IL-8 binding to CXCR1 stabilizes Snail via the ELMO1-NF-κB signaling pathway. (A) Expression levels of transcription factors Snail, Twist1, Slug, Zeb1 and Zeb2 in Scr/U251 and SiELMO1/U251 cells with or without IL-8 for 24 h were detected by Western blot. β-actin was used as a loading control. (B) Expression levels of Snail mRNA in Scr/U251 and SiELMO1/U251 cells with or without IL-8 for 24 h were detected by Real time PCR. Bars, standard deviation. (C) Nuclear expression level of Snail in Scr/U251 and SiELMO1/U251 cells with or without IL-8 for 24 h was examined by Western blot. Nucleolin was used as a loading control. IL-8, 10 ng/mL. (D) U251 cells were pretreated with various inhibitors(all 20uM), BAY11–7082 (inhibitor of IκBα), SB203580(inhibitor of p38MAPK), U0126(inhibitor of ERK1/2) and LY294002(inhibitor of PI3K)for 1 h, DMSO was used as negative control. The expression level of Snail was detected by Western blot. IL-8, 10 ng/mL. (E) Expression levels of P-IκBα in Scr/U251, SiELMO1/U251, Scr/U87 and SiELMO1/U87 cells with or without 10 ng/mL IL-8 were examined by Western blot. IκBα was used as a loading control. IL-8, 10ng/mL. Quantification of relative protein levels is shown below the blots. Data were collected in this set of figures from a representative of at least 3 independent experiments (A–E).