Figure 7.

Ets-1 regulates INPP4B in colon cancer cells. (a) Total RNA from the FHC normal colon epithelial cell line and indicated colon cancer cell lines were subjected to qPCR analysis of Ets-1 mRNA expression. The relative abundance of Ets-1 mRNA in FHC cells was arbitrarily designated as 1. Data are represented as mean±s.e.m. of three individual experiments. (b) Whole-cell lysates from the FHC normal colon epithelial cell line and indicated colon cancer cell lines were subjected to western blotting analysis of Ets-1 and GAPDH (as a loading control). Data are representative of three individual experiments. (c) Whole-cell lysates from WiDr and HCT116 cells transfected with the control siRNA or two individual Ets-1 siRNAs (Ets-1 siRNA1 and Ets-1 siRNA2) were subjected to western blotting analysis of Ets-1, INPP4B and GAPDH (as a loading control). Data are representative of three individual experiments. (d) Whole-cell lysates from SW620 and the FHC cells transfected with the vector alone or Ets-1 cDNA were subjected to western blotting analysis of Ets-1, INPP4B and GAPDH (as a loading control). Data are representative of three individual experiments. (e) Formaldehyde-cross-linked chromatin of WiDr and HCT116 cells were subjected to immunoprecipitation with an antibody against Ets-1. The precipitates were subjected to PCR amplification using primers for the −279/−26 region of the INPP4B promoter. Data are representative of three individual experiments. (f) WiDr and HCT116 cells were transiently transfected with the pGL3-basic-based reporter constructs (pGL3-vector, pGL3-INPP4B wt or pGL3-INPP4B mut as shown in Supplementary Figure S8B) as well as pRL-TK Renilla luciferase control vector. Twenty-four hours later, cells were subjected to the measurement of the luciferase activity. Data are represented as mean±s.e.m. of three individual experiments. **P<0.01, Student's t-test.