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. 2014 Jul 15;9(7):e102041. doi: 10.1371/journal.pone.0102041

Figure 3. WT1 transcriptionally upregulates Snail in VHL-deficient cells.

Figure 3

(A) ChIP assays for WT1 were performed in the isogenic SN12C and ACHN cell lines. WT1 was immunoprecipitated and the bound DNA fragments were analyzed by PCR amplification for Snail. Histone H3 and rabbit IgG were used as positive and negative controls, respectively. Human RPL30 exon3 primers amplified a 160 bp fragment from immunoprecipitation of H3. Snail primers amplified a 120 bp fragment from immunoprecipitation using WT1 antibody. (B) SNAI1 promoter luciferase activity was measured in the isogenic SN12C and ACHN cells. (C) SN12C-VHL and ACHN-VHL cells were cotransfected with either scrambled or siRNA-WT1 oligonucleotides, and SNAI1 promoter luciferase activity was measured. Graphs depict mean±SD of one representative of three independent experiments. (D) Top, schematic representation of WT1 binding sequence within the Snail promoter with mutated residues highlighted with asterisks (***). Bottom, HEK293T cells were cotransfected with GFP or GFP-WT1 and either wild-type or mutated SNAI1 promoter constructs and luciferase activity was measured. Graph depicts mean±SD of three independent experiments. *, P<0.05.