Figure 3: ZNF304 associates with ITGB1 promoter and regulates β1 integrin expression.

(a) Western blot analysis of basal protein expression levels of ZNF304 and β1 integrin in HIO180 (non-transformed ovarian cell line) and six ovarian cancer cell lines. (b) The ten predicted binding sites of ZNF304 in the ITGB1 promoter on the basis of support vector machine scores using an online tool, which is available at http://compbio.cs.princeton.edu/zf/. (c) ITGB1 promoter with ten predicted binding sites (in red) and six primer sets were designed for the ten predicted binding sites. (d) Chromatin immunoprecipitation (ChIP) analyses with ZNF304 antibody in HeyA8 cells. Relevant sequences were quantified by PCR with six pre-designed primers subsequent to ChIP assay. (e) Densitometric analysis of ChIP data. Sequence and antibody-specific controls were included. Data are presented as percentage of input. (f) Luciferase activity in control siRNA (black)- or ZNF304 siRNA (grey)-treated HeyA8 cells. Fold induction was calculated after normalization with empty vector. Data are presented as mean±s.e.m. of n≥3 experimental groups. *P≥0.05, **P≥0.01,***P≥0.001 (Student’s t-test). Luciferase activity was inhibited after control siRNA treatment or ZNF304 siRNA treatment in BS1-vector-, in BS2-vector- and in BS-3-vector-transfected cells. (g) Luciferase activity increased after transfection of ZNF304-expressing vector into BS1-, BS2- and BS3-vector-transfected HeyA8 cells. Data are presented as mean±s.e.m. of n≥3 experimental groups. *P≥0.05, **P≥0.01,***P≥0.001 (Student’s t-test). NS, not significant.