Fig. 2

Hyperactive mTORC1 aggravates preosteoblast senescence and age-related bone loss. a PCR analysis of Tsc1 allele recombination in tissues from 12- and 18-month-old ΔTsc1 mice. Primers for GAPDH were used as a loading control. b Immunostaining of GFP, which indicated Osx-Cre expression, in bone sections of the mice in a. Scale bar, 200 μm. c Double immunostaining of Osx and pS6 and quantification of pS6⁺ preosteoblasts (pS6⁺ Osx⁺) relative to total preosteoblasts (Osx⁺) in bone sections of the mice in a. Scale bar, 100 μm. d Representative microcomputed tomography (μCT) images of 12- and 18-month-old ΔTsc1 tibias compared with those of the littermate controls, Scale bar, 500 μm. e Quantification of Tb. BV/TV, Tb.N, Tb.Sp, and Tb.Th. f Representative images of calcein labels and quantification of the mineral apposition rate (MAR) in femurs from the 18-month-old ΔTsc1 mice. Scale bar = 50 μm. Double immunostaining of Osx plus p16 (g) and Osx plus Ki-67 (h) in tibias of the 18-month-old ΔTsc1 mice. Double positively stained cells were quantified. Scale bar: 100 μm. In primary calvarial osteoblasts isolated from neonatal ΔTsc1 and Tsc1^fl/fl mice, senescence was induced by ROS. After 3 days, (i) the cells were immunostained for EdU and analyzed for EdU⁺ cells relative to total cells. Scale bar, 50 μm. j Representative images of SA-β-gal staining and quantification of the proportion of SA-β-gal-positive cells in the senescence-induced cells. Scale bar, 100 μm. k After induction of osteogenic differentiation, cells were subjected to AR-S staining on Day 14 after differentiation induction. 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