Fig. 4
NET-DNA promoted the progression of CCA via NKκB signaling pathway. A Heat map of DEGs in CCA cells treated with NET-DNA or vehicle (5ng/μl) for 3 days. B KEGG enrichment analysis of most differentially expressed genes (absolute -log10 fold change >2). C The expression of p-NFκB was measured by IHC of CCA organoids treated with or without NET-DNA (5ng/μl, 18h), Scale bar=10μm. D IHC detection of p-NFκB from liver sections of CCA mouse model in the indicated group, Scale bar=30μm. E Quantification of the p-NFκB staining scores in tumor sections (n=5) from PAD4 KO and WT mice. F IF analysis of p-NFκB (red), tubulin (green) in indicated cells with or without BAY11-7082 treatment (5μM). Scale bar=5μm. G Quantification of p-NFκB in the indicative group. H Proliferation rate after BAY11-7082 treatment was analyzed by CCK-8 assay of indicated CCA cells. I Representative bioluminescence imaging demonstrated that downregulated NFκB signaling pathway with BAY11-7082 (10mg/kg/i.p.) by every 2 days prevented CCA progression induced by NETs, photos were taken 21days after the hydrodynamic liver injection; the statistical analysis of bioluminescence signal in the below panel; IHC analyzed the expression of p-NFκB in the indicative group. Data are means ± SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001