Thus, we speculated that TIMMDC1 knockdown inactivates AKT/GSK-3/-catenin signaling pathway may be related to the inhibition of ROS production. I but not complex II~ IV, and caused an obvious inhibition in mitochondrial respiration and ATP-linked oxygen consumption. Besides, the glycolysis pathway was also attenuated by TIMMDC1 knockdown, and the ATP content in the group of shTIMMDC1 cells was significantly lower than that in the shCont cells. The expression levels of phosphoylated AKT(Ser473) and GSK-3 (Ser9), as well as the downstream protein -catenin and c-Myc were also markedly reduced in the group of shTIMMDC1 cells. Taken together, these findings suggest that TIMMDC1 may play an important role in human gastric cancer development, and its underlying mechanism is not only associated with mitochondrial complex I inhibition and reduced mitochondrial respiration, but is also associated with reduced glycolysis activity and the AKT/GSK3/-catenin signaling pathways. as well as evidences that TIMMDC1 knockdown inhibits human gastric cancer cells growth and metastasis. Materials and Methods Reagents Rotenone, carbonyl cyanide ptrifluoromethoxyphenyl- hydrazone (FCCP), oligomycin, and 2-deoxy-D-glucose (2-DG) were from Sigma (St Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM) was from Gibco (USA). Fetal bovine serum (FBS) was from NATOCOR (Argentina). Cell Counting Kit-8 (CCK-8), BCA protein assay kit, ATP assay kit, ROS assay kit and crystal violet dye were obtained from Beyotime Institute of Biotechnology (Nanjing, China). XF assay medium and XF calibrant solution were obtained from Seahorse Bioscience (USA). RNA Extraction Kit, PrimeScript RT reagent kit, and SYBR VU 0364439 Premix Ex Taq were from TakaRa Biotechnology (Dalian) Co, Ltd (Dalian, China). Animals All experiments using animals were performed in accordance with the National Institutes of Health Guide for the Care and Use VU 0364439 of Laboratory Animals. Male nude mice of the age of 3-4 weeks (10-12g) were used, and they were purchased from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China, approval No. SCXK 2012-0002). The animals had access to standard chow and received water ad libitum. Cell culture and transfections Human gastric cancer cell line SGC-7901 and BGC-823 were obtained from the Institute of Cell Biology, Chinese Academy of Sciences (Shanghai) and China Center for Type Culture Collection, CCTCC (Wuhan), respectively. Cells were cultured in DMEM supplemented with 10% FBS and antibiotics (100 U/mL penicillin G and 100 g/mL streptomycin) at 37C in a humidified atmosphere of 5% CO2. The cell lines SGC-7901 and BGC-823 were transfected with small interfering (si) RNAs (TIMMDC1-siRNA or nonsilencing control siRNA) using Lipofectamine 3000 (Invitrogen) according to manufacturer’s instructions. VU 0364439 Quantitative real-time polymerase chain reaction (PCR) Total RNA was isolated with Trizol reagent according to the manufacturer’s guidelines and was quantified by Nanodrop spectrophotometry. cDNA was synthesized from 2 g total RNA using the PrimeScript RT reagent kit according to the manufacturer’s instructions. Real-time quantification PCRs of TIMMDC was performed using SYBR Premix Ex Taq. All expression values of target genes were calculated using the 2-Ct method 11,12. The primers used for the experiment were as follows: TIMMDC1 (Fw: 5′-AGTTACTGAGCACCTCCCT-3′; Rev: 5′-GCATTCAT CGGACATGGCAG-3′), -actin (Fw: 5′-CCCTGGCACCCAGC AC-3′; Rev: 5′-GCC GATCCACACGGAGTAC-3′). Western blot analysis The cells were Cav2 collected and lysed in Western and IP lysis buffer containing PMSF for 5 min on ice, followed by centrifugation at 13,000 for 25 min at 4C. The supernatant was harvested, and the protein concentration was quantified using a BCA protein assay kit. Western blot analysis was carried out by standard protocol. The following antibodies were used: rabbit anti-TIMMDC1 antibody (1:1000), rabbit anti-Phospho-AKT antibody (1:1000), rabbit anti-Phospho-GSK-3 antibody (1:1000), rabbit anti–catenin antibody (1:1000), rabbit anti-AKT antibody (1:1000), rabbit anti-GSK-3 (1:1000), rabbit anti-c-Myc antibody (1:1000) were from Abcam Inc. Mouse anti–actin antibody (1:1000, AA128), HRP-labeled goat anti-rabbit IgG (1:500, A0208), and HRP-labeled goat anti-mouse IgG (1:500, A0216) were from Beyotime Institute of Biotechnology (Nanjing, China). Cell proliferation assay SGC-7901 and BGC-823 cells in the control, shCont and shTIMMDC1 group were plated onto 96-well plates at a equal density of 3 103 cells/well. From the second day after plating, the Cell Counting Kit-8 (CCK-8) was.