Fig. 7

Effects of BV02 treatment on NMDA-induced neuronal excitotoxicity. A Treatment with the small-molecule 14-3-3 inhibitor BV02 (40 μM) promotes endogenous Eag1 protein level in cultured cortical neurons. Neurons (DIV12) were pretreated with the indicated concentrations of BV02 for six hours, followed by immunoblotting analyses with the indicated antibodies. B–D BV02 (40 μM) treatment averts NMDA-mediated reduction of endogenous Eag1 protein level and rescues NMDA excitotoxicity in cultured cortical neurons. Neurons were pretreated with DMSO or BV02, and then subject to 6-h treatment of 20 μM NMDA (in the absence or presence of 30-min 50-μM AP5 pretreatment), followed by immunoblotting analyses (B), MTT assay (C), or immunofluorescent inspections (D). Cell viability is expressed as the relative optical density at 540 nm of formazan with respect to the non-NMDA-treatment (Untreated) control of DMSO-treated neurons. Statistical comparisons were performed with respect to the untreated group of DMSO-treated neurons (*, P < 0.05; n = 5). For immunofluorescent experiments, neurons were stained with the anti-MAP2 antibody (red) and the nucleus counterstain DAPI (blue). NMDA treatment led to a significant diminishment of immunofluorescent signals of MAP2 (but not those of DAPI), which was prevented by pretreatment with AP5 or BV02. Scale bar, 10 μm