Gst π

Human gastric cell line SGC7901 was obtained and cultured in Research Center, The Fourth Hospital of Hebei Medical University. GES-1 cell line was obtained from Shanghai Institutes of Biochemistry and Cell Biology, CAS. Adriamycin-resistant GC MDR cell line SGC7901/ADR was a gift from academician Fan Daiming in Digestive Disease Research Institute of The Fourth Military Medical University. RPMI 1640 culture medium and trypsin were purchased from Gibco Company; TRIzol reagent and Lipofectamine™ 2000 transfection reagent were obtained from Invitrogen. Fluorescence quantitative RT-PCR reagents and Reverse Transcription Kit were purchased from Promega. PCR primers and siRNA were synthesized at the Shanghai Sangon Biotech. Protein extraction kit was purchased from Beyotime Company, China; antibody for ZNF139 was provided by Sigma, U.S.A.; MDR1/P-gp, GST-π, MRP, Bcl-2, TS and β-actin antibodies were purchased from Santa Cruz Biotechnology, U.S.A.; MTT was bought from Sigma, U.S.A.

Cells (3 × 10⁵) were seeded into 6-well plates and incubated for 24 h, lysed, centrifuged at 4℃ at 14,000 × g for 5 min. The concentration of protein in the supernatant was measured by a Bradford assay. A 100 μg protein sample was separated on a 12% SDS-PAGE gel and transferred onto a PVDF membrane. The membrane was blocked with 5% skim milk for 2 h at room temperature. Monoclonal antibodies against MDR1 (1:800, Santa Cruz), MRP1 (1:800, Santa Cruz), GST-π (1:800, Santa Cruz), CDX2 (1:800, Santa Cruz), Bcl-2 (1:800, Santa Cruz), Survivin (1:800, Santa Cruz), Topo-Ⅱ(1:800, Santa Cruz), p-AKT (1:500, Santa Cruz) and β-actin (1:5000, Sigma) were added and incubated at 4°C overnight in 5% BSA. The membrane was washed 3 times with Tris-buffered saline (TBS) containing 0.1% Tween 20. Horseradish peroxidase (HRP)-labeled secondary antibody (1:1000, Sigma) was added and incubated at room temperature for 1 h. After the last membrane wash, protein was visualized using a DAB kit (Millipore).

The cells were cultured in a 6-well plate for 24 h, then incubated with different ratios of CIK (10:1 or 20:1) for 72 h. The cells were then digested, resuspended and lysed. Next, centrifugation at 10,000 × g was performed for 10 min at 4°C, and the supernatant was extracted to obtain the total protein. Electrophoresis was performed in 12% SDS polyacrylamide gel and the protein was transferred to polyvinylidene fluoride membranes. The membranes were blocked by 5% skimmed milk overnight at 4°C, then monoclonal rabbit anti-human MDR1, MRP1, GST-π, Bcl-2, Survivin, p-AKT and β-actin antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were added and incubated at 4°C overnight. The membranes were washed and incubated for 1 h with peroxidase-labeled anti-rabbit immunoglobulin G. Finally, the membranes were exposed to the Immobilon™ western chemiluminescent horseradish peroxidase substrate for 1 min and visualized.