Cell culture conditions and reagents
HCT-116 colonic adenocarcinoma cells were purchased from American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 (Welgene bioscience, Daeu, Korea) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Sigma Chemical Company, St. Louis, MO), 50 unit/ml penicillin (Sigma Chemical Company), and 50 µg/ml streptomycin (Sigma). During incubation with chemicals, cells were cultured in serum-free RPMI 1640 media. All the chemicals were purchased from Sigma.
Cellular viability assay
Colorimetric analysis of cell growth was performed with Thiazolyl Blue Tetrazolium Bromide (MTT, Fluka, USA) assay. Cells (5 × 104/well) were cultured in 96-well plate for each time and the MTT (20 µl from 5 mg/ml stock solution) was treated onto cells for 2 h. Supernatant was removed and dissolved with 200 µl dimethyl sulfoxide (DMSO). Optical density at 560 nm was measured and was the background OD at 670 nm was subtracted from OD at 560 nm. Corrected optical density was directly correlated with cell quantity.
Western immunoblot analysis
Levels of protein expression were compared using Western immunoblot analysis using rabbit polyclonal anti-human Actin antibody (Santacruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-human TSP-1 antibody (Ab-11, NeoMarkers, Fremont, CA), and rabbit polyclonal anti-p-p44/42 MAP kinase antibody (anti-ERK1/2 antibody, Cell signaling technology, Beverly, MA). Cells were washed with ice-cold phosphate buffer, lysed in boiling lysis buffer [1% (w/v) SDS, 1.0 mM sodium ortho-vanadate, and 10 mM Tris, pH 7.4], and sonicated for 5 s. Lysates containing proteins were quantified using BCA protein assay kit (Pierce, Rockford, IL). Fifty micrograms of protein was separated by Bio-Rad gel mini-electrophoresis. Proteins were transferred onto PVDF membrane (Amersham Pharmacia Biotech, Piscataway, NJ) and the blots were blocked for 1 h with 5% skim milk in Tris-buffered saline plus Tween 0.05% (TBST) and probed with each antibody for 2 h at room temperature or overnight at 4 °C. After three times washing with TBST, blots were incubated with horseradish-conjugated secondary antibody for 1 h and washed with TBST three times. Protein was detected by Enhanced Chemiluminescence (ECL) substrate (Amersham Pharmacia Biotech, Piscataway, NJ).
Traditional reverse transcription-polymerase chain reaction (RT-PCR)
RNA was extracted with Trizol reagent (Invitrogen) according to the manufacturer’s instructions. RNA (100 ng) from each sample was transcribed to cDNA by Prime RT-Premix RNase H-Reverse-transcriptase (Genet Bio, Nonsan, Korea). The amplification was performed with HS Taq DNA Polymerase (Genet Bio, Nonsan, Korea) in Mycycler Thermal Cycler (Bio-Rad Laboratories Inc., Hercules, CA) using the following parameters: denaturation at 94 °C for 2 min and 25 cycles of reactions of denaturation at 98 °C for 10 s, annealing at 59 °C for 30 s, and elongation at 72 °C for 45 s. An aliquot of each PCR product was subjected to 1.2% (w/v) agarose gel electrophoresis and visualized by staining with ethidium bromide. The 5′ forward and 3′ reverse-complement PCR primers for amplification of each gene were as follow: human thrombospondin-1 (5′-AGAATGCTGTCCTCGCTGTT-3′ and 5′-TTTCTTGCAGGCTTTGGTCT), and human GAPDH (5′-TCAACGGATTTGGTCGTATT-3′ and 5′-CTGTGGTCATGAGTCCTTCC-3′).
Construction of plasmids
Human TSP-1 promoter (from −954 to 147) was cloned into pGL3 basic vector that were generated. After PCR of promoter region with PfuTurbo DNA polymerase (Stratagene, La Jolla, CA) from human genomic DNA, the fragment was cloned into the TA vector (Invitrogen, Carlsbad, CA), sequenced, and further cloned into the pGLBasic3 vector. To inhibit ERK1/2 activity, MEK1 dominant negative plasmid was used. pMEV (control vector) and pMEV-MEK1-DN (dominant negative) were provided from Biomyx Technology (San Diego, CA). The activated form of RhoA expression plasmid (RhoAV; Q63V substitution) and dominant negative RhoA plasmid (RhoAN; T19N substitution) was provided from Upstate Biotech (Lake Plasid, NY).
Cells were transfected with mixture of plasmids using Trans-LT1 transfection reagent (Mirus, Madison, WI) according to the manufacturer’s protocol. For transfection of the luciferase reporter gene, a mixture of 1.5 µg firefly luciferase reporter and 0.15 µg renilla luciferase, pRL-null vector (Promega, Madison, WI) per 4.5 µl of Trans-LT1 reagent was applied for a 6 well culture plate. For the luciferase assay, at 18 h after transfection, cells were exposed to chemicals for the next 24 h and lysed for dual-luciferase reporter assay system (Promega, Madison, WI). All transfection efficiency was maintained at around 50 to 60%, which was confirmed with pMX-enhanced GFP vector. To create the stable cell lines, cells were transfected with single plasmid (1.5 µg DNA per 35 mm dish cells) using Trans-LT1 reagent. After 48 h, the cells were subjected to selection for stable integrants by exposure to 400 µg/ml G418 (Invitrogen, Carlsbad. CA) in complete medium containing 10% fetal bovine serum. Selection was continued until monolayer colonies formed. The transfectants were then maintained in medium supplemented with 10% fetal bovine serum and 200 µg/ml G418.
Cells were washed with cold PBS, lysed with passive lysis buffer (Promega, Madison, WI) and then centrifuged at 12,000 g for 4 min. The supernatant was collected isolated and stored at −80 °C until assessment of luciferase activity. Luciferase activity was measured with a dual-mode luminometer (Model TD-20/20, Turner Designs Co., Sunnyvale, CA) after briefly mixing the supernatant (10 µl) with 50 µl firefly luciferase assay substrate solution, followed with 50 µl stopping renilla luciferase assay solution (Promega, Madison, WI). The firefly luciferase activity was normalized against renilla luciferase activity using the following formula: firefly luciferase activity/renilla luciferase activity.
Data were analyzed using SigmaStat for Windows (Jandel Scientific, San Rafael, CA). For comparison of two groups of data, Student’s t test was performed. For comparison of multiple groups, data were subjected to ANOVA and pairwise comparisons made by the Student–Newman–Keuls (SNK) method. Data not meeting normality assumption were subjected to Kruskal–Wallace ANOVA on ranks and then pairwise comparisons made by the SNK method.