Sfrp1 vector

sFRP1 vector was purchased from OriGene Technologies, Inc. (Rockville, MD, USA). The green fluorescent protein-fused wild-type (WT) Rac1, constitutively active (CA) mutant Rac1 (Q61L), dominant-negative (DN) mutant Rac1 (T17N) (27 (link)), Tag5Amyc-GSK3β WT (28 (link)), pCS2 Flag Smad3 S204A (29 (link)), pCMV5B-Flag-Smad3 (30 (link)) and pCMV5 Smad2-HA (31 (link)) were purchased from Addgene, Inc. (Cambridge, MA, USA). Top-flash luciferase plasmid (BPS Bioscience, San Diego, CA, USA) is a luciferase reporter plasmid that contains two sets of 3 copies of the wild-type T-cell factor (TCF) binding regions. If the canonical Wnt signaling is activated, β-catenin will translocate to the nucleus to associate with TCF/lymphoid enhancer factor transcription factors to activate transcription of Wnt target genes. pSV-β-Galactosidase control vector was used as an internal control for transfection and was purchased from Promega Corporation (Madison, WI, USA). For plasmid transfection, SGC-7901/vector cells were seeded into 6-well plate and allowed to grow 24 h prior to transfection. Cells in each well were transfected with 4 µg plasmid using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 6 h at 37°C and the medium was changed subsequent to transfection.

The transcriptional activity of β-catenin was measured by co-transfection with Top-flash luciferase plasmid and sFRP1 vector (OriGene Technologies, Inc.) or the control vector using Lipofectamine^® 2000 for 6 h at 37°C. TOPFlash encoding the LEF/TCF binding sites (insert gene) linked to firefly luciferase and reflecting Wnt/β-catenin signaling activity was used. After 24 h incubation, the luciferase activity was measured and normalized to β-galactosidase activity (Promega Corporation). The Luciferase Reporter Gene Detection kit (Promega Corporation) and GloMax^®-Multi+ Detection system (Promega Corporation) were used according to the manufacturer's protocol. The data presented were the mean value of three independent experiments.