The effect of p38 silencing on Wnt/β-catenin pathway signal transduction.

<p>(A) One representative western blot used to quantify p38-siRNA interference efficiency and β-catenin 24 h of transfection. Total cellular proteins (20 µg/lane) were subject to 10% SDS-PAGE. Variation of loading was determined by blotting with anti-β-tubulin antibody. Western blot assays are representative of at least three experiments. (B) Luciferase activity of TopFlash reporter plasmid in cells transfected with p38-siRNA and NS-siRNA. Luciferase activity was assayed after 24 h of transfection. Firefly luciferase activity, normalized to the corrisponding renilla luciferase activity was expressed as fold change compared with control cells. Values represent mean±SD of three representative experiments performed in duplicate. (C) Semi-qunatitative real-time PCR was used to measure Wnt/β-catenin-target genes Axin2, Lef1 and Wisp1 mRNAs expression in B16-F0 cells after 24 h of transfection with p38-siRNA. The graphs show fold differences in transcript abundance in comparison with non-specific siRNA-treated cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033021#s2" target="_blank">Results</a> shown were normalized by the β-actin mRNA levels. The data show the mean±SD of three experiments performed in triplicate. *P≤0.05 versus control.