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. 2017 Dec 19;7:17753. doi: 10.1038/s41598-017-18101-7

Figure 6.

Figure 6

Snail is essential for expression and promoter activation of MMP9 and ZEB1. HepG2 cells were transfected with none (MOCK) or plasmids encoding indicated Snail shRNAs (A,B,C) and control shRNA of Lamin (A), (B) or GFP (C) for 24 h followed by untreated (MOCK) or treated with 50 nM TPA for 4 h (A); 4 and 6 h for ZEB1 and MMP9, respectively (B); or 12 h (C). HCC340 were transfected with pcDNA3 vector or Snail expressing plasmid (p-Snail) for 36 h (D) and (E). RT-PCR (A), (D) and quantitative RT-PCR (B), (E) of Snail, MMP9 and ZEB1 and promoter assay of MMP9-950 (C, upper panel) and ZEB1-1079 (C, lower panel) were performed. In (A,D), the numbers below each figure are the ratios of relative mRNA based on RT-PCR of indicated transcriptional factor vs GAPDH, taking the data of MOCK (A) and pcDNA3 (D) as 1.0. The results are average of 3 reproducible experiments with C.V. of 7.5%. In (B) and (E), the relative mRNA was calculated based on real time RT-PCR, taking the data of MOCK (B) and pcDNA3 (E) as 1.0. In (C), the relative dual luciferase activity of MMP9-950 or ZEB1-1079 was calculated, taking MOCK as 1.0. The data in (B), is average of two representative experiment with C.V. of 12%. In (C) and (E), (**,*) represent the statistically significant difference (p < 0.005, N = 3), (p < 0.05, N = 3) between the indicated samples and the control GFP shRNA (C) and pcDNA3 (E) group.