FIG. 1.

Inhibition of Akt sensitizes SH-SY5Y cells to H₂O₂ toxicity. (A) Western blotting of ser473-phosphoAkt and of total Akt in homogenates of SH-SY5Y cells exposed or not to 200μM H₂O₂ for the time indicated. One representative gel out of four independent experiments is shown. The filter was stripped and probed for actin as a marker of protein loading. The densitometry ratio of pAkt/total Akt is reported. (B) SH-SY5Y cells were preincubated or not with an Akt inhibitor and cultivated for up to 120 min in the absence or the presence of 200μM H₂O₂ (perox). At designated time-points, the adherent viable (trypan blue excluding) cells were counted. Cell density data obtained from three independent experiments in triple. (C) SH-SY5Y cells were preincubated or not with an Akt inhibitor and cultivated on sterile coverslips for 30 or 120 min in the absence or the presence of 200μM H₂O₂ (perox). At the end, the cells were stained with mitotracker and for immunofluorescence detection of active bax. Bax-positive/mitotracker-negative cells amounted to 15 ± 5% at 30 min and to 40 ± 15% (of the adherent cells) at 120 min in cultures exposed to H₂O₂ in the absence of the Akt inhibitor and to 42 ± 12% and to > 80% in the parallel cultures incubated in the presence of the Akt inhibitor. Data reproduced in three independent experiments. Bar = 10 μm.