on vector pEGFP C1, and transfection was verified by DNA sequencing. Transfected cells were seeded in 6 cm diameter dishes at 5 105 cells dish, and transfected with either pEGFP SIRT1 or empty vector using Lipofectamine 2000, according www.selleckchem.com/products/AP24534.html to the manufacturer s protocol. Transfected cells were further e amined in cell proliferation assays. OECM1 cells were transiently transfected with small interfering RNA against SIRT1, or with a nontargeting control in Opti MEM I reduced serum medium containing Lipofectamine RNAiMA . Transfection efficiency was assessed by western blot. RNA isolation and quantitative real time PCR For gene e pression analysis, pairs of tumor and normal marginal tissues were obtained from 21 OSCCs. The tis sues were frozen and stored in liquid nitrogen at ?196 C until use.
Total RNA obtained from cultured cells and human tissue was e tracted using TRIzol reagent. cDNA was then reverse transcribed and ampli fied by PCR using a Transcriptor First Strand cDNA Synthesis kit. Quantitative RT PCR was jperformed using the FastStart Universal SYBR Green Master mi and an Applied Biosystems ABI 7900 RealTime PCR System. The oligonucleotide primers used for human SIRT1, Smad4, MMP7, and glyceraldehyde 3 phosphate dehydrogenase Gene e pression levels were normalized using GAPDH as an internal reference gene, and the average relative change was calculated from triplicate or quintuplicate determinations made by relative quantification, and applying the delta delta cycle threshold method.
The protocol for this study was approved by the Institutional Review Board of the Department of Oral and Ma illofacial Surgery of Chi Mei Medical, Liouying, Taiwan. Cell chemotactic migration and invasion assay The chemotactic migration of cells was evaluated using a 24 well chemota is chamber equipped with 8 um pore size membranes. Cell invasion ability was assessed using Falcon Cell Culture Inserts with Matrigel. Samples containing 1 105 cells were resuspended in serum free medium with 0. 1% BSA, and then plated onto the transwell chamber. The chambers were incubated for 24 h with complete culture medium added in the lower chamber. Non mobile cells were removed, and the chambers were stained with crystal violet. Photo micrographs of 5 regions were captured from duplicated chambers. The numbers of cells were counted and nor malized to the control.
All e periments were performed in triplicate and repeated Drug_discovery three times. Cytosolic, nuclear isolation and immunoprecipitation Cytosolic and nuclear e tracts were prepared using a NE PER Nuclear and Cytoplasmic E traction Reagents kit following the manufacturers protocol. Isolated nuclear e tracts http://www.selleckchem.com/products/VX-770.html were lysed with RIPA buffer, and then subjected to direct western blot analysis or immunoprecipitation. Then, 2 mg of pro tein from each sample was used for immunoprecipitation with a Pierce Crosslink IP Kit, and the results were analyzed by western blot. Western blot analysis Cells were lysed directly in RIPA buffer containing