Supplementary Figure Legends
Supplementary Figure 1. Cartilage-specific Dicam transgenic (Col2-Dicam Tg) mice
(A) Schematic map of the Col2-Dicam-pNASSb expressing vector. (B) Control pNASSb and Col2-Dicam vector were transduced into HeLa cells and their transduction efficiency was assessed with βGal staining and Western blot analysis for Dicam. (C) RT-PCR for Dicam expression in Col2-DicamTg mice and their WT littermates confirmed the successful generation of transgenic mice. (D) Gross appearance of Col2-DicamTg and WT littermates at P0.
Supplementary Figure 2.Dicam expression by chondrogenic growth factors and during chondrogenic differentiation from MSCs
(A) Primary chondrocytes from E15.5 (5 x 105/well/6 well plate) cultured for 2 days were treated with indicated growth factors after overnight starvation. After 24 h, their RNA and protein was harvested and subjected to RT-qPCR and Western blot analyses for assessing the expression of Dicam. Col2 and Col10 were used as an indicator for the effect of each growth factor. (B) Expression pattern of Dicam during chondrogenic differentiation was analyzed by high density micromass culture derived from E11.5 limb bud MSC as described in supplementary methods. mRNA level of Dicam was increased until day 7 and then decreased, protein was hightest at day 9.
Supplementary Figure 3.Dicam expression in articular cartilage and growth plate in postnatal 8 weeks
Expression of Dicam in articular cartilage and growth plate of 8-week-old mouse tibia was assessed with immunohistochemistry. Dicam is mainly expressed in articular cartilage and not detected in growth plate chondrocyte in 8-week-old mouse tibia. Scale bar = 100μm.
Supplementary Figure 4. Immunofluorescence staining of FAK and Dicam in primary chondrocytes
GFP-tagged Dicam expressing plasmid (pEGFP-N1-Dicam) was transfected into primary chondrocyte and analyzed the expression of FAK (Red) by immunofluorescence staining. Expression of FAK was increased in the GFP-positive Dicam-overexpressing cell, but not colocalized with Dicam. DAPI (blue) indicates the nucleus. Scale bar = 10 μm.
Supplementary Table 1. Primers for real time qPCR used in this study
Gene / Primer / Sequence 5' to 3'Dicam / Forward / CAGCGGAGGATGCAAGAC
Reverse / TATGGAGCCTGATCCCTTGT
Sox9 / Forward / CAGCAAGACTCTGGGCAAG
Reverse / TCCACGAAGGGTCTCTTCTC
Col2 / Forward / CGGTCCTACGGTGTCAGG
Reverse / TTATACCTCTGCCCATTCTGC
Aggrecan / Forward / CCAGCCTACACCCCAGTG
Reverse / GAGGGTGGGAAGCCATGT
Col10 / Forward / GCATCTCCCAGCACCAGA
Reverse / CCATGAACCAGGGTCAAGAA
MMP3 / Forward / TTGTTCTTTGATGCAGTCAGC
Reverse / GATTTGCGCCAAAAGTGC
MMP13 / Forward / GCCAGAACTTCCCAACCAT
Reverse / TCAGAGCCCAGAATTTTCTCC
Runx2 / Forward / GCCCAGGCGTATTTCAGA
Reverse / TGCCTGGCTCTTCTTACTGAG
Osterix / Forward / CTGGCCTCACATCATTTTCTCA
Reverse / CTTCGGGAAAACGGCAAATA
Ihh / Forward / TGCATTGCTCTGTCAAGTCTG
Reverse / GCTCCCCGTTCTCTAGGC
Ptch1 / Forward / GGAAGGGGCAAAGCTACAGT
Reverse / TCCACCGTAAAGGAGGCTTA
Pthlh / Forward / GGTTCAGCAGTGGAGTGTCC
Reverse / CAGACACAGCGCGTTTGA
Gli1 / Forward / CTGACTGTGCCCGAGAGTG
Reverse / CGCTGCTGCAAGAGGACT
Gli2 / Forward / GCAGACTGCACCAAGGAGTA
Reverse / CGTGGATGTGTTCATTGTTGA
Gli3 / Forward / CACCAAAACAGAACACATTCCA
Reverse / GGGGTCTGTGTAACGCTTG
Id1 / Forward / GCGAGATCAGTGCCTTGG
Reverse / CTCCTGAAGGGCTGGAGT
Id2 / Forward / CCCGGTCTTCCTCCTACGA
Reverse / CTGTGGTCCGACAGGCTGTT
Hhip / Forward / CCCAGACTCACAATGGAAAACTCT
Reverse / GGTGTAGTAGCCGTTTCGACAGA
Zfp521 / Forward / AAGCAAGCGAAACCGAGAT
Reverse / TTCTGGCCTCTTCTTGCAGT
Dusp6 / Forward / TTTTCCCTGAGGCCATTTCTT
Reverse / GATACCTGCCAAGCAATGCA
Axin2 / Forward / GACGCACTGACCGACGATT
Reverse / TTCTTACTCCCCATGCGGTAA
Hes1 / Forward / CCAGCCAGTGTCAACACGA
Reverse / AATGCCGGGAGCTATCTTTCT
Supplementary methods.
Micromass culture
Limb buds from 10.5 dpc embryos were digested with Dispase II (Roche, Indianapolis, IN), and suspended cells in growth media composed of DMEM mixed with Ham’s F12 at a 2:3 ratio, 10% FBS, and 2mM L-glutamine. The cells were concentrated at 2.5 x 107 cells/ml, and a 10 μl drop was spotted on the culture plate. After 1 day for stable attachment to the culture dish, media were changed to differentiation media that containing with 10 mM of β-glycerophosphate and 50 μg/ml of ascorbic acid in the growth media, and cultured for 13 days. Media were changed every other day.
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