Fig. 6

Circ-YAP is transcriptionally activated by YAP. A, B. qRT-PCR analysis of total and nascent circ-YAP levels in YAP-silenced or verteporfin-treated cells. C. Luciferase reporter assay testing the effect of YAP activation on circ-YAP promoter activity. D. Two putative YAP/TEAD binding motifs on circ-YAP promoter. E, F. The promoter activity of circ-YAP was tested in HT29 and SW480 cells co-transfected with p-YAP-5SA and wild-type or mutant luciferase reporter vectors. G, H. qRT-PCR analysis of circ-YAP expression in YAP-activated cells transfected with the indicated siRNAs. I-K. ChIP assay using the indicated antibodies, followed by qPCR analysis of the enrichment of YAP, PoI II and p-PoI II on circ-YAP promoter. L, M. ChIP-re-ChIP assay using anti-YAP/TEAD1 antibody, followed by qPCR analysis of the enrichment of YAP and TEAD1 on circ-YAP promoter. N. DNA pull-down assay using the wild-type or mutant biotinylated circ-YAP promoter probe, followed by western blot analysis of YAP and TEAD1 protein levels in CRC cells. *P < 0.05, **P < 0.01, ***P < 0.001. Data are the mean ± SD of three independent experiments carried out in triplicate. The uncropped western blot data are provided as a Original Blot Image file