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. 2014 Dec 22;6(3):1422–1434. doi: 10.18632/oncotarget.2805

Figure 6. WNK2 negatively regulates IL-6 expression.

Figure 6

(A) IL-6 expression was analyzed by qRT-PCR. IL-6 expression levels were normalized to GAPDH expression and results are presented as fold differences relative to SW1088 C- or A172.Ev cells. Data correspond to the mean values ± standard errors and are representative of, at least, three independent experiments. ***, significantly different from SW1088 C- or A172.Ev cells (p < 0.001). (B) The cells were treated for 24 hours with DMSO or the chemical inhibitor of JNK (SP600125; iJNK). IL-6 mRNA levels were measured by Real-time PCR. Relative expression changes are presented as fold increase of IL-6/GAPDH in comparison with A172 or SW1088 shW2 treated with DMSO. Data on graphs represent the mean values ±. standard errors and are representative of, at least, three independent experiments. **, significantly different from SW1088 shW2 treated with DMSO (p < 0.01), ***, significantly different from SW1088 C-or A172 cells treated with DMSO (p < 0.001). (C) The cells were treated for 24 hours with DMSO or the chemical inhibitors of JNK (SP600125; iJNK). After the treatment the conditioned medium was collected and analyzed using an IL-6 ELISA assay. IL-6 levels are expressed as fold-increase over the levels in control conditions. Data on graphs represent the mean values ±. standard errors and are representative of, at least, three independent experiments. *, significantly different from A172 cells treated with DMSO (p < 0.05), ***, significantly different from SW1088 shW2 cells treated with DMSO (p < 0.001). (D) Cells were treated for 3 hours with H2O (−), or with a chemical inhibitor of Rac1 (NSC23766; iRac) (+). After treatment the cell lysates were analyzed by Western Blot, immunostained with anti-phospho Thr183/Tyr185 JNK antibody, and after stripping, the same membrane was immunostained for total JNK, and α-tubulin. Graphs correspond to the mean values ± standard errors and are representative of, at least, three independent experiments. *, significantly different from SW1088 C- cells (p < 0.05), **, significantly different from SW1088 C- or A172.Ev cells (p < 0.01).