The liposomes were added to the cells and siRNA treatment was continued for 24 h, and then, the cells were treated with H. pylori at a multiplicity of infection (MOI) of 100 for 24 h and finally exposed to either a solvent (ethanol, <0.1% final concentration) or 1 nmol/L 1α,25(OH)2D3 for the indicated time periods. GES-1 cells were treated with siVDR or 1α,25(OH)2D3 the next day with or without H. pylori for 24 h for a time-course study. At the end of the incubation period, cells were washed with PBS. The cells PD0325901 were scraped
into the lysis buffer (Sangon Biotech Inc., Shanghai) and centrifuged at ~14,000 ×g for 10 min to pellet the cell debris. Total protein was quantified using the Bradford assay, and equal amounts of protein were separated by 12% SDS–PAGE and then transferred to a polyvinylidene difluoride GDC0449 membrane (Milipore, Buckinghamshire, UK). The membranes were blocked at room temperature for 1 h with 5% nonfat milk in 1 × TBST (TBS + 0.05% Tween
20) and subsequently incubated with mouse anti-VDR at 1:500 (sc-13133, Santa Cruz Biotechnology Inc.), mouse anti-CAMP at 1:1000 (sc-130552, Santa Cruz Biotechnology Inc.) or mouse anti-GAPDH at 1:10,000 (sc-130301, Santa Cruz Biotechnology Inc.) at 4 °C overnight. After washing with TBST, the membranes were incubated with the appropriate HRP-conjugated secondary antibody at room temperature for 1 h, and the antibody binding was visualized using the ECL detection system (MultiSciences). GES-1 cells were infected with H. pylori SS1 (1 × 108 bacteria/mL) in the presence or absence of siVDR, siCAMP or 1,25(OH)2D3. After incubation for 2 h at 37 °C in an atmosphere
containing 5% O2, 10% CO2, and 85% N2, GES-1 cells were washed two times and treated with 150 μg/mL gentamicin for 2 h to kill extracellular bacteria. The infected cells were washed two times and then incubated with gentamicin-containing (25 μg/mL) medium before the samples were harvested. The cells were lysed with 1 mL of 0.01% saponin ALOX15 in Dulbecco’s phosphate-buffered saline (DPBS) and diluted and then plated on Columbia agar plates; the number of visible colonies was counted after 3–5 days of incubation. The CFU data are derived from triplicate wells in three independent experiments using three separate donors. All data are expressed as the mean ± standard deviation (SD) value from at least three independent experiments. Statistical analysis was performed using two-tailed unpaired Student’s t-test or one-way ANOVA with Bonferroni’s multiple comparison test or two-tailed Pearson’s correlation coefficient analysis to analyze statistically significant differences between groups by the SPSS software (version 16.0). Differences at p < .05 were considered significant. A total of 33 outpatients (mean age, 53.