Effect of PI3K inhibition on TGF-β-induced apoptotic and HGF-induced anti-apoptotic activities in oval cells. A.

<p>Mouse Met^flx/flx oval cells were serum-starved and incubated in the absence or presence of TGF-β (1 ng/ml) ± HGF (40 ng/ml) and/or LY294002 (7.5 µM) for 24 hours. Apoptotic index was calculated by counting apoptotic nuclei after PI staining under a fluorescence microscope. A minimum of 1000 nuclei was counted per condition. Data are mean±S.D. of a representative experiment performed with triplicate dishes. <b>B.</b> Mouse Met^flx/flx and Met^−/− oval cells were serum-starved and incubated in the absence or presence of TGF-β (1 ng/ml) ± LY294002 (7.5 µM) for different periods of time. Fluorescence intensity was measured in a FACScan flow cytometer and the percentage of annexin V positive/PI negative cells was calculated. Data are expressed as fold induction over untreated cells and are mean±SEM of four independent experiments. <b>C, D and E.</b> Mouse Met^flx/flx oval cells were serum-starved and incubated in the absence or presence of TGF-β (1 ng/ml) ± LY294002 (7.5 µM). <b>C.</b> After 8 hours of treatment, cells were collected for spectrophotometric determination of intracellular glutathione. Results are expressed as fold change over untreated cells and are mean±SEM of two independent experiments run in duplicate. <b>D and E.</b> After 12 hours of treatment RNA was isolated and <i>Nox4</i> and <i>Bmf</i> mRNA levels were analyzed by qRT-PCR and normalized to the housekeeping gene <i>Gusb</i>. Data represent fold change relative to untreated samples and are mean±S.E.M of three independent experiments. <b>Black bars</b>, Met^flx/flx cells. <b>White bars</b>, Met^−/− cells. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001 (treated versus untreated); #<i>P</i><0.05 (T+LY treated versus T treated).