Pcdna3

The Smad2 gene was cloned into the vector pcDNA3.1 (TranSheep Bio Co. Ltd.). The chimeric genes of the IL‐11 promoter plasmids for transfection experiments were constructed in a pGL4.1‐basic vector (TranSheep Bio Co. Ltd.) by ligating the luciferase gene at the 5′‐flanking regions of the gene upstream. MRC‐5 cells were plated into 24‐well culture plates 24 h before transfection. The mixtures of 1 μg each of pcDNA3.1‐basic and pGL4.1‐basic, overexpressed pcDNA3.1 and pGL4.1‐basic, overexpressed pcDNA3.1 and pGL4.1‐mutant (mutating the binding sequence “ATCTCTGTCTCCC” into “AAAAAAAAAAAAA”), overexpressed pcDNA3.1 and pGL4.1‐promoter, overexpressed pcDNA3.1 and pGL4.1‐mutant (with or without TGF‐β1), and overexpressed pcDNA3.1 and pGL4.1‐promoter (with or without TGFβ1) were cotransfected with Firefly luciferase (Fluc)–Renilla luciferase (Rluc) into human MRC‐5 cells with the X‐tremeGENE HP DNA Transfection Reagent (Roche Diagnostics Corp.). Two days later, a commercial kit (Promega Corporation) was used to measure the promoter‐driven luciferase activity.

According to the results of CHIP, four, one and five putative KBSs, respectively, were predicted from the promoter regions of human BCL2 (positions −1201 to −1), BAX (positions −645 to −317) and BIK (positions −1200 to −1). The above promoter regions were amplified from genomic DNA and were subcloned into firefly luciferase reporter vector pGL3-Basic (pGL3b) or pGL4.1 (Promega, Madison, WI) to get pGL3b-BCL2, pGL4.1-BAX and pGL4.1-BIK. Constructs with mutations of the putative KBSs were generated using mutagenic oligonucleotide primers according to the manual of the GeneTailor Site-Directed Mutagenesis System (Invitrogen). HUVECs were cotransfected with firefly luciferase reporter constructs and Renilla luciferase reporter vector pRL-TK (E2241, Promega) together with pCDNA3.1-Kaiso or pCDNA3.1 empty vector using FuGENE HD Transfection Reagent (Roche, Mannheim, Germany). Then, 48 h after transfection, relative luciferase activity represented as the ratio of firefly to Renilla was measured with a Dual-Luciferase Reporter Assay System (Promega). All primers used for genomic DNA amplification and site-directed mutation are listed in Supplementary Tables S4–S5.

The gastric cancer cell lines AGS and SNU-1 were purchased from ATCC (American Tissue Culture Collection, Manassas, VA). HGC-27 were purchased from AMSBIO (Amsbio, United Kingdom). Cells were maintained in Ham’s F-12 with 10% fetal bovine serum (Invitrogen Life Technologies) and incubated at 37 °C in 5% CO₂. The establishment of AGS cells stably-expressing human TFF1 was described before [23 (link)]. Briefly, the human TFF1 coding sequence was amplified by PCR, and cloned in-frame into the mammalian expression vector pcDNA3.1 (Invitrogen). Using Fugene-6 (Cat# E2691 Roche Applied Science) and following the manufacturer’s protocols, AGS cells were transfected with pcDNA3.1-TFF1 or pcDNA3.1 empty vector as a control. Stable cell lines were selected using G418 (Cat# 10131035 Invitrogen) (0.5 mg/ml). For the transient expression of TFF1, SNU-1 cells were transfected with the mammalian expression plasmid, pTT5 [28 (link)] in frame with TFF1, or PTT5 empty vector using Fugene-6 for 48 h. Protein expression of TFF1 in these cells was confirmed by using TFF1 Antibody (Origene, Rockville, MD).