Figure 2. Inhibition of mTORC1 prevents chondrocyte proliferation while promoting differentiation.

(a) CCK8 proliferation assay of chondrocytes cultured in normal growth medium or with 1, 10 or 50 nM rapamycin treatment. OD₄₅₀values were converted to cell numbers. One-way analysis of variance (ANOVA) and Dunnett's multiple comparison test; NS, not significant, *P<0.05, n=5; Error bars indicate s.d. (b) qPCR and western blot analysis of primary chondrocytes cultured in growth medium or ITS medium. Cells were treated with or without rapamycin from day 6 and were harvested on day 14 after confluence. One-way ANOVA and Dunnett's multiple comparison test, *P<0.05, n≥3; Error bars indicate s.d. (c) HE staining, pS6 immunofluorescence and MMP-13 in situ mRNA analysis of Tibia sections of mice at P0 whose maternal mice receiving a daily intraperitoneal injection of rapamycin (1.5 mg kg⁻¹ per day) or 0.9% saline for 3 days before sacrifice. Scale bar, 100 μm. PZ, proliferative chondrocyte zone; HZ, hypertrophic chondrocyte zone. (d) Quantification of Tibia PZ and HZ length in P0 mice receiving rapamycin. Student's t-test, *P<0.05, n≥3; Error bars indicate s.d. (e) Western blot of pure cartilage tissues harvested from long bone mice receiving rapamycin.