Wells containing growth medium without cells served while the blank. (Hercules, California). Chemiluminescence reagents (Western Pico Super Transmission) were purchased from Pierce (Rockford, Il). stilbenoid (compounds 9-12) that was consequently converted to its isomer (compounds 5-8) by photo-isomerization. Namely, compound 5 was acquired by photo-isomerization of 9 in turn obtained by reaction of the diethyl (4-methoxybenzyl)-phosphonate (prepared from 4-methoxybenzylchloride) with 3,4-dimethoxybenzaldehyde. Compound 6 was prepared by photo-isomerization of 10 in turn obtained by reaction of diethyl (3,5-dimethoxybenzyl)-phosphonate (prepared from 3,5-dimethoxybenzylbromide) with 3,4-dimethoxybenzaldehyde. Compound 7 was acquired by photo-isomerization of 11 in turn obtained by reaction of the diethyl (4-methoxybenzyl)-phosphonate (prepared from 4-methoxybenzylchloride) with 3,4,5-trimethoxybenzaldehyde. Compound 8 was acquired by photo-isomerization of 12 in turn obtained by reaction of diethyl (3,5-di-methoxybenzyl)-phosphonate (prepared from 3,5-dimethoxybenzylbromide) with the 3,4,5-trimethoxybenzaldehyde. Irradiation experiments were performed using a Rayonet photochemical reactor, as previously reported (34). All photo-isomerizations were acquired with 80-82% conversion to the isomer. Each combination was resolved by silica gel PLC column chromatography using CACNA2 a gradient of EtOAc-product from the residual isomer. Spectral data of compounds 5 – 8 are in perfect agreement with those reported in the literature (35-37). Cell-based Assays Cell tradition B16 cells (F1 and F10) were cultured in 60-mm or 100-mm Falcon dishes comprising RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 1% penicillin/streptomycin and 0.1% fungizone. Cells were cultivated Galangin at 37C inside a 5% CO2 atmosphere and sub-cultured twice per week. In the same growth medium, immortalized mouse melanocytes (melan-a cells) were cultured at 37C with 10% CO2 in the presence of 200 nM tetradecanoyl phorbol acetate (TPA). Prior to the experiment, cells were plated and incubated over night. The next day, the cells were washed with PBS, and new medium was restored. Each compound was added to cells Galangin in the indicated concentration and incubated at 37C and 5% CO2 prior to the experiment. Preparation of cell lysates Cells were lysed in 0.25 mL lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100) containing phosphatase inhibitors (2.5 mM sodium pyrophosphate, 1 mM -glycerophosphate, 1 mM Na3VO4, 1 g/l leupeptin) and protease inhibitors. Cells were sonicated three times for 10 s and centrifuged at 10000 g for 10 min to remove insoluble material. The producing lysate was analyzed for protein content using the Bio-Rad reagent and bovine serum albumin as standard. Western blot analysis Cell lysates were denatured by heating in sample buffer at 95 C for 5 min. Samples were resolved by 7.5% SDS-PAGE gel and transferred to a PVDF membrane (Millipore; Billerica, MA). The membrane was incubated over night with antibodies to -tubulin. After washing the membrane with TBST (150 mM NaCl, 20 mM Tris pH 7.4, 0.1% Tween 20, v/v), the appropriate Galangin horseradish-peroxidase conjugated secondary antibody was added and the membrane was incubated at 4C for 1-2 h. After washing three times with TBST, the membrane was visualized with reagents for chemiluminescence. Motility assay Measurements were carried out having a 10-well glass slip and a pre-chilled cell sedimentation manifold (Creative Scientific Methods Inc., Phoenix, AZ). Cells (4 103/well) were pipetted into the manifold in triplicate enabling their sedimentation onto the slip as a small concentric circle. After incubation for approximately 16 h at 37 C and 5% CO2, the manifold was eliminated (t=0) permitting the cells to radiate outwardly. At this time the press in each well was replaced with media comprising 10 M of a resveratrol analogue or DMSO (0.1%, v/v). Both at t=0 and following incubation with the analogues for 18 h at 37 C, images of the cells were recorded and analyzed using Motic Image 2.0 software. The motility of each cell sample was judged from the increase in area occupied from the cells and averaging the ideals for triplicate wells. Wound healing assay Cells were plated inside a 6-well plate (Falcon) and cultivated for 24-48 hours until a dense monolayer was accomplished. Two parallel lines were drawn with coloured markers within the undersurface of the plate to identify the area to be analyzed. A scuff wound of standard width was created in this region by drawing a sterile micropipette tip across.