DEV025825 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Reduced osteoclastogenesis in primary osteoblasts from Bmpr1a cKO mice. (A) Quantitative RT-PCR for the RANKL-OPG pathway and Sost using primary osteoblasts. Expression of Bmpr1a and Rankl was reduced 70% and 40%, respectively, whereas Opg increased 2.5-fold in cKO osteoblasts (CRE) compared with control (Mock), resulting in reduced relative ratio of Rankl to Opg by 80% (right panel). Scale bars: 20 µm. (B) Osteoclast formation assay in vitro using primary monocytic cells (upper panel) or bone marrow cells (lower panel). Analysis of monocytic cells showed that osteoclast number was unchanged between wild type and cKO. Analysis of bone marrow cells showed that osteoclast number was significantly reduced in cKO compared with wild type. (C) Relative expression of Bmpr1a in osteoclast cultures from spleen cells (left bars) and combined cultured of osteoblast and osteoclast from bone marrow cells (right). The asterisk indicates a statistically significant difference between cKO and WT from three independent experiments. (In A-C results are, mean±s.d., Student’s t-test; *P<0.01; *P<0.01). Details of the materials and methods can be provided on request.

• Supplemental Figure S2 -

  Fig. S2. Microarray analysis using cKO and wild-type calvariae at E18.5 from four independent females. The asterisk indicates a statistically significant difference between cKO (n=4) and wild type (n=4) (−5.68-fold, P=3.27E⁻²⁴). Sost expression was the most downregulated in the entire mouse genome examined. Details of the materials and methods can be provided on request.

• Supplemental Figure S3 -

  Fig. S3. Suppressed expression of sclerostin by Noggin treatment ex vivo. (A) Calvarial bones from mice carrying TOPGAL were treated with Noggin (100 ng/ml). broken line, areas of parietal bones (P). (B) Quantitative RT-PCR analysis for Sost in wild-type bones treated with Noggin ex vivo. The asterisk indicates a statistically significant difference between Noggin treatment (WT+Noggin) and non-treatment (WT) from three independent experiments (mean±s.d., Student’s t test; *P<0.01).

• Supplemental Figure S4 -

  Fig. S4. Expression of bone formation markers in caBmpr1a bones at E18.5. (A) Schematic representation of the transgenic constructs. Mice conditionally expressing a constitutively active form of human Bmpr1a (caBmpr1a, Q207D), which has a mutation in the GS domain of BMPR1A resulting in ligand-independent activation of Smad signaling after Cre recombination, were generated using a transgenic construct similar to the one previously reported (Fukuda et al., 2006). A CAG promoter for ubiquitous expression, a lacZ with a nuclear translocation signal for detection of transgene expression and an IRES-EGFP for monitoring the Cre-dependent recombination were assembled as shown. An HA-tag was added to caBmpr1a for direct detection of the mutant protein. (B) Quantitative RT-PCR for bone formation markers (Runx2, Sp7, Ibsp, Akp2 and Bglap2) was performed using caBmpr1a calvariae (black bar: Cre+, caBmpr1a) and control littermates (open bar: Cre-, caBmpr1a) at E18.5. Expressions of these markers were increased in the caBmpr1a, consistent with evidence that Smad signaling positively controls the expression of bone formation markers. Values are expressed relative to control at E18.5, and represent mean±s.d. from three independent experiments. Student’s t test; *P<0.05.