Figure 6

p38 MAP kinase downregulates the transcription and the translation of E-selectin by modulating miR-146a and miR-31. (A) Endothelial cells were pretreated with 10 μM of p38 inhibitor SB203580, before being treated with IL-1β (20ng/ml) for four hours. Western blotting monitored the expression of E-selectin and the activities of p38 (anti-phospho-HSP27 (S⁸²)), NF-κB (anti-phospho-p65 (S⁵³⁶)) and c-Jun (anti-phospho-c-Jun (S⁶³)) signalling pathways. GAPDH was used as loading control. (B) Endothelial cells were transfected with 50 nM of miRNA inhibitors or control, before being pretreated with 10 μM of p38 inhibitor SB203580, and treated with IL-1β (20 ng/ml) for four hours. Analysis as in (A) The p-values are calculated comparing to the “antagomir ctrl/DMSO/IL-1β” group. (C) Endothelial cells were treated as in A. RT-qPCR monitored the level of E-selectin mRNA relative to GAPDH mRNA. (D) Endothelial cells were treated as in b., then analyzed as in (C,E) Endothelial cells were treated as in (B) Western blotting monitored the expression of E-selectin and the activities of p38 (anti-phospho-HSP27 (S⁸²) signalling pathway. GAPDH was used as loading control. The Western blots represent three independent experiments. The quantification are the mean values of three independent experiments, the error bars represent standard errors of three independent experiments, and the significance was analyzed using a Student’s t-test (*p < 0.05; **p < 0.01). N.D.: non detectable.