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Copyright by Eunyoung Park 2007 - The University of Texas at Austin

Copyright by Eunyoung Park 2007 - The University of Texas at Austin

described above.

described above. Antibody (10 μg/ml) was added to the upper portion of the chambers and 20 ng/ml of HGF was added to the lower portion of the chamber, containing medium supplemented with 10% FCS, to serve as a chemo-attractant. After 24 h, cells that had moved through the membrane were visualized by propidium iodide staining and quantitated. RNA extraction and quantitative real time RT-PCR Cells were plated at a density of 1x 10 6 cells/100 mm dish. After 24 h, cells were treated with 0 (ethanol vehicle control), 1, or 10 μM retinol. Twenty-four h later total RNA was extracted with RNAstat (Tel-Test, Inc, Friendswood, Texas). Reverse transcription was performed from 2 μg of total RNA using oligo-dT and AMV reverse transcriptase (Promega, Madison, WI) according to manufacture’s instructions. The RNA was incubated for 10 min at 70°C and subsequently subjected to reverse transcription for 15 min at 42°C, followed by heating at 95°C for 5 min. The sequences of the primers used are displayed in Table 1. The identity of the amplified PCR product was confirmed by sequence analysis. Quantitative real time-reverse transcriptase (RT)- PCR was performed with SYBR Green dye (Perkin-Elmer-Applied Biosystems, Foster City, CA) using an ABI 7900HT (Perkin-Elmer-Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Briefly, an initial denaturation step (10 min at 95°C) was followed by a two-step PCR (15 s at 95°C; 1 min at 60°C, 40 cycles). PCR reactions were performed in duplicate. Relative amounts of MMP cDNA were 47

calculated by the comparative CT method (97). CT values obtained for the different MMP were normalized to corresponding CT values of GAPDH. Zymography HCT-116 and SW620 cells were plated at a density of 2 x 10 6 cells/100 mm dish. Cells were washed with PBS twice and treated with 0 (ethanol vehicle control), 1, or 10 μM retinol in serum free media for 24 h. MMP-2 activity was detected in serum-free media concentrated using a centricon column with a 10 kDa cutoff value (Millipore, Volketswil, Switzerland). MMP-9 activity was measured in serum-free media incubated with MMP-9 antibody at a 1:500 dilution and 20 μl of protein A/G plus agarose bead/mL (#SC-2003, Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. The following day the beads were washed with 1% NP-40 three times and then once with PBS. Sample buffer (0.1 M Tris⋅HCl (pH 8.6), 10% Glycerol, 0.0025% bromophenol blue) was used to elute MMP-9 from the agarose beads prior to zymography. Zymograms were prepared as described previously (98). Equal amounts of protein were separated using a 0.1% gelatin/10% SDS-PAGE gel. After electrophoresis, the gels were washed in 2.5% Triton X-100 for 1 h at room temperature and incubated overnight with developing buffer (Invitrogen, Carlsbad, CA) at 37°C. Gels were stained with 0.25 % Coomassie Blue Dye R250 and distained with 30% methanol and 10% acetic acid. 48

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