Fig. 5

NET-DNA mediated NFκB signaling pathway activation via ITGAV. A Membrane protein extracted from RBE cells were incubated either with uncoupled beads or with beads coupled to biotin-NET-DNA. ITGAV, indicated by the arrow. B Immunoprecipitated bound proteins were blotted with anti-ITGAV antibody. C Representative images of ITGAV detected by IHC in CCA patients. Scale bar=50μm. Ci IHC staining score was used to compare the protein expression of ITGAV between CCA tissues (n=100) and the adjacent normal bile duct (n=100). D GEO databases (GSE107943) showed that mRNA levels of ITGAV were magnificently increased in CCA tissues (n=27) compared with the paired adjacent normal tissues (n=27). E ITGAV overexpression was positively correlated with p-NFκB upregulation in CCA organoid sections, Scale bar=50μm. F Representative images of IHC staining analysis of ITGAV and p-NFκB expression in CCA tissues from mouse model. G IHC scores were used to exam correlation of protein expression between ITGAV and p-NFκB (n=32, p=0.007, Spearman’s coefficient r=0.47). H Proliferation rate after transfection with short hairpin RNAs was analyzed by CCK-8 assay of indicated CCA cells. I Transwell migration and invasion assays for indicated cell lines. Data are means ± SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001