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. Author manuscript; available in PMC: 2016 Jun 27.
Published in final edited form as: Mol Cancer Res. 2011 Oct 17;9(12):1644–1657. doi: 10.1158/1541-7786.MCR-11-0371

Figure 4. Snail1 transcriptionally represses Twist1 expression.

Figure 4

(A) Western blotting for indicated proteins of WCL from MCF10A cells transduced with a lentivirus expressing either the vector pBABE or Snail1. (B) Schematic of the human Twist1 promoter showing 13 consensus E-boxes grouped into 3 main clusters A to C. Green box: minimal Twist1 promoter; Red box: E-box; ATG: Transcription start site; Numbered blue double arrows: primer sets 1–7. (C) Luciferase reporter assay in HEK293T cells using mutants (P1-P4) of the human Twist1 promoter fused with the luciferase reporter plasmid pGL2 (left), and co-transfected with either a pCMV-3Flag-Snail1 plasmid or the empty vector (right). hEcad: Human E-cadherin promoter used as a positive control for Snail1-dependent repression. Fold changes in luciferase activity over the empty pGL2 vector are shown. Each experiment was performed in triplicate. Error bars represent means +/− s.e.m. Representatives of 3 independent experiments are shown. *, p < 0.05, Student’s T-test. (D) Upper: Chromatin immunoprecipitation using a Snail1 monoclonal antibody in nuclear extracts from MCF10A cells either untreated or treated with 2ng/ml TGFβ1 for 2 days. Immunoprecipitated chromatins were subjected with PCR using primer sets 1 to 7 as shown in (B). Lower: PCR using primer sets 1 to 7 of total chromatin inputs. Representatives of 3 experiments are shown. See also related Figure S8.