with the lentiviral luciferase construct and selected stable clones using puromycin. Clonal populations of cells were expanded in vitro and tested for their luciferase activities. Several bright clones were repeatedly selected again and pooled together for cells with high luciferase expression to be used in vivo. In vitro and in vivo Bioluminescent Imaging In vitro and in vivo bioluminescence was performed using an IVIS Spectrum. Images and detection of BL signals were acquired and analyzed using Living Image Software V. 4.1. For in vitro imaging, cells tagged with luciferase were serially diluted in a black, clear bottom 96well plate. D-luciferin was added to the wells, the plate was incubated at 37uC, 5% CO2 for 10 min after which images were taken. For in vivo BL imaging, anesthetized mice were injected intraperitoneally with 150 mg/kg D-luciferin and placed inside the camera box for 10 min. Continuous exposure to 2% Tipifarnib chemical information isoflurane sustained sedation during imaging. Acquisition time was usually maximum 3 min with auto-exposure depending on the BL of tumors. Measurements of emitted light were performed for regions of interest and quantified as photon flux and were characterized in vitro and in vivo. Vectors contained green fluorescent protein and a puromycin resistance gene. Although cells demonstrated high GFP signals in vitro, the in vivo experiments 9726632 were unsuccessful due MTA1-Mediated Anticancer Effects of Pterostilbene sr). Normalization was done for all images at the 
end of the experiment. Gray scale images of mice were also collected at each session. Detection of metastasis during the experiment was contaminated by strong primary signal resulting in false-positive signals. Therefore, at the end of experiment, we secluded the primary signal by excising the prostate and surrounding muscle tissue to expose organs in abdominal cavity. Kidneys, livers and lung/heart from each mouse were isolated and ex vivo imaged by increasing binning and F/stop. Orthotopic PCa Xenografts Seven-week-old male nude mice were fed phytoestrogen-free AIN-76A diet from the day received. Animals were randomly assigned to two major groups prior to surgery and 5 MTA1-Mediated Anticancer Effects of Pterostilbene 6 MTA1-Mediated Anticancer Effects of Pterostilbene subsequently injected with either Du145-EV-Luc or Du145MTA1shRNA-Luc cells. During the surgery mice were anesthetized with 2% isoflurane, a 79 mm abdominal midline incision was made, and the prostate was exposed. 2.56106 cells of each cell line, in 20 ml PBS and Matrigel, were injected into anterior prostate. The wound was sutured, and the skin was closed with autoclips. Animals were closely monitored after surgery and clips were removed at day 10. Tumors colonized and grew for 14 days. Mice that developed tumors in each group were randomized into three subgroups with daily i.p. administration of 10% DMSO sham control; 50 mg/kg resveratrol 1700309 or PTER. Both compounds were soluble in 10% DMSO when warmed-up using water bath. Each week fresh solutions were prepared for injections. Body weights and BL signals were monitored weekly. The mice were sacrificed at week 8 after cell inoculation. At necropsy, prostates and other organs were excised, ex vivo imaged and fixed with 10% neutral-buffered formalin. Serum was also collected and stored at 280uC. temperature for 0.5 min, increased to 246uC at the rate of 0.5uC/ min and held for 0.5 min, increased to 280uC at the rate of 20uC/ min and held for 2 min, then final
