Figure 6.

Flv induces neuroprotection by increasing Sig-1R protein expression. (a) Tm (1 μg/ml) was added to mock-transfected HEK293 cells and Sig-1R-overexpressing HEK293 cells. For cytotoxicity comparisons, the free LDH activity was measured over the time course shown in the figure. Cytotoxicity was significantly reduced at 6, 12, and 24 h in Sig-1R expressing cells. Values are the mean±S.D. (*P<0.05, Student's t-test; n=3). (b) Tm (1 μg/ml) or Tm (1 μg/ml) and Flv (10 μg/ml) were added to Sig-1R^+/+ and Sig-1R^−/− MEFs. The free LDH activity in each treatment condition was measured over the time course shown in the figure, and the cytotoxicities were compared. After Flv treatment, a significant reduction in toxicity was observed in Sig-1R^+/+ MEFs but not in Sig-1R^−/− MEFs. Values are the mean±S.D. (*P<0.05, Student's t-test; n=3). (c) In the upper figure, Tm (1 μg/ml) or Tm (1 μg/ml) and Flv (10 μg/ml) were added to Sig-1R^+/+ MEFs, and caspase-12 processing was monitored over the time course shown. Caspase-12 processing was significantly reduced with Tm+Flv treatment compared with processing after treatment with Tm alone, indicating that Flv suppressed the induction of cell death. In the lower figure, Tm (1 μg/ml) or Tm (1 μg/ml) and Flv (10 μg/ml) were added to Sig-1R^−/− MEFs, and caspase-12 processing was monitored over the time course shown. Caspase-12 processing in Sig-1R-deficient cells was not affected by the addition of Flv. (d) Processed caspase-12/total caspase-12 in (c) was measured with densitometry. Black bar: Sig-1R^+/+ MEFs treated with Tm; dark gray bar: Sig-1R^+/+ MEFs treated with Tm+Flv; light gray bar: Sig-1R^−/− MEFs treated with Tm; white bar: Sig-1R^−/− MEFs treated with Tm+Flv. Processed caspase-12 was significantly reduced by Flv treatment in Sig-1R^+/+ MEF cells but not in Sig-1R^−/− MEF cells, demonstrating that the ability of Flv to suppress cell death induction was lost in the absence of Sig-1R. Values are the mean±S.D. (*P<0.05, Student's t-test; n=3)