Tgfbi Deficiency Leads to a Reduction in Skeletal Size and Degradation of the Bone Matrix

Tgfbi deficiency leads to a reduction in skeletal size and degradation of the bone matrix

Jung-Mi Lee, Eun-Hye Lee, In-San Kim, Jung-Eun Kim

J.-M, Lee, E.-H. Lee, J.-E. Kim

Department of Molecular Medicine, Kyungpook National University School of Medicine, Daegu, 700-422, Republic of Korea

I.-S. Kim

Department of Biochemistry and Cell Biology, Kyungpook National University School of

Medicine, Daegu, 700-422, Republic of Korea

Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Republic of Korea

J.-M, Lee, E.-H. Lee, I.-S. Kim, J.-E. Kim

Cell and Matrix Research Institute, Kyungpook National University, Daegu, 700-422, Republic of Korea

J.-M, Lee, I.-S. Kim, J.-E. Kim

BK21 Plus KNU Biomedical Convergence Program, Department of Biomedical Science,

Kyungpook National University, Daegu, 700-422, Korea

Correspondence: J.-E. Kim

Department of Molecular Medicine, Cell and Matrix Research Institute, Kyungpook National University School of Medicine, Daegu, 700-422, Republic of Korea

Tel: +82 53 420 4949, Fax: +82 53 426 4944, e-mail:

Supplemental Materials and Methods

Reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNA was isolated from the long bone, kidneys, and liver at 4 weeks of age using TRIzol reagent (Sigma-Aldrich). RT was performed using a Superscript™ III First-Strand Synthesis kit with an oligo dT primer (Invitrogen). Tgfbi primers (548-bp, sense 5'-TGA AAA GGT CCC AGG AGA GA-3' and antisense 5'-CCT CCA GCA CGG TAT TGA GT-3') and GAPDH primers (983-bp, sense 5'-TGA AGG TCG GTG TGA ACG GAT TTG GC-3' and antisense 5'-CAT GTA GGC CAT GAG GTC CAC CAC-3') were used for PCR. PCR for Tgfbi and GAPDH was performed with 35 amplification cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 60 sec and 61°C for 60 sec, respectively, and extension at 72°C for 60 sec. PCR products were separated electrophoretically on 1% agarose gels and visualized with ethidium bromide staining.

Supplemental Figure Legends

Fig. S1. Tgfbi expression in mouse tissues. With RT-PCR analysis, Tgfbi mRNA expression was not detected in the long bones, kidneys, and liver of Tgfbi knockout mice.

Fig. S2. Microarchitecture parameters of vertebrae from Tgfbi knockout mice. (A) Histomorphometric analysis of the fourth lumbar vertebra, as shown in Fig. 2C and 2D showed no significant differences in mineralized cancellous bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), or trabecular separation (Tb.Sp). (B) The lumbar vertebrae of mice at P15 were analyzed with the von Kossa stain, which stains mineralized bones black. No differences in BV/TV, Tb.Th, Tb.N, and Tb.Sp were observed. WT, wild-type; KO, knockout.

Fig. S3. Altered localization and expression level of MMP13 and aggrecan proteins in Tgfbi knockout mice at P60. (A) MMP13 was widely detected in chondrocytes of the superficial and middle zones of articular cartilage in Tgfbi knockout mice. (B) The expression level of aggrecan was increased in Tgfbi knockout mice compared to wild-type. WT, wild-type; KO, knockout.