After transfecting T24, RT-4 and BMCs with the above plasmids, cells were processed with lysis buffer, and subsequently, luciferase activities were assessed with the Dual-Luciferase reporter system (Promega, WI) according to the manufacturers’ DZNeP instructions. Cell viability assay 1 × 104 T24 and RT-4 cells, 1.5 × 104 primary bladder cancer cells or 2 × 104 BMCs were cultured in each well of www.selleckchem.com/products/azd5582.html 96-well plates. Adenoviruses of indicated MOIs were added to cell cultures. After 6d, 50 μl of MTT (1 mg/ml) was added, and 4 h later, MTT-containing media was replaced with 150 μl of DMSO. The
spectrophotometric absorbance was assessed on a model 550 microplate reader (Bio-Rad Laboratories, Hercules, CA) at 570 nm with a reference wavelength of 655 nm. Cell viability = absorbance value of infected cells / absorbance value of uninfected BVD-523 in vitro control cells. Animal experiments Procedures for animal experiments were all approved by the Committee on the Use and Care on Animals in Qingdao Municipal Hospital (Qingdao, China). 2×106 T24 cells were inoculated at the left flanks of 5-week-old female BALB/c nude mice (Institute of Animal Center, Chinese Academy of Sciences, Shanghai, China). When tumors reached 7–9 mm in diameter, 24 mice were equally assigned into 4 groups (n=6). 100 μL of PBS with or without 2×108
pfu of Ad-EGFP, Ad-TRAIL and Ad-TRAIL-MRE-1-133-218 was directly administrated into tumors by injection, respectively. The administrations were performed every other day for five times with a total dosage of 1×109 pfu of adenoviruses. T-24 cancer xenograft was established by incubating 1.5×106 cells at the right flanks of 5-week-old female BALB/c nude mice. 24 mice were equally divided into 4 groups (n=6). The doses of used adenoviruses and injection procedures were the
same as those on T24 tumor xenograft. We periodically measured tumor diameter using calipers. Tumor volume (mm3) = maximal length mafosfamide (mm) × perpendicular width (mm) 2 / 2. Liver function evaluation To evaluate the hepatoxicity induced by adenovirus treatment, BALB/c mice (n=5) were intravenously injected with 1×109 pfu of indicated adenoviruses every other day for five times. On day 11, their blood (600 mL/mice) was harvested by cardiac puncture, followed by being incubated with 12 U of heparin. Alanine aminotransferase (ALT) levels in blood were detected at the Clinical Laboratory, Qingdao Manucipal Hospital (Qingdao, China). Histological staining On day 7 after adenovirus injection, one mouse was sacrificed from each group and its tumor, brain and liver were collected and fixed according to the routine procedures. Histological staining was then performed on formalin-fixed, paraffin-embedded tumor, brain and liver tissue sections using the streptavidinbiotin peroxidase complex method. Anti-TRAIL antibody (Santa Cruz Biotechnology, CA) was used to specifically recognize TRAIL protein. The sections were finally counterstained with hematoxylin.