Figure 3

Autophagy mobilized during differentiation of immortalized human mesenchymal stem cells (ihMSCs). Cultured ihMSCs were stimulated to undergo osteogenesis to form osteoblasts for 30 days at 21% and 4% oxygen. LC3I and LC3II levels were assessed via immunoblot every 10 days and normalized to β-actin to derive average LC3II band densities (A). Levels of Runx2 (B) and Osteocalcin (C), markers of early and late osteogenesis, respectively, were assayed via quantitative polymerase chain reaction (^#P <0.05, *P <0.01). A Von Kossa stain was employed every 10 days to visualize bone spicule deposition at both oxygen concentrations (D). The representative of experiments repeated at least three times is shown, and statistical significance is noted for difference from day 0 and between the two different ambient oxygen concentrations. Immunoblot normalized integrated densities were calculated and averaged by using a ratio of LC3II to beta-actin via sum of replicates for each blot. LC3, light chain 3.