HIMF-induced NF-κB activation and upregulation of Flk-1 are PI-3K/Akt pathway dependent. SVEC 4–10 cells were pretreated with signal transduction inhibitors or co-transfected with luciferase constructs and PI-3K dominant-negative mutant, then stimulated with HIMF (40 nmol/L) for various periods as indicated. (7A) HIMF strongly induces phosphorylation of Akt at Ser473 and Thr308. The Akt phosphorylation is detectable at 30 minutes and sustained for 360 min. HIMF also induced phosphorylation of ERK1/2 and p38 MAPK, but not JNKs, in SVEC 4–10 cells. The figures indicate the relative density compared to control. (7B) The PI-3K inhibitor LY294002 (10 μmol/L), but not SB203580 (5 μmol/L), PD098059 (5 μmol/L) or U0126 (5 μmol/L), abolished HIMF-induced Akt phosphorylation and upregulation of Flk-1 in SVEC 4–10 cells. (7C) Transfection of Δp85 into SVEC 4–10 cells abolished HIMF-induced phosphorylation of IKK and IκBα, prevented NF-κB activation and production of Flk-1. The symbol (*) indicates a significant increase from SVEC 4–10 controls without HIMF treatment (P < 0.05). The symbol (#) indicates a significant decrease from SVEC 4–10 cells treated with HIMF only (P < 0.05). Triplicate experiments were performed with essentially identical results (n = 3).