Nzhen, China). Klotho gene fragmentQIAamp DNA Mini Kit (QIAGEN, Valencia, CA, USA) was applied to extract genomic DNA from cultured cells by following the user manual. For bisulfite therapy, EZ DNA Methylation-Gold Kit (ZYMO Research, Orange, CA, USA) was utilized. Following purification, methylated genomic DNA was subjected to PCR amplification of Klotho gene promoter. The methylated DNA was amplified by Klotho(M)-F: 50-ATGAATTTGAGCGTTTACG AAAC-30, and Klotho(M)-R 50-ACTCCGCTAACAAT AATTACCTACG-30 primers, whilst the unmethylatedan kV kl ot ho -V 3M A + kl ot ho -ADBlklotho p-IGF-1R IGF-1R p-IRS-1 IRS-1 p-PI3K PI3K p-Akt Akt p-mTOR mTOR GAPDHVXie et al. Cancer Cell International 2013, 13:18 http://www.cancerci.com/content/13/1/Page 7 ofFigure 6 The roles of apoptosis and autophagy inhibitors on apoptosis and cell cycle. A) Relative cell viability in GC-7901 cell transfected with blank vector (blank-V), klotho expression vector (klotho-V), or klotho vector plus 3-MA (k-V + 3-MA), or Z-VAD-PMK (k-V + ZVP) incubation. p 0.01 vs. klotho-V. N = 5. B) Flow cytometry of cell apoptosis in GC-7901 cells transfected with blank vector (blank-V) or klotho expression vector (klotho-V), transfected with klotho expression vector plus 3-MA (K-V + 3-MA), or Z-VAD-PMK (k-V + ZVP) incubation. C) Percentage of apoptotic cells in B). p 0.001 vs. Alt Inhibitors Related Products blank-V, p 0.05 vs. klotho-V. #p 0.01 vs. klotho-V. N = five. D) Flow cytometry of cell cycle analysis. E) Percentage of sub-G0/G1 cells in D). p 0.001 vs. blank-V, p 0.05 vs. klotho-V. #p 0.01 vs. klotho-V. N = five.DNA was amplified by Klotho(U)-F: 50-ATGAATTTGA GTGTTTATGAAATGT-30, and Klotho(U)-R: 50-TCCA CTAACAATAATTACCTACAAA-30 primers. The amplified fragments have been 219 bp.Western blotThe anti-klotho, anti-Akt, anti-phospho-Akt1, anti-IGF-IR, anti-phospho-IGF-IR, anti-GAPDH, and HRP-conjugated second antibodies have been purchased from Santa CruzXie et al. Cancer Cell International 2013, 13:18 http://www.cancerci.com/content/13/1/Page 8 ofFigure 7 The roles of apoptosis and autophagy inhibitors on autophagy. A) Immunofluorence staining of LC3-II expression. The LC3-II good staining (green) located within the cytoplasm. The blue nuclear was stained by DAPI. GC-7901 cells have been transfected with blank vector (blankV), klotho expression vector (klotho-V), or klotho vector plus 3-MA (k-V + 3-MA), or Z-VAD-PMK (k-V + ZVP). B) Western blot of LC3-I and LC3-II expression.Biotechnology (Santa Cruz, CA, USA). The anti-LC3C-I antibody (Cat#: 6976?) was bought 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 had been purchased from Cell Signaling Technology (Danvers, MA, USA). Protein concentrations had been Antioxidants Inhibitors Related Products measured using BCA Protein Assay kit (Beyotime, Shanghai, China). Western blot was performed as previously described [26]. Briefly, 20 to 30 g of total protein were loaded onto a 10 or 12 SDS-PAGE gel and transferred to nitrocellulose membranes. Soon after blocking with 5 non-fat milk for 1 hour, membranes were incubated with primary antibody for two hrs at roomtemperature or overnight at 4 and subsequently incubated with HRP-labeled secondary antibody (1:two,000 dilution) for two hrs at room temperature. Reactive proteins have been detected utilizing chemiluminescent reagents (Pierce, Rockford, IL, USA). To handle for loading efficiency, the blots were stripped and reprobed with GAPDH antibody. Expressions of all proteins had been evaluated relative to GAPDH.