Fig. 2

SNX27 negatively regulates TNFα-induced NF-κB signalling activation. A SNX27 overexpression inhibits TNFα-induced NF-κB signalling activation. HeLa cells stably expressing control vector or SNX27 were treated with TNFα for indicated time points. Phosphorylated IκBα, IKKα/β and p65 were used to exam TNFα-induced NF-κB signalling activation. The relative protein expression level that normalised to loading control was calculated by ImageJ and labelled below each blot. The value of time 0 of control cells was set as 1. Immunoblotting was performed at least twice, and one representative figure was shown. B, C Knockout of SNX27 potentiates TNFα-induced NF-κB signalling activation. HeLa cells with CRISPR/Cas9 mediated SNX27 knockout were treated with TNFα for indicated time points. Immunoblotting was performed as A (B) to check the phosphorylation of IκBα, IKKα/β and p65. Immunoblotting was performed and the relative protein expression was calculated as A. Quantitative real-time PCR was used to check the expression of target genes of TNFα signal (C). Significant differences compared to control were calculated using multiple t-tests. The graphs showed mean ± SD, n = 3. ns indicates not significant; * indicates p < 0.05, ** indicates p < 0.01; *** indicates p < 0.001. D, E SNX27 negatively regulates TNFα-induced secretion of IL-6 and IL-8. HeLa cells with SNX27 overexpression (D) or knockout (E) were treated with TNFα for indicated time points. Cell medium was collected and then the secreted IL-6 and IL-8 were quantified by ELISA kit as described in "Methods". Significant differences were calculated as described in C