Figure 2
MiR-24 and p16INK4aexpressions during chondrogenesis and role of p16INK4ain chondrocyte cell cycle arrest. Human primary mesenchymal stromal cells (MSCs) were placed in three-dimensional (3D) culture conditions for 21 days. RNAs were harvested at indicated time points. (A-D) Gene expression analysis was performed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for Collagen 2b, matrix metalloprotease 13 (MMP13), p16INK4a, and miR-24. Data are shown as mean ± standard deviation (SD) (n = 3) normalized to D21. Immunohistochemistry (IHC) was performed on sections of formalin fixed paraffin-embedded long bones of transgenic mice deficient in p16INK4a or wild-type at the age of 1 month. (E,F) Growth plate was marked by Safranin-O staining and proliferating cell nuclear antigen (PCNA) (P, proliferative zone; H, pre-hypertrophic/hypertrophic zone). Images were taken with ×20 objective. (G) Quantification of the percentage of hypertrophic non-proliferative terminally differentiated cells on total cells within the growth plate in transgenic and wild-type mice (n = 4) was carried out by using ImageJ software. Data were normalized to 1 for wild-type and are shown as mean ± SD. **P <0.01, ***P <0.001.