Fig. 4
mTORC1 inhibition enables preosteoblast escape from senescence in vitro. a Primary calvarial osteoblasts isolated from neonatal ΔRaptor and Raptorfl/fl mice were induced to undergo senescence by ROS. After 3 days, the cells were analyzed for senescence marker (p16 and p53) expression and mTORC1 activity (pS6) with western blotting. b Immunostaining of EdU in the senescence-induced cells in a. Scale bar, 50 μm. c Quantitative analysis of EdU+ cells relative to total cells. d Representative images of SA-β-gal staining of the senescence-induced cells in a. Scale bar, 100 μm. e Quantification of the proportion of SA-β-gal-positive cells in each population. f qPCR analysis of IL-6 and Cxcl1 mRNA in the primary preosteoblasts in a. The cells in a were then induced to undergo osteogenic differentiation and subjected to (g) ALP staining or (h) AR-S staining on Day 7 or 14, respectively, after induction of differentiation. Data are shown as the mean ± SD. The numbers of samples (n) are indicated in each figure panel. P values were determined with two-tailed Student’s t test for single comparisons
