Fig. 5

mTORC1 activation in subchondral preosteoblasts produce Cxcl12 to promote chondrocyte hypertrophic differentiation MSCs osteogenic differentiation. a Western blot and quantitative PCR analysis of Cxcl12 in primary preosteoblasts. b–d) Cxcl12 assessed by ELISA in the cultured medium of primary preosteoblasts or in the serum at 6 weeks post ACLT surgery. e Representative immunostaining and quantitative analysis of CXCR4⁺ cells in articular cartilage and subchondral bone in ΔTSC1, ΔRaptor mice, and their littermates (Ctrl). Scale bars, 100 μm. Quantitative analysis of CXCR4⁺ cells out of total cartilage chondrocytes. f, g Quantitative PCR analysis of Col2, ACAN, Col X, MMP-13 mRNA in ADTC5 cells treated with the cultured medium of primary preosteoblasts and Cxcl12-neutralizing antibody for 7 days. h, i Western blot analysis of Col2, Col X, MMP-13, and Toluidine blue staining in ADTC5 cells treated with CM of primary preosteoblasts and Cxcl12-neutralizing antibody for 7 days. j Western blot analysis of Col 1α, Col X, MMP-13, and Alizarin red staining in ADTC5 cells treated with CM of primary preosteoblasts, recombinant murine Cxcl12 or Cxcl12-neutralizing antibody for 14 days. Data are shown as mean ± s.d. and analyzed by Student’s t test and one-way ANOVA . n ≥ 8, **P < 0.01