Significant lung metastasis was observed in control B16F10 but not in clone 2 cells injected mice

ay Reagents Resveratrol and LY294002 were purchased from Sigma Chemical, Co. Pre-miR-21 oligonucleotide, premiR negative control, PDCD4 siRNA and negative control siRNA were purchased from Ambion. Anti-PDCD4 antibody was from Epitomics, Inc, whereas, antibodies against phospho-Akt was purchased from Cell Signaling Equal numbers of cells were plated in each well of twelve-well culture plates. After the cells reached 70% to 80% confluence, a line was scratched in the middle of the well using a pipette tip to create a wound. Differential interference contrast images of the denuded area at three random fields per well were captured by a confocal microscopy just after the denudation. Cells were then treated with different reagents and incubated for Resveratrol and MicroRNA-21 another 24 h, and images were again recorded. For quantitation, the area of the closing wound is measured from the denuded area and normalized to vehicle-treated controls. To study the effects of the PDCD4 siRNA and pre-miR-21 on wound-healing of PC-3MMM2 cells, the cells were transfected with PDCD4 siRNA, premiR-21and their respective negative controls for 24 h after which the wound was created and the images were taken at time, 0 h. randomly chosen fields per treatment per insert. To study the effects of the PDCD4 siRNA and pre-miR-21, PC-3M-MM2 cells were transfected with PDCD4 siRNA, pre-miR-21and their respective negative controls for 24 h before seeding them on the top compartment. Western Blot Analysis At the end of the treatment, PC-3M-MM2 cells were washed with ice cold 1X PBS and homogenized using a sonicator in icecold lyses buffer containing 50 mM Tris HCl, 10 mM MgCl2 and 1 mM EDTA in the presence of protease inhibitors mixture and phosphatase inhibitor 1 . The wholecell lysates were then used for western blotting as described previously. Briefly, protein concentration was determined by Bradford method and equal amount of protein from each sample was mixed with solubilization buffer and heated on water bath at 95uC for 5 min. The samples were then resolved by SDS polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes, blocked in a solution containing 10X PBS, 10 mM EDTA, 20% of TritonX-100 and 5% low-fat skim milk for 1 h, and then incubated at 4uC overnight with the Modified Boyden Invasion Chamber Assay The ability of prostate cancer cells to SB-203580 site migrate through matrigelcoated membranes was measured using 24-well BD Biocoat Matrigel invasion chambers. PC3M-MM2 cells were suspended in the culture media without serum and were seeded on the top compartment of the invasion chamber followed by respective treatments. Complete media was added to the bottom chamber. At the end of 24 hours, the cell inserts were removed, and cells were carefully wiped from the top surface of the membrane with a cotton swab. The invasive cells adhering to the bottom surface of the membrane were stained with 100% methanol and 1% toluidine blue, respectively. The images were taken under a light microscope using a 20x objective. Total number of invaded cells was manually counted in four 3 Resveratrol and MicroRNA-21 primary antibody. After three washes in blocking solution, blots were incubated with horseradish peroxidase-labeled species specific IgG secondary antibody for 1 h at room temperature, washed three times with 1X TBS containing 1% Tween20. This was followed by three washes with 1X TBS, without Tween 20. The blot was treated with ECL plus reage

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