Figure 6
T1-targeted siRNA experiments in MC3T3-E1 cells and dot-blot analysis of T1KO tissues. (A) Quantitative real-time PCR analysis of siRNA experiments after 48 h in MC3T3-E1 cells. (B) Western-blot analysis of siRNA experiments after 72 h in MC3T3-E1 cells. The full-length blots are shown in Supplementary Fig. S9B. (C) Dot-blot analysis for proteins of WT and T1KO mice tissues. (A) The average expression of each gene in WT mice was defined as 1.0, and gene levels were compared with that of WT mice. mRNA expressions of collagen type 1, Wnt3a and aggrecan were downregulated by T1 gene knockdown, although T2 knockdown (T2KD) nor chondroitinase ABC (ChABC) treatment did not affect of them. The protein levels of collagen type 1 and Wnt3a after 72 h were significantly decreased by T1 gene knockdown by T1KD #a in MC3T3-E1 cells (B). T1KD #b, T2KD and ChABC did not suppress the protein expression of collagen type 1 and Wnt3a (B, Supplementary Fig. S9A). In P2 calvaria and 4W tendon tissues of T1KO mice, the protein amounts of collagen type 1 and Wnt3a were significantly reduced compared with that of WT mice (C). The average protein amount of each molecule in WT mice was defined as 1.0, and protein levels were compared with that of WT mice. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001, n = 5, Student’s t-test.
