In the present study, we further examined the tumor-suppressing function of ECRG4 gene, in terms of cell migration and invasion, Smad activation and explored possible molecular mechanism in ESCC. Materials and methods Construction of eukaryotic expression vector and stable transfection The coding region of ECRG4 cDNA was subcloned into constitutive mammalian expression vector pcDNA3.1 (Invitrogen). The cDNA was then fully sequenced to ensure that no mutation was introduced during

the PCR amplification. The resulting plasmid construct was named pcDNA3.1-ECRG4. The human esophageal squamous cell line EC9706 was established and studied by Han et al [9]. EC9706 cells were seeded in 6-cm dishes at 5×105 cells/dish and transfected with pcDNA3.1-ECRG4 Erismodegib and pcDNA3.1 using lipofectamine™2000 (Invitrogen), according to the manufacturer’s protocol. After NSC23766 culturing in medium containing 400 μg/ml of geneticin (Invitrogen) for 3 weeks, individual clones were isolated. Clones that expressed the ECRG4 cDNA coding region were maintained in medium containing 200 μg/ml of geneticin and used for further experiments. Cell proliferation assay EC9706 cells (pcDNA3.1 and pcDNA3.1-ECRGR4) were seeded into 96-well plates (1.5 × 103 cells/well). After culturing for various durations, cell proliferation was evaluated by thiazolyl blue tetrazolium bromide (MTT) assay, according to the manufacturer’s protocol (Sigma-Aldrich

Co., St. Louis, MO, USA). In brief, 10 μl MTT solution (5 mg/ml) was added to each well, then the cells were cultured for another 4 hours at 37°C, and 100 μl DMSO was added to each well and mix vigorously to solubilize colored crystals produced within the living cells. The absorbance at 570 nm was measured by using a multi-well scanning spectrophotometer Victor 3. In vitro cell migration and invasion assay Tangeritin Cells growing in the log phase were treated with trypsin and re-suspended as single-cell solutions. A total of 1 × 105 cells in 0.5 ml of serum-free RPMI 1640 medium were seeded on a 8 μm-pore polycarbonate membrane Boyden chambers insert in a transwell apparatus(Costar,

Cambridge, MA), either coated with or without Matrigel(BD Biosciences, San Jose, CA). 600 μl RPMI1640 containing 20% FBS was added to the lower chamber. After the cells were incubated for 12-24 hours at 37°C in a 5% CO2 incubator, cells on the top surface of the insert were removed by wiping with a cotton swab. Cells that migrated to the bottom surface of the insert were fixed in 100% methanol for 2 minutes, stained in 0.5% crystal violet for 2 min, rinsed in PBS and then subjected to microscopic inspection (×200). Values for invasion and migration were obtained by counting five fields per membrane and represent the average of three independent experiments. Cell adhesion assay Cells were plated on 100 ng/μl Matrigel-coated 96-well plates at a density of 5 × 104 per well.