siRNA silencing and ectopic overexpression of CDX2 in Caco-2 cells.

For CDX2 knockdown in Caco-2 cells, cells seeded in six-well plates and grown to ~60% confluency were transfected with a CDX2-specific double-stranded siRNA (siCDX2) or scrambled siRNA (both obtained from Thermo Fisher Scientific-Dharmacon) (Table 1) in a total concentration of 50 nM. The siRNA duplexes were used in a ratio of 1:5 relative to Lipofectamine 2000 reagent in OptiMEM. siRNA sequences are shown in Table 1. This procedure was repeated every 3rd day. Finally, cells were harvested on day 10 and processed for mRNA and protein analysis.

List of primers used in nonradioactive gel-shift and ChIP assays

ChIP, chromatin immunoprecipitation; DRA, downregulated in adenoma.

For CDX2 overexpression, Caco-2 cells were transfected with 1 μg of CDX2 expression vector containing two FLAG tags at the NH₂ terminus (kind gift from Dr. Taketo) or the corresponding empty vector (Qiagen, Germantown, MD) using Amaxa Nucleofector transfection reagents (Lonza, Walkersville, MD) according to the manufacturer’s instructions. Cell lysates were prepared 48 h after transfection, and overexpression was measured by quantitative RT-PCR and immunoblotting with respective antibodies.