMSO. Corresponding representative overlay histograms are displayed. (A–D) All results are representative of three independent experiments. Error bars indicate mean ± SD.