Fig. 6.

Ex vivo bone organ culture and in vitro chondrocyte analyses of the Igf2 null bone. (A) Representative images of metatarsal bones of WT and Igf2 null after 7 days of ex vivo culture. (B) Percentage of growth in length and width of metatarsal bones after 7 days of culture. (C) Alcian Blue and Hematoxylin staining of the metatarsal bones after 7 days of culture. The ratio of the lengths of hypertrophic zone (HZ)/total length of the cartilage template, as well as the ratio of the columnar zone (CZ)/HZ between the WT and Igf2 null bones were compared. For A-C, at least ten bones were analyzed for each group (WT and Igf2 null). Scale bars: 200 μm. (D) RT-qPCR analysis of Igf2 null and WT epiphyseal chondrocytes after 1 week of culturing in alginate beads. Data are presented as the ratio of Igf2 null/WT gene expression. (E) RT-qPCR analysis of epiphyseal chondrocytes treated with exogenous 10 ng/ml IGF2 and cultured in 3D alginate beads for 1 week. Data are presented as the ratio of gene expression from IGF2 treatment versus non-treated control in WT chondrocytes. (F) RT-qPCR analysis of selected genes after the treatment with an HIF2α inhibitor, HIF2 antagonist 2. For D-F, the experiments were conducted with biological triplicates and repeated three times. Mean±s.d. *P<0.05, **P<0.01, ***P<0.001 (D,E: unpaired t-test between WT and Igf2 null; F: ANOVA followed by Dunnett's test).