Fig. 4

ATF3 upregulated CYR61 by directly binding to its consensus binding sequence on the CYR61 gene. a qRT-PCR and western blot showed that SK-Hep1 and Li-7 cells overexpressing ATF3 had increased CYR61 mRNA and protein expression. b qRT-PCR and western blot showed that SMMC-7721 and Huh-7 cells with ATF3 knockdown had decreased CYR61 mRNA and protein expression. c Upregulated expression of CYR61 protein in tissue samples from xenografts was detected by western blot. d Potential ATF3 binding sites next to the transcriptional start site of the CYR61 sequence were identified with the JASPAR database (http://jaspar.genereg.net/). e The relative luciferase activities of the full CYR61 sequence and the truncated construct in 293 T, SMMC-7721 and Huh-7 cells. f The sequence logo of a potential ATF3 binding site in JASPAR and a diagram of mutant sites in the CYR61 sequence. g The relative luciferase activities of the truncated and mutant constructs of the CYR61 sequence in 293 T, SMMC-7721 and Huh-7 cells transfected with either ATF3 or pWPXL. h Assessment of ATF3 binding to the CYR61 sequence in 293 T, SMMC-7721 and Huh-7 cells was performed by Ch-IP using an antibody against ATF3 and a negative control (IgG). Agarose gel electrophoresis was used to analyze the crosslinking status. Data are shown as the mean ± S.D. from experiments with three replicates. *P < 0.05, **P < 0.01 and ns: no significance