Osteoblast adhesion to CTGF is via integrin receptors and through fourth domain of CTGF.

<p><b>(A)</b> Adhesion assay to analyze osteoblast adhesion to various concentrations of recombinant CTGF, used to coat the wells. The number of cells adhered to CTGF is compared to cell adhesion to 1% BSA (negative control). <b>(B)</b> Adhesion assay comparing osteoblast adhesion to 2 μg/ml CTGF, 2 μg/ml fibronectin, or 1% BSA (negative control) coated wells. <b>(C)</b> Adhesion assay comparing osteoblast adhesion to 2 μg/ml of full-length CTGF, third domain of CTGF, fourth domain of CTGF, or 1% BSA (negative control) coated wells. <b>(D)</b> Adhesion assay analyzing effect of osteoblast treatment with fourth domain of CTGF prior to culture on full-length CTGF coated wells. <b>(E)</b> Adhesion assay analyzing effect of divalent cation chelation by EDTA on osteoblast adhesion to full-length CTGF, and reversing this effect by adding excessive amount of divalent cations to the culture. <b>(F)</b> Adhesion assay studying the effect of blocking heparin binding site of CTGF molecule via adding heparin to osteoblast cell suspension prior to culture on CTGF coated wells. n = 6, *p<0.05; **p<0.01; ***p<0.001. Adhesion assays were repeated three times with similar results.</p

Osteoblast adhesion to CTGF matrix enhances osteoblast maturation and matrix mineralization.

<p><b>(A)</b> Alkaline Phosphatase (ALP) staining of osteoblasts cultured on 2 μg/ml CTGF or 1% BSA coated plates for 14 days. Scale bar = 2 mm. <b>(B)</b> ALP activity quantified at day 14 of culture and normalized to total protein content; n = 9 wells. <b>(C)</b> Alizarin red staining of osteoblasts cultured on 2 μg/ml CTGF or 1% BSA coated plates for 35 days. Same magnification as in A. <b>(D)</b> Number of nodules formed after 35 days of culture; n = 9 wells. <b>(E)</b> Area of nodules measured by ImageJ software. **p<0.01; ***p<0.001. Experiments were repeated three times with similar results.</p

Osteoblast adhesion to CTGF matrix induces Rac activation and cell spreading.

<p><b>(A)</b> Immunofluorescence staining of F-actin in osteoblasts cultured on 1% BSA, 2 μg/ml of CTGF or fibronectin coated slides for 8 hours at 37°C. Scale bar = 50 μm. <b>(B)</b> Cell spreading area of osteoblasts cultured on BSA, CTGF or fibronectin for 8 hours and stained for actin were measured by ImageJ; n = 50. **p<0.01; ***p<0.001. <b>(C)</b> Western blot analysis of active Rac1, total Rac1 and actin to study Rac1 activation levels. Osteoblasts were cultured for 2 hours on uncoated plates or plates coated with BSA, CTGF or fibronectin. Abbreviations: fibronectin (FN) and negative control (Cont). Experiments were repeated three times with similar results.</p

Inhibition of ERK prevents osteogenic differentiation in cells cultured on CTGF matrix.

<p>Alkaline Phosphatase (ALP) staining of osteoblasts cultured on 1% BSA <b>(A)</b> or 2 μg/ml CTGF coated plates in the absence of ERK inhibitor <b>(B)</b> and in the presence of ERK inhibitor <b>(C)</b> for 14 days. Scale bar = 2 mm. <b>(D)</b> ALP activity of osteoblasts cultured on BSA or CTGF coated plates, quantified at day 14 of culture and normalized to total protein content; n = 9 wells.</p

Osteoblast adhesion to CTGF is mediated by α_vβ₁ integrin receptor.

<p><b>(A)</b> Adhesion assay of osteoblasts treated with different blocking integrin antibodies prior to culture on CTGF or BSA (negative control) coated wells. The number of adhered cells in each treatment group was compared to the adhesion level of untreated cells. Adhesion to CTGF matrix after treatment with β₃ or β₅ integrin antibodies was not significantly different from untreated cells. <b>(B)</b> Western blot analyzing CTGF levels following immunoprecipitation of different integrin heterodimers from osteoblast cell lysates and control blot analyzing β₁ integrin levels to confirm that comparable amounts of integrin heterodimers were pulled down during the immunoprecipitation using their respective antibodies. IgG was used as negative control for integrin antibodies. <b>(C)</b> Immunofluorescence staining of osteoblasts cultured on CTGF coated slides for 24 hours at 37°C. Cells stained for α_vβ₁ (green), F-actin (red in left panel), or vinculin (red in right panel). Lower panel: Scale bar = 50 μm. Upper panel: Scale bar = 10 μm. n = 6, *p<0.05; **p<0.01; ***p<0.001. Experiments were repeated three times with similar results.</p

Osteoblast adhesion to CTGF induces Runx2 transcriptional activation of osteogenic markers.

<p><b>(A)</b> ChIP assay performed on osteoblasts cultured on CTGF coated, BSA coated or uncoated plates for 7 days while treated with osteogenic medium. Runx2 antibody or acetyl-Histone H4 antibody (positive control) used for chromatin immunoprecipitation. Quantitative PCR was performed using osteocalcin gene promoter primers. n = 3, ***p<0.001. ChIP assay repeated three times with similar results. <b>(B)</b> Quantitative PCR performed on osteoblasts cultured on CTGF or BSA coated plates for 7 days while treated with osteogenic media. OCN = osteocalcin, ALP = alkaline phosphatase. n = 3, **p<0.01; ***p<0.001. Experiments repeated three times with similar results.</p

Ets-1 synergizes with TGF-β1 for CCN2 promoter induction in osteoblasts.

<p>(<b>A</b>) Osteoblasts were plated in 96 well tissue culture plates and transfected with either 0.4 µg of an empty vector control (−) or the Ets-1 expression construct (+). All samples were co-transfected with 0.4 µg of our previously described CCN2 promoter luciferase reporter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035258#pone.0035258-Arnott2" target="_blank">[22]</a> and 0.2 µg of a renilla luciferase expression vector as an internal control. The cells were serum starved for 24 hrs and then treated with TGF-β1 (5 ng/ml) (+) or mock treated (−) with TGF-β1 diluent for 24 hrs. Luciferase activity was then assessed and expressed as a ratio of firefly/renilla luciferase (+SEM, n = 6). A = p<0.05 compared to +TGF-β1 only or +Ets-1 only. (<b>B</b>) Osteoblasts were plated in 96 well tissue culture plates and transfected with either 100 nM of Ets-1 siRNA (Ets-1) or control siRNA (C) for 48 hrs. All samples were co-transfected with 0.4 µg of our previously described CCN2 promoter luciferase reporter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035258#pone.0035258-Arnott2" target="_blank">[22]</a> and 0.2 µg of a renilla luciferase expression vector as an internal control. The cells were serum starved for 24 hrs and then treated with 5 ng/ml of TGF-β1 (+) or mock treated (−) with TGF-β1 diluent for 24 hrs. Luciferase activity was then assessed and expressed as a ratio of firefly/renilla luciferase (+SEM, n = 6). Star symbol indicates p<0.05 compared to control siRNA.</p

Ets-1 binds to EBE sites in the CCN2 promoter in osteoblasts.

<p>Electro-mobility shift assays (EMSA) from nuclear lysates were generated from osteoblasts that were treated with TGF-β1 (5 ng/ml) for 2 hrs. (<b>A</b>) Nuclear protein binding to the wild type E-E-E (lanes 1–6) (this probe contains EBE # 6-8; for probe design see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035258#pone-0035258-g006" target="_blank">Figure 6</a>) probe was assessed using 5 µg of nuclear lysates for each reaction. In some reactions, Ets-1 antibody was added at increasing concentrations (1 ug of antibody in lane 4; Two micrograms of antibody in lane 5) to test for Ets-1/probe interaction. Control antibody (2 ug) was also used (lane 6; C). In some cases, probe only (lane 1) or a molar excess of unlabeled probe (lane 3) was also used to demonstrate specificity. (<b>B</b>) Nuclear protein binding to the wild type S-E-T (lanes 7–14) (this probe contains EBE#5; for probe design see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035258#pone-0035258-g006" target="_blank">Figure 6</a>) probe was assessed using 5 µg of nuclear lysates for each reaction. In some reactions, Ets-1 antibody was added at increasing concentrations (1 ug of antibody in lane 10; 2 ug of antibody in lane 11) to test for Ets-1 protein/probe interaction or Smad 3 antibody (1 ug of antibody in lane 12; 2 ug of antibody in lane 13) to test for Smad 3 protein/probe interaction. Control antibody (2 ug) was also used (lane 14). In some cases, probe only (lane 7) or a molar excess of unlabeled probe (lane 9) was also used to demonstrate specificity.</p

EBE sites are required for CCN2 promoter activation by TGF-β1 in osteoblasts.

<p>Osteoblasts were plated in 96-well tissue culture plates and transfected with 0.4 µg of either EBE mutation construct 1–8, pGL3-Basic (negative control) or W787 (positive control) and all were co-transfected with 0.2 µg of a renilla luciferase expression vector (internal control) for 24 hrs. The cells were serum starved for 24 hrs and then treated with TGF-β1 (5 ng/ml) for 24 hrs. Luciferase activity was assessed, and expressed as a % of activity obtained using the full length W787 construct. (+SEM, n = 6). A = p<0.05 compared to W787.</p

Mutation of EBE sites prevents protein complex binding.

<p>(<b>A</b>) Electro-mobility shift assays (EMSA) probes were created that were homologues to the CCN2 promoter and contained either mutated or unmutated EBE sites (#5-8) as indicated. Each probe was dsDNA and 5′ biotinylated. (<b>B</b>) Electro-mobility shift assays (EMSA) from nuclear lysates were generated from osteoblasts that were treated with TGF-β1 (5 ng/ml) for 2 hrs. Nuclear protein binding to the wild type and mutated EBE sites in the CTGF promoter was assessed using 5 µg of nuclear lysates. The lane number above each well corresponds to the probe used for that reaction. The experiment was repeated four times with similar results.</p