FIG.5. Differential expression of chondrogenic and apoptotic marker genes.

(A). qRT-PCR was performed to examine following chondrogenic and apoptotic marker genes using total RNAs prepared from mouse hind limbs at E17.5 stage. As illustrated, Sox9 (2.30-fold, p=0.0297) is significantly upregulated in TG mice compared to their WT littermates. Meanwhile Bcl-2 (2-fold, p = 0.016), and Opn (1.93-fold, p = 0.0097) were also significantly upregulated in TG mice compared to littermate controls, while Bax (1.42 fold, p=0.1837) is slightly upregulated but without statistical significance. (B). Relevant marker genes were also examined at the P1 stage. As illustrated, Bcl-2 (2.3-fold, p = 0.002), Opn (1.69-fold, p = 0.0361), and Sox9 (17.88-fold, p=0.0484) were also significantly increased in TG mice compared to WT littermates. Similar to the result of E17.5, Bax (1.18 fold, p=0.2892) did not show significant changes between TG and WT controls. Bars denote means ± SE for marker gene expression. TG: transgenic; WT: wild-type. “*”: p<0.05; “**”: p<0.01. (C). Western blotting assay was performed using protein extracts prepared from TG or WT mouse hind limbs at the P1 stage and Runx2 or Sox9 antibodies. Both Runx2 and Sox9 are highly expressed in TG or WT mouse limbs (top). Anti-Lamin B antibody was used as an internal control. Densitometry analysis showed that Sox9 increased ~50%, while Runx2 was slightly decreased in TG mouse limbs (bottom), but not statistically significant. (D). Western blotting was also conducted using protein extracts from primary cultured chondrocytes of TG or WT mice at P1 stage and the antibodies as mentioned above. The results showed that Runx2 is weakly expressed, while Sox9 is highly expressed in all the primary chondrocytes examined (top). Densitometry analysis suggested that both runx2 and Sox9 are slightly increased in TG groups but without statistical significance (bottom).