Fig. 2

Blockage of NF-κB does not rescue cell death caused by CM in WT or A30P a-syn overexpressing cells. a LDH release 24 h after CM treatment. Graph shows mean ± SEM of six independent experiments. The results are expressed as a percentage of maximal LDH release obtained after complete cell lysis. Two-way ANOVA followed by Bonferroni test: *P < 0.05 vs non-treated cells; ^# P < 0.05 vs WT α-syn treated with CM or CM + NaSal; ⁺ P < 0.05 vs pHRIG treated with CM. b Densitometry analysis of p65/p50 heterodimers of the nuclear extracts of SH-SY5Y cells. The data represent the mean ± SEM of five independent experiments. Two-way ANOVA followed with Tukey test: *P < 0.05, **P < 0.01 and ***P < 0.001 in NT, CM + NaSal and NaSal only treated cells. c Representative EMSA autoradiography. The NF-κB-specific band is indicated by an arrow. NS represents non-specific binding. d Supershift and competition assay were performed on nuclear extract of SH-SY5Y overexpressing WT α-syn. First lane (from left to right) represents the WT α-syn treated with CM. Fifth lane represents the presence of unlabelled specific oligonucleotides (NF-κB consensus sequence, fivefold molar excess). Lane 6 represents the presence of non-specific oligonucleotide (TFIID consensus sequence at fivefold molar excess). Supershift assay was performed in the absence and presence of antibodies against NF-κB subunits p65, p50 and cRel, as indicated. e Cells treated with CM present with significant cell death compared to DMSO-treated groups (*P < 0.05 vs DMSO). There is no reduction in cell death (WT and A30P) after treatment with SN50 concomitantly with CM. Two-way ANOVA followed with Sidak test: ^# P < 0.05 vs WT α-syn + CM, ⁺ P < 0.05 vs WT α-syn + CM. Graph shows mean ± SEM of four to eight independent experiments