Smad4 sirna

The FAM modified 2′-O-me-oligonucleotides were synthesized by GenePharma (Shanghai, China). The sequences of the 2′-O-me-miR-146a mimics and 2′-O-me-miR-221 inhibitor, as well as a negative control of miRNA mimics (negative mimics) or inhibitors (negative inhibitors), were as follows: 5′-UGAGAACUGAAUUCCAUGGGUU-3′, 5′-AACCCAUGGAAUUCAGUUCUCA-3′ and 5′-UUGUACUACACAAAAGUACUG-3′. Smad4 siRNA (Smad4 siRNA: sc-29484; control siRNA: sc-37007) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). When the cells were grown to 70–80% confluence, transfection was performed using the Lipofectamine™ 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. After 4 h of transfection, the medium was replaced with fresh medium (DMEM/F12) containing 10% fetal bovine serum.

Rapamycin was obtained from LC Laboratories (Woburn, MA, USA). The phosphatidylinositol-3-kinase (PI3K) inhibitor, LY294002, and Wortmannin were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Total Smad2 (product number 5339S, monoclonal rabbit IgG), total Smad3 (product number 9523P, monoclonal rabbit IgG) and Smad4 (product number 9515, monoclonal rabbit IgG) primary antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA), and the glyceraldehyde 3-phosphate dehydrogenase antibody was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Smad4 siRNA and non-targeted negative control siRNA duplexes were obtained from Santa Cruz Biotechnology Inc. Smad4 primers were designed and synthesized by IDT (Coralville, IA, USA). A DNeasy Blood and Tissue kit was obtained from Qiagen (Hilden, Germany) and a Phusion High Fidelity DNA Polymerase kit was obtained from New England Biolabs (Ipswich, MA, USA).

SMAD4 and CDX2 were silenced by transfection with SMAD4 siRNA and CDX2 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA). In a six-well tissue culture plate, 2×10⁵ cells per well were seeded in 2 ml antibiotic-free normal growth medium supplemented with FBS. Cells were incubated at 37°C in a CO₂ incubator until the cells were 60–80% confluent. For each transfection, 4 µl of siRNA duplex which gives final concentration of 80 nM siRNA (Stock 20 µM siRNA) in 100 µl siRNA transfection medium (solution A) and 6 µl of siRNA transfection reagent in 100 µl siRNA transfection medium (solution B) were mixed and incubated for 45 min at room temperature. For each transfection, 0.8 ml siRNA transfection medium was added to each tube, mixed gently, overlaid onto washed cells and incubated for 5–7 hr at 37°C in a CO₂ incubator. After incubation, 1 ml of normal growth medium containing twice the normal serum and antibiotic concentration (2× normal growth medium) was added without removing the transfection mixture. Cells were incubated for an additional 18–24 hr. Medium was replaced with 1 ml of fresh 1 x normal growth medium and used for further studies.