Figure 1.

c-Abl^−/− osteoblasts show reduced proliferation potential in vivo and undergo premature senescence ex vivo. (a) c-Abl^−/− bone sections showed a reduced number of osteoblasts. Femur bones of 2-month-old c-Abl^−/− and WT mice were used for histomorphometric analysis. (b) c-Abl^−/− bone sections (newborn pups) showed a decrease in the number of S-phase cells. BrdU-positive cells (brown) were counted in equal areas underneath the growth plate. (c) Growth of c-Abl^−/− and WT osteoblasts. Calvarial osteoblasts were cultured as described and the numbers of cells are plotted against passage numbers. (d) The effects of c-Abl deficiency, c-Abl reconstitution, p16^INK4a deficiency and ectopic expression of Id1 on the lifespan of osteoblasts. Primary calvarial osteoblasts were isolated from p16^INK4a−/−, c-Abl^−/− p16^INK4a−/− mice and their control littermates. The doubling times (until senescence) were counted after passage 3. For retroviral expression of c-Abl, see Supplementary Fig. S2a. (e) Quantification results of cells positive for histochemical staining of senescence-associated SA-β-Gal. WT, c-Abl^−/− and c-Abl reconstituted or empty vector infected c-Abl^−/− osteoblasts were cultured, fixed and stained at a neutral pH for SA-β-Gal. Percentages of SA-β-Gal-positive cells at each passage of mutant and control osteoblast cultures are shown. Scale bar, 50 μm. Data are means ± s.e.m. (a, n = 5; b, n = 4; c–e, n = 3). *P < 0.05 when compared to WT counterparts. **P < 0.05 when compared to c-Abl^−/− osteoblasts or mice.