Fig. 4: EN2 directly regulates CCL20 expression in CRC cells.

a Nuclear and cytoplasmic fractionation assay confirmed the nuclear localization of endogenous EN2 in SW480 cells. Nuclear and cytoplasmic extracts were analyzed by western blotting. b The localization of EN2-Flag in HCT8 cells was identified by nuclear and cytoplasmic fractionation assay and western blotting. c Volcano plot of differentially expressed genes (DEGs) of SW480 cells after EN2 knocked down. The horizontal line at P-value = 0.05; vertical line at |log2FC| = 1. d, e The expression of CCL20 was determined by qRT-PCR (e) and ELISA (f) after EN2 was knocked down. f The EN2 binding motif was predicted from JASPAR matrix models. g The schematic structures of EN2 putative binding sites in the CCL20 promoter. h ChIP analysis of EN2 binding sites to the CCL20 promoter. i The CCL20-P-WT and CCL20-P-MUT luciferase activity in 293 T cells was confirmed by luciferase reporter assay after EN2 was overexpressed. j, k Promoter luciferase reporter assays of CCL20 in HCT8 cells with EN2 overexpressed (j) and in SW480 cells with EN2 knocked down (k) were performed. l The protein expression of EN2 and CCL20 detected by Immunohistochemistry staining in CRC tissues. Scale bar: 50 µm. m The analysis of correlation between EN2 and CCL20 expression levels in CRC tissues in GSE9348 and TCGA database. n ROC curves for the diagnosis of CRC patients in GSE9348. Data are presented as mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.