Quantitative real-time PCR MRP1-Mediated GSH Efflux in RPE Cells calculating 22DDCT

tamine 2000 following the manufacture’s manual. For pDisrup 8 clone selections, cells were selected with Blasticidin S.HCl at 25 mg/ml. Western Blot After washing with PBS twice, cells were extracted with cold lysis buffer and centrifuged at 15,000 g for 15 min at 4uC. Protein concentration of the supernatants was determined with Bradford assay. 1040 mg of samples was separated by electrophoresis on 816% SDS-PAGE and transferred to Polyvinylidene fluoride membrane. After blocking with 5% skimmed milk for 1 h, membranes were incubated with different specific primary antibodies in either 5% skimmed milk or 5% bovine serum albumin . After washing with PBST for 30 min, the membranes were further incubated with corresponding HRP-conjugated secondary antibodies and developed with Pierce’s West Pico chemiluminescence substrate. All results were obtained from 3 independent experiments. Materials and Methods Cell Culture and Reagents Murine melanoma B16F10, B16F0 cells, and NIH 3T3 cells were obtained from American Type Culture Collection. Human melanoma cells were kindly provided by Dr Jean-Pierre Abastado. Cells were cultured in Dulbecco’s Modified Eagle Medium with 10% fetal calf serum , and 1% Penicilin/Streptomycin mix and maintained at 37uC in a humidified atmosphere containing 5% CO2. Specific inhibitor for AKT was obtained from Calbiochem. Lipofectamine 2000 was purchased from Invitrogen. Cell Attachment Assay 96-well tissue culture plates were coated with Collagen Type IV followed by washing with PBS Scopoletin chemical information before blocking with 0.5% BSA. 16105 melanoma cells were seeded onto the pre-coated 96-well plates and incubated for 15, 30, 60 and 120 min. After incubation, the unattached cells were removed and the plate was stained with 0.5% crystal violet in 20% methanol for 20 min at room temperature and then washed with tap water. Cell attachment was evaluated spectrophotometrically by dissolving the stain with 20% acetic acid and measured at a wavelength of 570 nm with Tecan. Plasmids and DNA Constructs The pDisrup 8 vector was a gift from Dr. Han Jiahuai. The short small interfering RNA was constructed with a sequence specifically targeted to mouse Dph3 gene:. Target and scrambled control oligonucleotides duplexes were cloned into pSilencer4.1-CMV vector according to the manufacturer’s instructions. The Dph3 and its truncated colones were cloned into the sites of EcoRI and XhoI of pIRES2-EGFP vector containing a Myc-tag with gene specific primers. The primers used were as follows: Dph3, Dph3 140aa, Dph3 21-60aa, Dph3 4182aa, Dph3 ahelix deletion assay Dph3 Potentates the Metastasis of Melanoma Cells in vitro according to the manufacturer’s instructions. In brief, 1 6 105 cells with 500 ml in serum-free medium were added into the upper chamber and 750 ml of NIH-3T3 fibroblast conditioned medium was added into the lower chamber, serving as chemoattractant. After incubation in humidified tissue culture incubator, 37uC, 5% CO2 atmosphere for 24 h, the non-invasive cells in the upper surface of the membrane were removed by “scrubbing”with cotton tipped swab and the invasive cells migrating to the lower surface of the membrane were fixed and stained with 0.5% crystal violet for 30 minutes. Cell counting was then carried out by photographing the membrane through the microscope. 20 random fields under microscope at 20X magnification are taken. The migration assay was performed with the same strategy, just that the chamber membrane was not coated with matri