MNK-45, AGS, and GC-7901 cells are human gastric cancer cell lines, and GES-1 is a normal gastric epithelial cell line. All cells were obtained from the Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in either RPMI 1640 with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37°C, 5% CO2.
RT-PCR of Klotho gene expression
Cultured cells were homogenized in Trizol reagent (Invitrogen, Carlsbad, CA). Total RNA was isolated following the user manual. Reverse transcription was performed using First Strand cDNA Synthesis Kit (Fermentas China, Shenzhen, China). Klotho gene fragment was amplified using forward primer: 5′- CACGGCAAGGGTGCGTCCAT -3′ and reverse primer: 5′-TCGCGCCCACGAGATGGAGA-′3. The GAPDH gene was amplified using forward primer: 5′-CTCATGACCACAGTCCATGC-3′ and reverse primer: 5′-TTCAGCTCTGGGATGACCTT-3′. PCR products were visualized on 1.5% agarose gel containing 0.5 μg/ml of ethidium bromide.
Genomic DNA isolation, sodium bisulfite treatment and PCR amplification
QIAamp DNA Mini Kit (QIAGEN, Valencia, CA, USA) was used to extract genomic DNA from cultured cells by following the user manual. For bisulfite treatment, EZ DNA Methylation-Gold Kit (ZYMO RESEARCH, Orange, CA, USA) was used. After purification, methylated genomic DNA was subjected to PCR amplification of Klotho gene promoter. The methylated DNA was amplified by Klotho(M)-F: 5′-ATGAATTTGAGCGTTTACGAAAC-3′, and Klotho(M)-R 5′-ACTCCGCTAACAATAATTACCTACG-3′ primers, while the unmethylated DNA was amplified by Klotho(U)-F: 5′-ATGAATTTGAGTGTTTATGAAATGT-3′, and Klotho(U)-R: 5′-TCCACTAACAATAATTACCTACAAA-3′ primers. The amplified fragments were 219 bp.
The anti-klotho, anti-Akt, anti-phospho-Akt1, anti-IGF-IR, anti-phospho-IGF-IR, anti-GAPDH, and HRP-conjugated second antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-LC3C-I antibody (Cat#: 6976–1) was purchased from Epitomics (Burlingame, CA, USA). The anti-LC3B-II (Cat#: 3868), anti-IRS, anti-phospho-IRS, anti-PI3K, anti-phospho-PI3K, and anti-phospho-mTOR antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Protein concentrations were measured using BCA Protein Assay kit (Beyotime, Shanghai, China). Western blot was performed as previously described . Briefly, 20 to 30 μg of total protein were loaded onto a 10 or 12% SDS-PAGE gel and transferred to nitrocellulose membranes. After blocking with 5% non-fat milk for 1 hour, membranes were incubated with primary antibody for 2 hrs at room temperature or overnight at 4°C and subsequently incubated with HRP-labeled secondary antibody (1:2,000 dilution) for 2 hrs at room temperature. Reactive proteins were detected using chemiluminescent reagents (Pierce, Rockford, IL, USA). To control for loading efficiency, the blots were stripped and reprobed with GAPDH antibody. Expressions of all proteins were evaluated relative to GAPDH expression.
Cell viability assay
Cell Counting Kit-8 (CCK-8) (Beyotime, Shanghai, China) allows sensitive colorimetric assays for cell viability. Briefly, GC-7901 cells were seeded into 96-well plates at 1 × 104 cells per well 24 hrs before transfection. Cells were transfected with klotho expression vector, blank vector, or no vector (PBS) using lipofectamine 2000 according to the user manual (Invitrogen, Grand Island, NY, USA). Cells were then continually cultured in growth medium for 72 hrs. Ten μl of reagent provided with the kit were added to the cells and incubated for 1 h. Cell viability was assessed using the microplate reader at 450 nm. All results were normalized to OD values measured from an identically conditioned well with only growth medium.
Flow cytometry assay
GC-7901 cells were seeded in 10-cm dishes at a density of 2×106 cells per dish. After cells reached 70% confluency, cells were transfected with klotho expression vector, blank vector, or PBS as described above. Cells were then trypsinized and suspended with 500 μl of binding buffer containing 5 μl of Annexin V-FITC and 5 μl of Propidium Iodide (Abcam, Cambridge, MA, USA). After incubation in the dark for 1 hour, cells were subjected to flow cytometry assay.
Construction of klotho gene expression vector
The klotho gene was amplified from a cDNA library established from GES-1 cells. The open read frame (ORF) of klotho cDNA sequence was amplified by a forward primer containing Bgl II sequence (italic): ACTCAGATCTGAGCCGGGCGACGGCGCGCAGA and reverse primer containing a BamHI site (italic): CGGTGGATCCCCTATTTGTAACTTCTTCTGCC. The amplified klotho ORF was then cloned into pZsGreen1-C1 vector at Bgl II/Bam HI sites (Clontech, Mountain View, USA). The klotho ORF was fused with GFP at the C-terminal of GFP. The pZsGreen1-C1 vector without insertion was used as a blank vector control.
To identify the location and expression of LC3-II protein, we performed immunofluorescent staining in GC-7901 cells. Briefly, cells in 24-well plates were fixed by 10% paraformaldehyde for 30 min at 4°C. After cells were rinsed with PBS for 3 × 5 min, they were permeabilized with 0.5% Triton X-100 for 15 min. After a light rinse with PBS for 3 times, cells were incubated with 10 mM citrate buffer (pH 3.0) for antigen retrieval for 30 min and then incubated with 10% goat serum for 1 h to block nonspecific staining. Subsequently, the cells were incubated with rabbit-anti-LC3-II antibody (Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. After washing cells with PBST (PBS plus 0.05%Tween-20) for 3 × 5 min, cells were continually incubated with goat-anti-rabbit, FITC conjugated antibody (1:600, Cell Signaling Technology) for 1 h at room temperature, and then cells were washed with PBST and mounted with anti-fade medium. The staining was examined using a fluorescence microscope.
Cell treatments with autophagy and apoptosis inhibitors
GC-7901 cells at 70% confluency were transfected with klotho expression vector, blank vector, or PBS as described above. Cells transfected with klotho expression vector or PBS were incubated with 10 mM of autophagy inhibitor 3-methyladenine (3-MA) or 20 μM of apoptosis inhibitor Z-VAD-FMK for 24 hours. Cells were then harvested for Western blot and/or flow cytometry assay.
Data was analyzed using the SPSS 13.0 (statistical package for the Social Sciences Version 13.0). Two samples were compared using student t-test. A p < 0.05 was considered statistically significant.