MDA-MB-435 (referred to as 435), a gift from Dr. Janet Price (University of Texas-M. D. Anderson Cancer Center) is a human metastatic breast carcinoma cell line . (Note: There is a debate about the origin of this cell line (melanoma or breast cancer) [25–27], however BRMS1 has been shown to suppress metastasis of both these cancers. Hence we used this cell line as it is the best model for the regulation study of OPN. BRMS1-transfected 435 (435/BRMS1) were generated by us and the culture conditions have been described previously . For all functional and biological assays, cells with >95% viability were used at 70–90% confluence. All the lines were routinely checked and found negative for Mycoplasma spp. using the TaKaRa Mycoplasma detection kit (TaKaRa Bio, Otsu, Shiga, Japan).
Plasmids and transfections
pCMV-myc-BRMS1 was constructed by cloning the BRMS1 ORF in pCMV-myc (BD-Clontech, CA, Palo Alto, USA). The human OPN promoter construct was a gift from Dr. Iizuka, Hokkaido University, Japan . PCR-generated deletions of the OPN promoter (indicated in Fig.2A) were cloned in the pGL3-basic vector (Promega, Madison, WI, USA). A 3XOPN/NF-κB construct, pGL3-3XOPN/NF-κB, was made by cloning commercially synthesized oligomers bearing the OPN/NF-κB site repeated three times in tandem into pGL3-Control vector (Promega). The NF-κB site in the OPN promoter was disrupted and replaced with a Not I site (OPN/NotI) using the oligos 5'-GATCGATCGTGCGGCCGCAAATTCTAAGGAAAAATATTTTTAATTGTAATGCTG-3' and 5'-GATCGATCGTGCGGCCGCATGTTTTTCAGCTGAATGCACAAC-3' with pGL3-OPN as a template for inside-out PCR.
The pcDNA3-FLAG-HDAC3 plasmid was a gift from Dr. Edward Seto, University of South Florida, FL, USA .
To determine OPN expression, 4 × 106 cells were seeded in 5% FBS containing medium. After 24 hours, the medium was replaced with serum-free medium and assayed 24 hours later for OPN expression. The cell-free medium was resolved using a 12.5% SDS-PAGE. Proteins were transferred to a PVDF membrane and probed with the anti-human OPN mouse monoclonal antibody  (1:1000) followed by secondary antibody conjugated to horseradish peroxidase (Amersham Biosciences, Piscataway, NJ, USA) and detected using chemiluminescence (Amersham BioSciences). OPN is seen at ~55–65 kDa. Cell lysates were prepared as described previously  and 30 μg was resolved using a 12.5% SDS-PAGE and immunoblotted with the respective primary antibody followed by detection using Supersignal West Dura (Pierce, Rockford, IL, USA). Epitope-tagged BRMS1 expression from 435/BRMS1 was detected using anti-901 epitope tag antibody, described previously by us . Densitometric analysis was performed using the digital densitometry analysis tool of AlphaEase®FC image analysis software.
COS-7 cells were co-transfected with pCMV-myc-BRMS1 and pcDNA3-FLAG or pcDNA3-FLAG-HDAC3 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) per the manufacturer's instructions. Transfected cells were lysed and immunoprecipitated with 1 μg anti-FLAG Ab (Sigma) and immunoblotted as described above. The membrane was probed with 1:500 dilution anti-myc Ab (BD Clontech, Palo Alto, CA, USA). For analysis of the acetylation status of p65, 300 μM Trichostatin A (Calbiochem, EMD Biosciences, La Jolla, CA) was added to the lysis buffer to halt HDAC activity released upon cell lysis. The cell lysate was immunoprecipitated with the anti-p65 antibody (Santa Cruz) followed by the rabbit anti-acetyl lysine antibody (1:1000) (Chemicon, Temecula, CA, USA) for immunodetection.
Luciferase reporter assays
COS-7 cells were transfected using Lipofectamine 2000 (Invitrogen) per the manufacturer's instructions. Total protein was harvested (Luciferase assay kit, Promega) and the luciferase activity measured using a Turner 20/20 luminometer (Turner Biosystems, Sunnyvale, CA, USA). The luciferase reading was normalized to the total protein concentration. Data is expressed as Relative luciferase activity, where control is 100%.
Electrophoretic mobility shift assay (EMSA)
Nuclear proteins were isolated from MDA-MB-435 cells grown until approximately 85% confluent in a 100 mm dish using the protocol of Zayzafoon, et al . Binding reactions containing either 10 μg nuclear extract or 100 ng or 66 ng, respectively, of recombinant p65 (Active Motif, Carlsbad CA) or p50 (Promega), 1 μg poly (dI-dC), and 20 fmol biotinylated oligonucleotide probe in buffer (10 mM Tris, 50 mM KCl, 1 mM DTT, 5 mM MgCl2) were incubated at room temperature for 30 minutes.
The oligonucleotide probes correspond to the OPN/NF-κB site (5'-GAATTTCATGGGGAAGTCCAAATTCTAAG) or Mut OPN/NF-κB (5'-GAATTTCATGC GGACT TCG AAATTCTAAG).
A consensus NF-κB probe (5'-AGTTGAGGGGACTTTCCCAGGC) served as a specific inhibitor. For antibody supershift, the samples were preincubated with 2 μg of anti-p65 antibody (Santa Cruz). Gel electrophoresis, blotting and development followed the manufacturer's protocol of the Chemiluminescent LightShift Assay Kit (Pierce). Four pmol of the consensus NF-κB probe (5'-AGTTGAGGGGACTTTCCCAGGC) served as a specific cold competitor.
Cells (435) were utilized for chromatin immunoprecipitation using the ChIP-IT kit (Active Motif) as directed by the manufacturer using p65 or p50 antibody. Parallel controls for each experiment included samples with no chromatin, no antibody, normal rabbit IgG (Santa Cruz Biotech), and the kit-provided positive (RNA polymerase II) and negative control antibodies. After elution and purification, the recovered immunoprecipitated DNA samples were used for PCR (Platinum Taq polymerase; Invitrogen) using primers [5'-CAGTTGCAGCCTTCTCAGC-3' (forward) and 5'-CCTTTGTTCCACAGGAGACC-3' (reverse)] to amplify a 201 bp segment of the OPN promoter containing the NF-κB site. PCR products were analyzed by agarose gel electrophoresis.
The specificity of the pull-down was confirmed by amplifying a region 1575 bp upstream from the PCR product containing the NF-kB site tested. The primers used were 5'-TTCCCCCTACCAAATGTTCA-3' and 5'-TGCTGCAAAAGTAATTGTGGTT-3'. The PCR generates a 151 bp product. This segment lacks a predicted NF-κB site .
Statistical analysis was done using the unpaired one-tailed Student's t-test (Graphpad Prism, San Diego, CA, USA).