Figure 2. Noggin silencing has no effect on cell proliferation and affects only minimally the expression of osteotropic factors, in vitro. A. In vitro proliferation of mock and Nog-KD clones was measured by BrdU incorporation for 4 days and compared to PC-3/ Fluc cells. Average values of 3 independent experiments performed in quadruplicate wells (+/2 SD). B. Expression of Dkk-1, PTHrP, RANKL, CSF-1 and IL-8 mRNA. mRNA expression levels (+/2 SD) are quantified by real-time RT-PCR and normalized to b-actin as endogenous control. The mRNA expression level in PC-3/Fluc cells is set as 100%; the mean of 2 to 3 independent experiments is shown. ***P,0.001, mock and Nog- KD 17 clones versus PC-3/Fluc and Nog-KD 14 clones. doi:10.1371/journal.pone.0016078.g002 tibiae xenografted with Nog-KD clones the residual bone interposed between areas of osteolysis showed some evidence of a more radio-dense aspect (Figure S2). In the first experiment, both the radiography and the m-CT reconstruction performed at the conclusion of the experiment (day 25) indicated further development of predominantly osteolytic lesions in PC-3/Fluc- and mock clone-bearing tibiae (Figure 3A and B). In contrast, in the tibiae xenografted with the Nog-KD clones an evident increase in radiodensity of the residual bone and, in addition, development of bone spiculae projecting outside the cortex was invariably observed (Figure 3A and B). In the second experiment the observation period was prolonged in order to allow progression of the bone response. The animals inoculated with the PC-3/Fluc clone needed to be sacrificed at day 23 due to severe osteolysis of the xenografted bones and swelling of the limb, and to signs of pain and distress. In contrast, bones xenografted with the Nog-KD clones showed a more preserved structural integrity and a less pronounced swelling of the limb, with no signs of pain and distress. Accordingly, the animals xenografted with Nog-KD 17 and Nog- KD 14 clones were kept for a period of 30 and 33 days, respectively. Consistently with the evolution of the bone response observed in the first experiment, tibiae xenografted with Nog-KD clones showed enhanced radiodensity of the residual bone and pronounced bone spiculae projecting outside the cortex (Figure S3A and B). Role of Noggin in Osteolytic Bone Metastasis Figure 3. Noggin silencing promotes increase in radiodensity and partial bone repair in advanced osteolytic lesions. A. Representative images of radiography and B. 3-D reconstruction (m-CT) of tibiae (top) and of 1 mm thick cross sections (bottom) of sham- operated and cancer cell-xenografted tibiae at day 25 after intra- osseous inoculation. doi:10.1371/journal.pone.0016078.g003 Modification of bone structural parameters induced in vivo by Noggin-silenced PC-3/Fluc cells Quantitative m-CT analysis confirmed that the tibiae xenograft- ed with the PC-3/Fluc and mock clones had significantly lower BV/TV ratio than the sham-operated ones. On the contrary, the tibiae inoculated with the Nog-KD clones had higher BV/TV ratio than with PC-3/Fluc and mock clones and were not different from the sham-operated ones (Figure 4A). Total bone mineral content measured by pQCT was lower in the tibiae xenografted with the PC-3/Fluc and mock clones, as compared with the sham-operated ones, but significance was reached only for mock 5-xenografted bones. In contrast total bone mineral content in the Nog-KD clones-xenografted tibiae was higher than in PC-3/Fluc and mock clones-xenografted ones, but reached significance only when compared to the mock clones (Figure 4B). Histomorphometry In the tibiae xenografted with PC-3/Fluc and mock clones there was a significant increase in osteoclast number and percentage of surface covered by osteoclasts, as compared to the sham-operated tibiae (Figure 5A and C). This was accompanied by a significant decrease in osteoblast number and percentage of surface covered by active osteoblasts (Figure 5B and D). Tibiae xenografted with the Nog-KD clones showed signifi- cantly lower number of osteoclasts and percentage of surface covered by osteoclasts than those xenografted with PC-3/Fluc and mock clones (Figure 5A and C). Conversely, they showed a significantly higher number of active osteoblasts and percentage of surface covered by osteoblasts than those inoculated with PC-3/ Fluc and mock clones. In Nog-KD clones-xenografted tibiae the number of osteoblasts was higher than in the sham-operated tibiae, but this increase was significant only for Nog-KD 14 (Figure 5B and D). Despite the fact that the mRNA expression of IL-8, known to stimulate osteoclast recruitment, was significantly up-regulated in vitro in the Nog-KD 17 clone, as compared to the Nog-KD 14 clone, there was no obvious difference in the osteoclast number between the bones xenografted with the two PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e16078