Fig. 4

A30P-aSyn-O induce NFAT translocation into the nucleus and cell death in differentiated N2a dopaminergic cells. Differentiated N2a cells were treated with 10 µM A30P-aSyn-O for 3 h. A Cells were immunostained with anti-NFATc3 and DAPI and imaged on a confocal microscope. The extent of NFAT translocation into nucleus was calculated by measuring the ratio of the red fluorescence intensity in the nucleus by the red fluorescence intensity in the cytosol. Scale bar = 50 μm. B Differentiated N2a cells were co-transfected with pGL4.30 plasmids containing firefly (reporter gene) and renilla luciferase (control gene). The firefly luciferase gene is under the regulation of 3× NFAT promoter, while renilla luciferase is under the regulation of a constitutive promoter, serving as a normalizing control. N2a cells were treated with ionomycin 3 µM (C) or A30P-aSyn-O 10 µM (D). After 18 h, a dual-luciferase assay was performed to evaluate NFAT activation measured by firefly luciferase luminescence. Cells were treated with 50–500 nM of OHT (Tamoxifen) as indicated in the X axis and after 48 h cytotoxicity was measured by LDH release (E). All experiments were performed at least three times and graphs are represented as mean and SD where *p > 0.05, **p > 0.01 and ***p > 0.001