Figure 1

PS protects mouse cortical neurons from tPA/NMDA-induced injury. (A) Dose-dependent neuroprotective effects of PS (12.5-200 nM) in a 14-day old neuronal cultures 24 h after tPA/NMDA treatment. Cell survival was quantified with a WST assay. (B) Representative fluorescent-TUNEL (green)/Hoechst (blue) staining in mouse cortical neurons 24 h after tPA/NMDA treatment in the presence or absence of PS. (C) The number of apoptotic TUNEL-positive cells quantified as described in the Methods in the presence of NMDA with and without tPA or PS. (D) Survival of mouse cortical neurons 24 h after a combined tPA/NMDA treatment with and without PS, caspase-9 inhibitor (z-LEHD-fmk 5 μM), caspase-8 inhibitor (z-IETD-fmk, 50 μM), or caspase-3 inhibitor (Ac-DEVD-CHO, 50 μM). (E) Caspase-8 activity in mouse cortical neurons 8 h after tPA/NMDA exposure in the presence or absence of PS and a caspase-8 inhibitor (z-IETD-fmk; 50 μM). (F) Caspase-3 activity in mouse cortical neurons 12 h after tPA/NMDA in the presence or absence of PS, caspase-9 inhibitor (z-LEHD-fmk 5 μM), caspase-8 inhibitor (z-IETD-fmk, 50 μM), or caspase-3 inhibitor (Ac-DEVD-CHO, 50 μM). (G) Western blots for FasL in whole-cell extracts from mouse cortical neurons 6 h after tPA/NMDA in the presence or absence of PS. Intensity of FasL signal was measured by scanning densitometry and normalized to β-actin. (H) Caspase-8 activity in mouse cortical neurons 8 h after tPA/NMDA in the presence of anti-FasL antibody (α-FasL; 10 μg/ml). Anti-FasL antibody was added simultaneously with tPA-NMDA. Caspase inhibitors were applied 1 h prior to tPA/NMDA treatment. In all studies murine PS was used at 100 nM, unless specified differently, and was added simultaneously with tPA (20 μg/ml) and NMDA (25 μM). Mean ± SEM, n = 3 independent cultures in triplicate. *p < 0.05 compared with values in the absence of PS. NS, non-significant.