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Fig. 3 | Journal of Experimental & Clinical Cancer Research

Fig. 3

From: CCR7 mediates the TNF-α-induced lymphatic metastasis of gallbladder cancer through the “ERK1/2 - AP-1” and “JNK - AP-1” pathways

Fig. 3

TNF-α enhanced the promoter activity and protein expression of the CCR7 gene through the “ERK1/2-AP-1 and JNK-AP-1” pathways. a Activity analysis of the CCR7 promoter. NOZ cells were transfected with 1 μg each of the CCR7 promoter constructs and 0.1 μg of pRL-TK. Each transfection was performed in duplicate. b Nucleotide sequence of the −243 to −108 region of the CCR7 promoter. The two putative AP-1 binding sites (CTCTGTCACC and ATGAGTCAG) were marked with red letters (named AP-1(1) and AP-1(2)). c Site-directed mutagenesis assay. The mutated construct PGL4-AP1mut2 but not PGL4-AP1mut1 exhibited lower activities than the non-mutated construct PGL4-462/+74. d ChIP assay. Chromatin from NOZ cells was immunoprecipitated with the anti-AP-1 antibody. The total extracted DNA (Input) and the immunoprecipitated samples were PCR-amplified using primers specific to the regions of the CCR7 promoter containing the AP-1(2) binding site (134 bp). A normal rabbit IgG and no antibody sample were also included as controls. eg The effect of the TNF-α⁄AP-1 signalling pathway on the promoter activity and protein expression of the CCR7 gene. After transfection with AP-1 siRNA, the promoter activity and protein level of CCR7 were accordingly reduced, irrespective of treatment with TNF-α. h The expression of p-ERK1/2, p-JNK and p-p38MAPK was increased in NOZ cells after treatment with TNF-α. i-k The effect of inhibition of MAPK pathway members on the promoter activity and protein expression of CCR7. When treated with PD98059 (50 μM), SP600125 (10 μM) or SB203580 (20 μM), the expression of AP-1 and p-AP-1 in NOZ cells was reduced. However, the promoter activity and protein expression of CCR7 were significantly reduced only in the PD98059- and SP600125-treated groups (*P < 0.05). All of the experiments were repeated three times

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