Additional Supporting Information may be found in the online version of this article.

jbmr1989-sm-0001-SuppFig-S1.tif182KFigure S1. Targeted disruption of the Siglec-15 gene. A. Schematic representations of the genomic region of the Siglec-15 gene (top line), the targeting vector (middle line), and the mutated allele (bottom line). Exons, represented by black boxes, are numbered (Ex1-6). The neomycin-resistance gene (Neo) is indicated. The probe used for Southern hybridization is indicated (P). The Neo cassette was inserted to replace exons 2 to 5. B. PCR genotyping of WT (+/+), heterozygous (+/−), and homozygous (−/−) mice using a common primer (WT-R) in combination with primers discriminating wild-type Siglec-15 alleles (WT-F) and targeted alleles (Neo-F). Another primer set (Neo-f and Neo-r) was used to detect the Neo cassette.
jbmr1989-sm-0001-SuppFig-S2.tif454KFigure S2. Decreased bone formation in vivo is not due to dysfunction of osteoblasts in Siglec-15−/− mice. A. Data of bone morphometric analysis of WT and Siglec-15−/− mice. For dynamic histomorphometry, tetracycline (20 mg/kg) and calcein (20 mg/kg) were injected subcutaneously 4 and 2 d before the mice were killed. Trabecular bone at the primary and secondary spongiosa of each tibia was observed by fluorescence microscopy to evaluate static and dynamic parameters of bone formation. Osteoblast surface (Ob.Pm), osteoid volume (O.Ar), bone volume (B.Ar), interlabel width, single- and double-labeled surface perimeters (sLS and dLS), and total bone surface (B.Pm) were measured. Ob.Pm/B.Pm, Mineral apposition rate (MAR), mineralized surface (mineralized surface/bone surface [Md.Pm/B.Pm]), and bone formation rate (BFR/B.Pm) were calculated from the sLS, dLS, and B.Pm according to Parfitt et al. [30]. B. Alkaline phosphatase activity of osteoblasts from WT or Siglec-15−/− neonatal calvariae at 14 d of culture.
jbmr1989-sm-0002-SuppTab-S1.docx15KTable S1. Sequence of primers used in quantitative RT-PCR.

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