Fig. 6

Effects of difopein expression on NMDA-induced neuronal excitotoxicity. A, B Difopein rescues NMDA excitotoxicity and averts NMDA-mediated reduction of endogenous Eag1 protein level in cultured cortical neurons. Neurons (DIV10) were subject to transfection with either R18 mutant or difopein. Two days post-transfection, neurons were stimulated with 20 μM NMDA for six hours, in the absence or presence of 30-min pretreatment with the NMDA receptor antagonist AP5 (50 μM), followed by MTT assays (A) or immunoblotting analyses with the indicated antibodies (B). Cell viability is expressed as the relative optical density at 540 nm of the mitochondria-produced formazan with respect to the non-NMDA-treatment (Untreated) control of YFP-R18-mut-transfected neurons. Statistical comparisons were performed with respect to the untreated group of R18 mutant-transfected neurons (*, P < 0.05; n = 9). C Proteasome inhibition prevents NMDA-mediated reduction of endogenous Eag1 protein level in cultured cortical neurons. Neurons (DIV12) were pretreated with the NMDA receptor antagonist AP5 (50 μM), the proteasomal inhibitors ALLN (10 μM) and MG132 (20 μM), or the caspase inhibitor zVAD-FMK (20 μM) for 30 min. Cells were then treated with 20 μM NMDA for 12 h in the presence of the specified inhibitors, followed by immunoblotting analyses with the indicated antibodies