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. 2019 May 31;9(14):4208–4220. doi: 10.7150/thno.33803

Figure 6.

Figure 6

EBF1 regulates USP5 expression. A. The schematic diagram of the USP5 promoter region (http://genome.ucsc.edu/). TSS: transcription start site; TIS: translation initial site. B. Different truncated fragments of USP5 promoter were cloned into pGL4 reporter. The luciferase activities in transfected cells were measured by using the dual luciferase reporter assays. C. The predicted binding sites for transcription factors in -230/+32 region of USP5 promoter (http://jaspar.binf.ku.dk/). D. After transfected with indicated siRNAs targeting the transcription factors for 72 hours, USP5 in HCT116 cells was analyzed by immunoblotting. GAPDH was used as a loading control. E & F. After transfected with siEBF1#1, siEBF1#2, siEBF1#3 or siNC for 72 hours, EBF1 and USP5 levels in HCT116 cells were assessed by immunoblotting (E) and qRT-PCR (F). **p<0.01. G. HCT116 cells were transfected with indicated amounts of Myc-EBF1-expressing plasmids and then analyzed by immunoblotting against USP5, Myc and GAPDH. H. ChIP assay with anti-EBF1 antibody was performed. The -230/-160 fragment in USP5 promoter region was preferentially pulled down in HCT116 cells. I. The expression of EBF1 and USP5 in seven colorectal cancer cell lines was examined by immunoblotting. J. The expression of EBF1 and USP5 in 2 representative fresh primary colorectal cancer tissues and individual normal tissues were assessed by immunoblotting. GAPDH was used as a loading control.