FIGURE 3.

B3C protects neuronal survival factor MEF2 from MPP⁺-induced inhibition in both nuclear and mitochondrial compartments. A, attenuation of MPP⁺-induced inhibition of MEF2 DNA binding activity in the nucleus. SN4741 cells were treated with 500 μm MPP⁺ with or without B3C for 24 h. Nuclear extracts adjusted for equal amounts of MEF2D (bottom panel) were used for EMSA assay (top panel: arrowhead, MEF2D supershift complex; arrow, MEF2D and probe complex; double arrow, free probe). wt, wild type MEF2; mt, mutant. B, attenuation of MPP⁺-induced inhibition of MEF2 transactivation activity. MEF2 reporter gene expression was measured after 24 h of 500 μm MPP⁺ treatment with or without B3C. The data from three independent experiments with each in triplicate were expressed as the percentage of control activity (n = 3; *, p < 0.05 versus MPP⁺ treated group or untreated control). C, attenuation of MPP⁺-induced inhibition of MEF2D in mitochondria. Lysates from purified mitochondrial extracts from 500 μm MPP⁺-treated SN4741 cells with or without B3C were probed for MEF2D, ND6 (MEF2D target gene), and VDAC (mitochondrial loading control). Quantification was expressed as the ratio relative to control (n = 4; *, p < 0.05 when compared with the MPP⁺-treated group, respectively). D, reverse of the MPP⁺-induced decline of complex I activity in mitochondria. SN4741 cells were preincubated with 10 μm B3C for 2 h and then exposed to 500 μm MPP⁺ for 24 h. Mitochondrial complex I activity was measured using a specific assay kit from Abcam. E, B3C does not significantly change the levels of MEF2A-C. SN4741 cells were preincubated with or without B3C at the indicated concentrations for 2 h and then exposed to 500 μm MPP⁺ for 24 h. Lysates of purified nuclei and mitochondrial extracts from these SN4741 cells were probed for MEF2A, -2B, -2C, and -2D, poly(ADP-ribose) polymerase-2 (PARP) (nuclei loading control), and VDAC (mitochondrial loading control).