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X.-B.W., R.L., and X.-H.L. a time-dependent way, and inhibited progression of cell cycle from G0/G1 to S-stage. When treated with siTCF-3, the Eca-109 cells exhibited improved apoptosis, with up-regulated cleaved caspase and Bax expressions, whereas Bcl-2 manifestation was down-regulated. The present study demonstrates gene silencing inhibits Eca-109 cell growth and proliferation, suppresses cell cycle progression, and promotes apoptosis, which might serve as a new objective for EC treatment. gene silencing and the proliferation and apoptosis of Eca-109 cells. Materials and methods Cell tradition The EC cell collection Eca-109, purchased from Cell Standard bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, was cultured in 1640 medium (Gibco BRL Co., Ltd., Gaithersburg, MD, U.S.A.) with 1% double antibody (100 U/l penicillin and 100 mg/l streptomycin, Gibco BRL Co., Ltd., Gaithersburg, MD, U.S.A.) containing 10% FBS (Tianhang Biological Systems Inc., Zhejiang, NDRG1 China), later on cells were cultured at 37C with 5% CO2. Reading of the cells was taken once every 2C3 days, and then cells in the logarithmic growth phase were collected for the following experiment. Cell treatment Eca-109 cells in the logarithmic growth phase were inoculated into six-well plates for 24 h, and then cultured in the medium without antibiotic. When the cell denseness reached 40C60%, cells were cultured in Opti-MEM medium without serum. Cells were transfected after reaching 70% convergence, and the compound of transfection reagent and siRNA (200 l) were arranged in accordance with the teaching of Lipofectamine 2000 Transfection Kit (Invitrogen Inc., Carlsbad, CA, U.S.A.), which was then added into the medium. A negative sequence without interference was transfected like a control. After transfection, the cells were cultured at 37C with 5% CO2, followed by the alternative of the medium with 1.5 ml of normal medium comprising serum after 6-h culture. The cells were divided into control group, bad control group (NC group, with non-specific siRNA-NC), and siRNA against TCF-3 (siTCF-3)-1 group, siTCF-3-2 group, and siTCF-3-3 group (with gene silenced by RNAi 1, RNAi 2, RNAi 3). All siRNA sequences were CA-4948 synthesized by Shanghai GenePharma Co., Ltd. Shanghai, China. Sequences of siTCF-3-1, siTCF-3-2, and siTCF-3-3 are demonstrated in Table 1. Forty-eight hours after the cell treatment, the mRNA level of TCF-3 in Eca-109 cells was determined by reverse-transcription quantitative PCR (RT-qPCR) to display the best siTCF-3 for later on experiments (Table 1). Table 1 The sequences of three siTCF-3 gene silencing within the growth of Eca-109 cell) was measured. At the same time, the changes in the morphology of the cells before culturing and after gene silencing were observed via an inverted microscope. BrDU CA-4948 assay The Eca-109 cells in the logarithmic growth phase were resuspended in the cell growth medium. Cell denseness was adjusted to 1 1.0 104 cells/ml, and then cells were seeded in 24-well plates with cover glass, 3 ml in each well. The medium was left behind and different siRNAs were added 24 h later on after incubation, with three duplicated well units. Twenty-four hours after incubation, the cover glass was washed thrice with PBS in 3 min. Then, immersed in 4% formaldehydum polymerisatum and incubated at space temp for 15 min, the cover glass was washed again thrice with PBS CA-4948 in 3 min. After osmotic treatment with 0.05% Triton X-100 (ST795, Beyotime Biotechnology Co., Ltd., Shanghai, China) for 20 min, the samples were incubated with 2 mol/l HCL at space temp for 1 h, and washed with PBS thrice in 5 min. Next, the samples were immersed in 0.1 M Na2B4O7 (equal to 2 mol/l HCL) at space temperature for 15 min, and washed with PBS thrice in 10 min. Then, PBS was blotted by absorbent paper and normal goat serum was added drop by drop. Sufficient amount of well-diluted main antibody of BrDU was added into each glass (1:50, ab187275, Abcam Inc., Cambridge, MA, U.S.A.) and incubated at 4C inside a damp box over night. The glass was washed with PBS comprising Tween (PBST) thrice in 3 min. Then, the surplus CA-4948 liquid was blotted by absorbent paper. Subsequently, the samples were added with diluted goat anti-rat IgG-CY3 (1:100, BA1031, Wuhan Boster Biological Technology Ltd., Wuhan, Hubei, China), placed in a damp package, and incubated at 20C37C for 1 h. The glass was washed again with PBST thrice in 3 min, and DAPI (C1002, Beyotime Biotechnology Co., Ltd., Shanghai, China) was added to incubate in the dark for 5 min, the nuclei of specimens were stained, and washed with PBST four instances in 5 min.