The Fas gene was subcloned to pAdTrack-CMV plasmid (a gift from Gang Huang, Third Military mTOR signaling pathway Medical University, Chongqing, China) and recombinants of pAdTrack-CMV-Fas Histone Methyltransferase inhibitor were generated by transformation the shuttle plasmid linearized with Pme I to BJ5183 cells with the adenoviruses backbone plasmid for homologous
recombination. The recombinant adenoviruses were packaged and propagated in 293 cells. Viral titers were determined by standard plaque assay after the Fas adenoviruses concentrated by CsCl ultracentrifugation using a standard method . H446/CDDP cells were transfected with 50 multiplicity of infection (MOI) of adenoviruses in serum free RPMI and maintained in complete medium at 37°C until post-transfection day 3. The transfectants overexpressing Fas were obtained and designated as H446/CDDP/Fas. H446/CDDP cells transfected with empty adenoviruses were indicated as H446/CDDP/Empty and used as negative control in all assays. Conventional RT-PCR analysis On post-transfection day 3, total RNAs were isolated from H446/CDDP, H446/CDDP/empty, Selleck Epacadostat and H446/CDDP/Fas cells using TRIzol reagent (TianGen, Beijing, China) and subsequently used for semiquantitative PCR. RT was performed with 1 μg of total RNA from each sample using oligo(dT) 18 primers and 200 units of SuperScript II RT (Life Technologies Inc., Gaithersburg, Md., USA) for cDNA synthesis. cDNA amplification was conducted in 20 μl solution
2 μl of diluted cDNA, 10 pmol primer pairs for Fas, GST-π, ERCC1 and GAPDH, respectively, and 10 μl of Taq PCR Master mix (TianGen, Beijing, China). The PCR consisted of initial denaturation at 94°C for 5 min, followed by 30 reaction cycles (30 seconds at 94°C, 30 seconds at 61°C, and 30 seconds at 72°C) and a final cycle at 72°C for 10 min. Primers used in PCR were listed in Table 1. GAPDH was used as internal control. All PCR products were electrophoretically separated on ethidium bromide-stained agarose gel and visualized with UV light. Table 1 PCR primer sequences and product sizes. Primersa Oligonucleotide Sequences Product Size (bp) PCR Cycles Fas F: 5′GTCCAAAAGTGTTAATGCCCAAGT 3′ 232 30 R: 5′ATGGGCTTTGTCTGTGTACTCCT 3′ GST-π F: 5′ CCGCCCTACACCGTGGTCTAT 3′ 260 30 R: 5′ GCTGCCTCCTGCTGGTCCTT 3′ ERCC1-2 F: 5′ ACGCCGAATATGCCATCTCAC Meloxicam 3′ 292 30 R: 5′ AGCCGCCCATGGATGTAGTCT 3′ GAPDH F: 5′ ACCCATCACCATCTTCCAGGAG 3′ 159 30 R: 5′ GAAGGGGCGGAGATGATGAC 3′ a All primers were designed using genetool software. Real-time quantitative PCR (RT-qPCR) RT-qPCR was performed with ABI 7500 Thermal Cycler and SYBR Green qPCR kit (Toyobo, Japan). PCR reactions were prepared in low-profile microplates with each well containing 10 μl of master mix, 2 μl of diluted cDNA, 10 pmol each of primers listed in Table 1 for Fas, GST-π, ERCC1 and control GAPDH, respectively, in a 20 μl reaction volume.