Figure 3

Effect of inhibitors of PI3K and Akt on the intracellular distribution of GAPDH. (A) HDFs were incubated in culture medium containing 10% FBS and treated with vehicle (DMSO) or various concentrations of the PI3K inhibitor LY 294002 (10-50 µM) for 2 days. Cells were immunostained against GAPDH and analyzed by confocal laser scanning microscopy, as described in Figure 1. The number of cells with cytosolic GAPDH alone (cytosol: black bar) and cells having nuclear GAPDH with or without cytosolic GAPDH (nuclear +/- cytosol: red bar) was counted and the percent distributions are plotted as means ± standard deviations. ^***P < 0.001, comparison between the FBS control and FBS + LY 294002 (µM). (B) HDFs were serum-depleted by incubating in SFM for 4 days, and then either 10% FBS, 100 ng/ml PDGF, or 30 µM LPA was re-added to serum-depleted cells in the absence (-) or presence (+) of 50 µM LY 294002 for 2 days. ^**P < 0.01 and ^***P < 0.001, comparison between the absence (-) and presence (+) of LY 294002. (C) HDFs were incubated in medium containing 10% FBS and treated with vehicle (DMSO) or various concentrations of the Akt inhibitor SH-5 (5-20 µM) for 2 days. ^**P < 0.01, comparison between the FBS control and FBS + SH-5 (µM). (D) HDFs were serum-depleted by incubating in SFM for 4 days, and then either 10% FBS, 100 ng/ml PDGF, or 30 µM LPA was re-added to serum-depleted cells in the absence (-) or presence (+) of 20 µM SH-5 for 2 days. ^**P < 0.01 and ^***P < 0.001, comparison between the absence (-) and presence (+) of SH-5. The results shown are representative of more than three independent experiments. Serum-starved HDF cells were pre-incubated with vehicle (-), 50 µM LY 294002 (E, +), or 20 µM SH-5 (F, +). Cells were treated with 10% FBS/DMEM for 30 min and then lysed. Total cell extract (30 µg per lane) was resolved by 10% PAGE and analyzed by Western blot analysis, using specific antibodies against phospho (Ser⁴⁷³)-Akt, total Akt, phospho (Ser⁸)-GSK-3β, total GSK-3β, phospho-Bad, total Bad, and β-actin as a control (1:1,000 dilutions).