Fig. 7. Chemical inhibition of the PI 3-kinase pathway blocks HGF/SF-induced Akt phosphorylation and inhibits HGF/SF-mediated VEGF/VPF gene transcription and protein expression. (A) Detection of phosphorylated Akt (at residue threonine 308, Thr³⁰⁸; upper panel; at serine residue 473, Ser⁴⁷³, lower panel) or of the respective total Akt protein expression (Akt; antibodies from Cell Signaling) by western blot analysis. HaCaT cells were left untreated or were exposed to HGF/SF (for 10 minutes at 100 ng/ml) after preincubation with the PI 3-kinase inhibitors wortmannin (Wort., at 100 nM, for 60 min) or LY 294002 (LY; at 10 µM, for 60 min) or with solvent only (DMSO, 0.1%) as indicated. Experiments were repeated three times with comparable results. (B) Analysis of CAT expression derived from a transiently transfected -88/+54 bp VEGF/VPF-promoter-based construct. HaCaT cells were cultured in the absence or presence of wortmannin (at 100 nM, starting 1 hour prior to HGF/SF treatment) without growth factor stimulation or with HGF/SF treatment (for 16 hour at 100 ng/ml) as indicated. Values represent the mean±s.d. of three triplicate assays. (C) VEGF/VPF protein of supernatants derived from confluent HaCaT cells. Cells were cultured in the absence or presence of LY 294002 (at 10 µM, for 60 minutes, starting 1 hour prior to HGF/SF treatment) without growth factor stimulation or with HGF/SF treatment (for 24 hours at 100 ng/ml) as indicated. Data from three triplicate experiments are expressed as ng secreted VEGF/VPF protein per mg total cellular protein (mean±s.e.m.). Statistical analyses were performed on data from three sets of experiments (Student's t test, ^**P<0.01, ^*P<0.05).