Tumor cell cycle arrest induced by shear stress: Roles of integrins and Smad -- Chang et al. 105 (10): 3927 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Chang et al. 10.1073/pnas.0712353105.

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Flow Apparatus.
The glass slide with cultured tumor cells was mounted in a parallel-plate flow chamber characterized and described in detail (1). The chamber was connected to a perfusion loop system, kept in a constant temperature-controlled enclosure, and maintained at pH 7.4 by continuous gassing with a humidified mixture of 5% CO₂ in air. The fluid shear stress (t) generated on the cells by flow was estimated to be 12 dynes/cm², unless otherwise noted, using the formula t= 6mQ/wh², where m is the dynamic viscosity of the perfusate, Q is the flow rate, and h and w are the channel height and width, respectively. Primer Designs.
OCN (sense: 5'-TGAGAGCCCTCACACTCCTC-3'; antisense: 5'-A CCTTTGCTGGACTCTGCAC-3'; product length, 98 bp), ALP (sense: 5'-CAACCCTGGGG AGGAGAC-3'; antisense: 5'-GCATTGGTGTTGTACGTCTTG-3'; product length, 78 bp), b-actin (sense: 5'-AAATCGTCCGTGACATCAAG-3'; antisense: 5'-GGAAGGAAGGCTGG AAGA GA-3'; product length, 180 bp). Flow-Cytometric Analysis.
The cells were harvested in PBS containing 2 mM EDTA, washed once with PBS, and fixed for 30 min in cold ethanol (70%). Fixed cells were washed and permeabilized with 0.1% Triton X-100 in PBS. They were then stained with 50 mg/ml propidium iodide (Roche) and 1 mg/ml RNase A for 30 min. Stained cells were analyzed with a fluorescence-activated cell sorter (FACS) Calibur (Becton-Dickinson), and the data were analyzed by using a mod-fit cell cycle analysis program. ALP-Specific Activity Assay.
Cell extract was prepared with 0.1% Triton X-100 after shear stress experiments. Cellular ALP activity was assayed at the end of the incubation with 10 mM p-nitrophenyl phosphate in 0.15 M sodium carbonate buffer (pH 10.3) and 1 mM MgCl₂ as described (2) and was normalized against cellular protein determined by the Bio-Rad protein assay. RNA Isolation and Quantitative Real-Time PCR.
The total RNA was isolated by the guanidium isothiocyanate/phenochloroform method and converted to cDNA as described (3). The cDNA was amplified through PCR on a LightCycler (Roche Diagnosticss) by using LightCycler FastStart DNA MasterPlus SYBR green I (Roche Diagnostics) with 0.5 mM primers of differentiation marker genes (i.e., OCN and ALP) or apoptosis index genes (i.e., Bcl-2 and Bax). PCR was performed in triplicate at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 5 sec, extension at 72°C for 8 sec, and single signal acquisition for 10 sec. b-Actin gene expression was used as an internal control. The PCR conditions were optimized to obtain a PCR product with a single peak on melting curve analysis on the LightCycler. Raw data collected from the LightCycler were analyzed by using LightCycler Software Version 3.5 (Roche Diagnostics). The gene expression levels were normalized with b-actin gene expression levels in the same sample. Western Blot Analysis.
The cells were collected by scraping and lysed with a buffer containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and a protease inhibitor mixture (PMSF, aprotinin, and sodium orthovanadate). The total cell lysate (100 mg of protein) was separated by SDS/PAGE (12% running, 4% stacking) and transferred onto a polyvinylidene fluoride membrane (Immobilon P, 0.45-mm pore size). The membrane was then incubated with the designated antibodies. Immunodetection was performed by using the Western-Light chemiluminescent detection system (Applied Biosystems). Immunoprecipitation.
The cells were scraped and lysed with a buffer containing 25 mM Hepes (pH 7.4), 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 0.125 M NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF, 10 mg/ml leupeptin, and 2 mM BGP. The cells were disrupted on ice by repeated aspiration through a 21-gauge needle. The same amount of protein from each sample was incubated with a designated antibody for 2 h at 4°C with gentle shaking. The immune complex was then incubated with protein A/G plus agarose for 1 h and collected by centrifugation. This agarose-bound immunoprecipitates were washed and incubated with boiling sample buffer containing 62 mM Tris·HCl (pH 6.7), 1.25% (wt/vol) SDS, 10% (vol/vol) glycerol, 3.75% (vol/vol) mercaptoethanol, and 0.05% (wt/vol) bromophenol blue. The samples were then subjected to SDS/PAGE and Western blotting. Treatments with RGD Peptides and mAbs.
The type I collagen contains the integrin-recognition tripeptide RGD (Arg-Gly-Asp) sequence (4). To block specific integrin-collagen interactions, the cells were preincubated with the tetrapeptide RGDS (Arg-Gly-Asp-Ser; 500 mg/ml), which blocks cell adhesion through the RGD sequence on ECM proteins, or the antibodies (10 mg/ml) against a_vb3 and b₁ integrins for 2 h before seeding onto the glass slides precoated with type I collagen and during the application of fluid flow. Reporter Gene Construct, DNA Plasmids, siRNA, Transfection, and Luciferase Assay.
The OCN promoter construct (OCN-Luc) contains 800 bp of OCN 5'-flanking DNA linked to the firefly luciferase reporter gene of plasmid pGL3 (Promega) (5). This fragment of OCN promoter contains Runx2-binding sites. DNA plasmids at a concentration of 1 mg/ml were transfected into MG63 cells at 60% confluence by using Lipofectamine (GIBCO). The pSV-b-galactosidase plasmid was cotransfected to normalize the transfection efficiency. The cells were kept as static controls or subjected to shear stress experiments 48 h after transfection. For siRNA transfection, MG63 cells at 70-80% confluence were transfected with the designated siRNA at various concentrations (5, 15, 30, and 40 nM) by using RNAiMAX transfection kit (Invitrogen). EMSA.
The cells were collected by scraping in PBS. After centrifugation of the cell suspension at 1,500 ´ g, the cell pellets were resuspended in cold buffer A (containing 10 mmol/liter KCl, 0.1 mmol/liter EDTA, 1 mmol/liter DTT, and 1 mmol/liter phenyl methylsulfonyl fluoride) for 15 min. The cells were lysed by adding 10% Nonidet P-40 and then centrifuged at 50,000 ´ g to obtain pellets of nuclei. The nuclear pellets were resuspended in cold buffer B (containing 20 mmol/liter Hepes, 1 mmol/liter EDTA, 1 mmol/liter DTT, 1 mmol/liter PMSF, and 400 mmol/liter NaCl), vigorously agitated, and then centrifuged. The supernatant containing the nuclear proteins was used for the EMSA or stored at -70°C until used. A sequence of 20-bp oligonucleotides containing the human OCN Runx2 site was synthesized (5'-CGTATTAACCACAATACTCG-3' and 5'-AATTGGTGTTATGAGCATGC-3') (6). The oligonucleotides were end-labeled with [g-³²P]ATP. The extracted nuclear proteins (10 mg) were incubated with 0.1 ng ³²P-labeled DNA for 15 min at room temperature in 25 ml of binding buffer containing 1 mg of poly(dI-dC). In the antibody supershift assay, an antibody against Runx2 (1 mg each; Cell Signaling Technology) was incubated with the mixture for 10 min at room temperature, followed by the addition of the labeled probe. The mixtures were electrophoresed on 5% nondenaturing polyacrylamide gels. The gels were dried and imaged by autoradiography.

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