For the experiments, GES-1 cells were seeded at a density of 5 × 105 cells/mL of medium Palbociclib purchase in six-well plates and grown to 80% confluence prior to the
experiments. Helicobacter pylori strain SS1 (both VacA+ and CagA+) was obtained from the National Institute for Communicable Disease Control and Prevention (NICDC), Beijing, China. The strains were grown in a microaerobic humidified atmosphere (5% O2, 10% CO2, 85% N2) on 10% lysed sheep blood Columbia agar at 37 °C. After 48–72 h, bacteria were harvested in phosphate-buffered saline (PBS) (pH 7.4) or in RPMI-1640 medium without antibiotics, resuspended to a concentration of 6 × 108 CFU/mL and used immediately. Subconfluent GES-1 cells were cultured alone or with various doses of freshly harvested H. pylori (1 × 104–6 × 108 CFU/mL) for various periods of time. At the end of the treatment, GES-1 cells were harvested and processed for the preparation of whole-cell extracts and western blotting. Total RNA was isolated from GES-1 cells
or gastric mucosa tissues using the Trizol reagent (BBI) according to the manufacturer’s instructions. The first-strand cDNAs were synthesized from total RNA using reverse transcriptase (Takara, Dalian, China) according to the manufacturer’s instructions. All PCR primers were synthesized by Bio Basic Inc. (Shanghai, China) (Table 1). cDNA samples in each treatment group were pooled in subsequent experiments and reactions d see more set in a 15-μL reaction mixture in 96-well plates. Real-time RT-PCR quantitation for individual target mRNA was performed on an ABI Model 7500 Sequence Detector (Applied Biosystems, Foster City, CA) using a TaKaRa real-time PCR kit. RT-PCRs were performed using the following parameters: 95 °C for 2 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 34 s and
72 °C for 15 s. For each sample, a melting curve was generated at the end of the reaction to ensure specificity. Gene expression levels were normalized to those of GAPDH, and the data were analyzed using comparative cycle MCE公司 threshold calculations. Data were expressed as fold changes relative to the control group. Each real-time PCR experiment was run three times. The comparative 2− ΔΔCT method was used for quantification and statistical analysis (the results were expressed as fold changes relative to normal controls). GES-1 cells were transfected with either nonspecific siRNA oligomers or siRNAs targeting the VDR mRNA (Invitrogen, Shanghai, China) by using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. The cells were seeded into 24-well plates and grown in phenol red-free RPMI1640 supplemented with 5% FBS.