Cell Culture and treatments
Normal bronchial and lung fibroblast (NL-20; WI-38, and IMR-90) and lung cancer cells (small cell lung cancer and non-small cell lung cancer cells: H720, and H1299) were cultured in growth medium as specified by American Type Culture Collection. The construction and procedure for wild type Runx2 or DNA binding mutant expressing adenovirus and lentivral transduction in normal and cancer cells are reported previously [24, 45].
Animals were maintained at the University of Massachusetts Medical School following procedures approved by the Institutional Animal Care and Use Committee (IACUC). Primary calvarial cells from Runx2 -/- mice were isolated as previously described .
Normal bronchial NL-20 or lung cancer H-1299 cells were transduced with lentivirus expressing shRNA-Runx2 target sequence 5’-AAGGTTCAACGATCTGAGATTTG-3’ sequence in pLVTHM vector under H1 promoter . Runx2 knockdown efficiency was confirmed by western blot and real time RT-PCR analysis.
Western blot analysis
Runx2 protein levels in normal bronchial, fibroblast and lung cancer whole cell lysates or nuclear lysates were detected by western blot analysis as described previously . Runx2 antibody (MBL Inc., Woburn, MA) or Suv39h1 (C-14, Santa Cruz Biotechnology Inc. Santa Cruz, CA) and HRP-conjugated secondary antibodies (Santa Cruz) were used to detect immunoreactive proteins.
Chromatin immunoprecipitation (ChIP) was performed as previously described . Protein-DNA complexes were immunoprecipitated using Runx2 antibody (M-70, Santa Cruz Biotechnology Inc.), Suv39h1 (Santa Cruz Biotechnology Inc.) and histone H3K9 (Abcam, Cambridge, MA) or IgG as a control. Purified DNA was subjected to real time PCR amplification with SYBR Green chemistry on an ABI real time thermocycler. BMP-3B promoter fragment containing Runx elements were amplified using forward primer: 5’ ACT TTG ATG AAT CCG CAA CC-3’ and reverse primer: 5’ TTG TCT TGC CTC TAGCAG GAT-3’.
Real time RT-PCR analysis
The mRNA levels of Runx2, BMP-3B, GAPDH and 28S in primary osteoblasts, normal lung fibroblast, bronchial and lung cancer cells were analyzed after adenovirus- or lentiviral-mediated Runx2 transduction. Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s specification. Purified RNA was oligo dT primed and cDNA synthesized at 42°C with SuperScript II RNA polymerase (Invitrogen). For PCR amplification, the following primers were used: Runx2, forward primer: 5’- CGG CCC TCC CTG AAC TCT -3’, reverse primer: 5’- TGC CTG CCT GGG GTC TGT A -3’, GAPDH, forward primer: 5’- ATG TTC GTC ATG GGT GTG AA -3’, reverse primer: 5’- TGT GGT CAT GAG TCC TTC CA -3’. BMP-3B, forward primer: 5’-AGC TGC TGG ACT TTG ACG AG-3’, reverse primer: 5’-TGA CAA TGC TCT GGA TGG TG-3’. 28S, forward primer: 5’- GAA CTT TGA AGG CCG AAG TG-3’, reverse primer: 5’-ATC TGA ACC CGA CTC CCT TT-3’. The gene expression levels were quantified by ΔΔCt method of relative quantification by normalizing the data with internal control and expressed relative to appropriate control cell line as indicated in the figure legends.
Wound healing assay
H1299 cells stably expressing Runx2 or empty vector treated control cells were cultured in triplicates in a 6 well dish with reduced serum conditions (0.2% serum) for overnight. The next day, a scratch was made approximately in the center of the monolayer by a sterile 200μl pipette tip. The detached cells and debris were washed with serum-free RPMI medium. The cells were then supplemented with or without TGF-β (5 ng/ml) containing RPMI medium. Five random images per well were photographed at 0h, 6h, 24h and 48h. The distance of the scratch was measured in ImageJ software at every time point. The wound distance at 0h was assigned as 100% and used to calculate percent wound closure at other time points. The P-value for statistical significance was calculated by unpaired T-test.
Cell proliferation assay
H1299 cells stably expressing Runx2 or empty vector treated control were counted in a hemacytometer and 1000 cells per well were seeded in a 96-well plate. To determine the changes in proliferation, the cells were indirectly assayed for cell number via a tetrazolium compound-based colorimetric assay (CellTiter 96 kit from Promega Inc. Madison, WI) according to manufacturer’s instructions. At indicated time points over a period of four days, the cell titer reagent (20μl/well) was added to the plate and incubated at 37°C for 1 hour. The quantity of color developed (formazan product from the tetrazolium compound) was measured by reading absorbance at 490 nm in a spectrophotometer (Fluostar Optima BMG Labtech Inc. Cary, NC).
Lung cancer H1299-WT-Runx2 or -shRunx2 cells were washed with ice-cold PBS and harvested in lysis buffer [50 mM NaCl, 50 mM Tris (pH 8.0), 1% NP- 40, 25 mM MG132, and 1× protease inhibitor mixture (Roche, Indianapolis, IN)]. Lysates were incubated overnight at 4°C with 3 μg of rabbit antibodies against Runx2 antibody (M-70, Santa Cruz Biotechnology Inc.), and Suv39h1 (Santa Cruz Biotechnologies). Lysates were then incubated with protein A/G beads for 2 h, followed by four washes with wash buffer [50 mM NaCl, 20 mM Tris (pH 8.3), 0.5% Na-deoxycholate, 0.5% Nonidet P-40, 2 mM EDTA, 25 mM MG132, and 1× protease inhibitor mixture]. The total cell lysates and immunoprecipitated protein complexes were resolved by 8% SDS/PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Billerica, MA). Blots were incubated with Runx2 (M-70) or Suv39h1 (C-14) antibodies. Membranes were then incubated with HRP-conjugated secondary antibodies against rabbit or mouse (1:2,000). Proteins bands were visualized with a chemiluminescence detection kit (Perkin–Elmer Life Sciences,Waltham, MA).
WI-38 and H1299 cells grown on gelatin coated cover slips were processed for immunofluorescence microscopy as previously described  using rabbit polyclonal Runx2 antibody (Santa Cruz Biotechnology, Inc.), followed by incubation with Alexa 488 conjugated secondary antibody (Molecular Probes, Eugene, OR). All images were taken using a Zeiss Axioplan digital microscope and analyzed using Metamorph software (Universal Imaging, Downingtown, PA).