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PubMed Central, Figure 5: Nat Cell Biol. 2012 Nov; 14(11): 1212–1222. Published online 2012 Oct 21. doi:  10.1038/ncb2607
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Nat Cell Biol. Author manuscript; available in PMC 2013 May 1.
Published in final edited form as:
Nat Cell Biol. 2012 Nov; 14(11): 1212–1222.
Published online 2012 Oct 21. doi:  10.1038/ncb2607

Figure 5

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Elf5 binds to the SNAI2 promoter and represses its expression

(a) Schematic depiction of the SNAI2 promoter with several putative ELF5 binding sites (red boxes) indicated. Primer sets for ChIP analyses are indicated by the arrows in the schematic diagram. (b) Evolutionary conservation of the ELF5 binding motif in the P2 promoter-proximal region of the SNAI2 gene. (c) ChIP analysis of HA-Elf5 binding to the SNAI2 promoter in MDA-231-Elf5 cells. Quantitative PCR was performed with primers specific to seven regions on the SNAI2 promoter as indicated in a. Primers against the -300 bp promoter region (P2) show significant enrichment after normalization to the GAPDH control. Experiments were performed three times, each with qRT-PCR in technical duplicate, and data presented as the mean ± SD. (d) Relative expression of WT or mutant (MT) SNAI2 promoter-driven luciferase reporters in control or Elf5-overexpressing MDA-231 cells. The data represented are shown as mean ± SD collected from 3 independent experiments. (e) Relative expression of a wild type (WT) SNAI2 promoter-driven luciferase reporter in MCF-7 cells transiently transfected with vector control or HA-Elf5 expression plasmids. The data represented are shown as mean ± SD collected from 3 independent experiments. (f) Western blot of Elf5 and SNAIL2 protein levels in MDA-231-Elf5 cells transduced with vector (control) or SNAI2. Uncropped images of blots are shown in Supplementary Fig. S9. (g) Phase contrast images showing morphological characteristics of control, Elf5, or HA-Elf5 and FLAG-SNAIL2 overexpressing MDA-231 cells. Outlined cells demonstrate spindle-like (left and right) or cobblestone-like (middle) morphologies. Size bar = 10 μm (h) Transwell migration assay with control, Elf5, or Elf5 and SNAIL2 overexpressing MDA-231cells. Data are shown as mean ± SD collected from 10 fields of 3 independent experiments. Student’s t-tests were performed to assess statistical significance in c, d, e, and h. * p < 0.05 by Student’s t-test; # p > 0.05.

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