Figure 3.

TPA induced binding of Snail, EGR and SP1 on MMP9 promoter. HepG2 cells were treated with 50 nM TPA for the times indicated, ChIP assay for binding of Snail, EGR1 and SP1 on MMP9-pro179 and MMP9-pro155 (A,B) and double ChIP of EGR (1^st)/Snail (2^nd) and Snail (1^st)/SP1 (2^nd) for amplifying MMP9-pro165 using quantitative PCR (C), were performed. In (A), ChIPs of histone binding on GAPDH promoter and E-cadherin on MMP9 promoter were used for positive and negative control, respectively. Input is for loading control for ChIP. (B) is the quantitative figure for (A). Relative amount of the indicated promoter fragments was normalized with the binding amount of histone3 on GAPDH promoter, taking the time zero group as 1.0. (*^{, #} and ^$) represent statistic significance (p < 0.05, N = 3) between the indicated sample with the time zero group. In (C), (**^,*) represent the statistically significant difference (p < 0.005, p < 0.05, respectively, N = 3) of relative MMP9-pro165 between each of the indicated samples and time zero group. Schematic maps of MMP9-pro179, MMP9-pro165 and MMP9-pro155 across the indicated regions on MMP9 promoter are demonstrated in supplemental Fig. 2A. (D), (E). HepG2 cells were treated with 50 nM TPA as indicated, Western (D) and IP/Western (E) blot of indicated proteins were performed. In (D), “>” indicated the position of Snail and EGR-1, whereas * indicated the un-identified protein with M.W. slightly larger than Snail. In (D), (E) the numbers below are relative intensities of indicated protein or Snail bound EGR1 vs GADPH as a loading control, and “Snail bound EGR1” vs immunoprecipitated Snail or EGR1as an input, taking the time zero sample as 1.0. The data were average of two reproducible experiments with C.V. of 0.05. In (E), I.P.: Immunoprecipitation, W.B.:Western blot. The lines between the images in (A), (D) and (E) are included for dividing gels of different ChIP assays, Western blot or IP, respectively, of indicated molecules.