Figure 3

ERK and AMPK inhibition prevent LPS-induced increase in TNF-α transcript and protein levels. (A) ERK pathway activation in rENSpc treated with LPS from two minutes to seven hours was measured by Western blotting using phospho-ERK antibodies (P-ERK). The relative amount of P-ERK was determined by normalizing with β-actin (four to eight independent experiments). Values represent the mean ± SEM as fold/mean t0 (one-way ANOVA test followed by Dunn’s post-hoc test; *P <0.05 as compared with t0 without LPS). (B) Localization of activated − ERK and − AMPK in rENSpc treated for two hours with LPS was performed by immunocytochemistry using P-ERK or phospho-Acetyl-CoA Carboxylase (P-ACC), with S100β and Sox10 (glial markers) or Hu and Tuj (βIII-tubulin, neuronal markers). Scale bar: 50 μm. Inserts show higher magnification images (40x for P-ERK and 100x for P-ACC). ERK- and AMPK-dependent pathway contribution to TNFα production were assessed using MEK1/2 inhibitor (U0126; 10 μM) and AMPK inhibitor (C compound, CC; 10 μM) co-treatment on rENSpc treated (+) or not (−) with LPS for seven hours. (C) Quantification of TNF-α mRNA by qPCR (six to 11 independent samples). (D) TNF-α secretion was assayed by ELISA with or without the presence of inhibitors (six independent samples). The independence of the ERK and AMPK pathways was determined by Western blotting. (E) ERK phosphorylation was not affected by CC and (F) ACC phosphorylation was not modified by U0126 (eight and three independent experiments, respectively). (Mann-Whitney U test; *P <0.05 as compared with control without LPS; #P <0.05 as compared with LPS without inhibitor). (G) The EFS impact on ERK and AMPK activation was measured on rENSpc treated (+) or not (−) with LPS and EFS for two hours by Western blotting using P-ERK and P-AMPK antibodies. Immunoblots are representative of five independent experiments with similar results.