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

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

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