Fig. 6. u50535 promotes CRC growth and metastasis via activation of CCL20 signaling.

a After transfecting siCCL20 to u50535 stable overexpression cells (PCDHu5OE), the expression of u50535 was not affected after down-regulation of CCL20, whereas the expression of CCL20 was significantly decreased. b Proliferation assay by RTCA showed that CCL20 knockdown can reverse the proliferation activity induced by u50535 overexpression (PCDHu5OE), especially in 120 h. c Transwell assay showed that CCL20 knockdown can inhibit the migration ability caused by u50535 overexpression. d Luciferase assay showed that relative luciferase activity of pGL4-CCL20-promoter was significantly increased by u50535 overexpression in DLD1 and HCT8, supporting that u50535 could positively regulate CCL20 transcription directly or indirectly. e CHIRP assay revealed that u50535 was significantly retrieved by u50535 antisense odd/even probes compared with negative control LacZ in DLD1 (left), while chromatin/DNA of CCL20 core promoter region was not significantly enriched (right), indicating an indirect regulation. f The mRNA expression of CCL20 and related genes were testified in xenograft tumor from control/u50535OE mice model. g u50535 overexpression in DLD1 cells can significantly promote CCL20, ERK, NFKBIA genes expression, while adding siRNA-CCL20 greatly inhibits the expression of CCL20, CCR6 and ERK. Data from three independent experiments were expressed as mean ± SD. * P < 0.05; ** P < 0.01