Journal: The Journal of Cell Biology
Article Title: Type XII collagen regulates osteoblast polarity and communication during bone formation
doi: 10.1083/jcb.201010010
Figure Lengend Snippet: Delayed terminal differentiation in type XII collagen–null osteoblasts. (A–E) Primary osteoblasts were derived from neonatal calvaria of Col12a1 +/+ and Col12a1 −/− mice and cultured in osteogenic medium. The cells are harvested after 0, 7, 14, and 21 d in culture and used for quantitative real-time PCR analysis. Each group represents the mean of triplicate determinations. mRNA was normalized to Gapdh expression. (A) Type XII collagen mRNA expression is stable during osteoblast development. (B and C) Similar expression patterns are detected in mRNA expressions of Runx2 (B) and type I collagen (C) in Col12a1 +/+ and Col12a1 −/− osteoblasts. (D and E) The expression of osteocalcin ( Ocn ; D) and osteopontin ( Opn ; E), which are terminal osteoblast differentiation markers, is significantly decreased in Col12a1 −/− osteoblasts as compared with wild-type controls. **, P < 0.01. Error bars are mean ± SD. (F) Immunoblot analysis of P30 femurs from Col12a1 +/+ ( n = 2) and Col12a1 −/− ( n = 3) mice was performed to validate the in vitro data. Opn and Ocn expression is decreased, whereas type I expression is stable in Col12a1 −/− femurs compared with the mRNA data.
Article Snippet: The whole tissue and cell lysate preparation and immunoblot were performed as previously described ( ) using rabbit anti–type XII collagen (1:1,000 dilution; KR33), rabbit anti-Cx43 (1:10,000 dilution; Sigma-Aldrich), rabbit anti–mouse collagen I (1:1,000 dilution; Millipore), rabbit anti-Cad11 (1:250 dilution; Invitrogen), rabbit anti–N-cadherin (1:1,000 dilution; Cell Signaling Technology), 0.1 µg/ml of goat anti–mouse osteopontin (R&D Systems), goat anti-osteocalcin (1:100 dilution; Santa Cruz Biotechnology, Inc.), mouse anti–β-actin (1:5,000 dilution; Millipore), and anti–mouse or anti–rabbit HRP-conjugated secondary antibodies (GE Healthcare).
Techniques: Derivative Assay, Cell Culture, Real-time Polymerase Chain Reaction, Expressing, Western Blot, In Vitro