Fig. 3

Dietary restriction enhances the self-replication of resting chondrocytes while inhibiting their differentiation. a Schematic diagrams of the fate-mapping analysis of Axin2⁺ cells in the proximal tibial growth plate in Axin2Cre^ERT2;R26R^ZsGreen mice (pulsed on P23–25 and traced for 11 days). b, c Representative images of descendants of initially labeled Axin2⁺ cells (ZsGreen⁺ cells) in the proximal tibial growth plate in the control (b) and DR (c) groups. d, e Quantification of the ZsGreen⁺ columns in the growth plate (d) and ZsGreen⁺ cells in the top 50 μm (e). Control group (n = 7), DR group (n = 9). ****P =< 0.000 1 (d), *P = 0.012 5 (e). f, g Representative images for in situ hybridization of Clu in the proximal tibial growth plate in the control (f) and DR (g) groups. h Quantification of Clu⁺ cells in the top 50 μm. Control group (n = 9), DR group (n = 10). ***P = 0.000 8. i, j Representative images of hematoxylin and eosin staining of the proximal tibial growth plate in the control (i) and DR (j) groups. k Quantification of column number per 100 µm width of the growth plate. Control group (n = 12), DR group (n = 11). *P = 0.024 5. l, m Representative images of EdU labeling in the proximal tibial growth plate in the control (l) and DR (m) groups. EdU (50 μg·g⁻¹ body weight) was injected two hours before euthanasia. n Percentage of growth plate cells with EdU. Control group (n = 6), DR group (n = 6). ****P =< 0.000 1. o, p Representative images for in situ hybridization of Ki67 and ZsGreen⁺ cells in the proximal tibial growth plate in the control (o) and DR (p) groups. Arrow heads, ZsGreen⁺ cells expressing Ki67 (o, p). q Percentage of Ki67⁺ cells among ZsGreen⁺ cells in the top 50 µm. Control group (n = 7), DR group (n = 9). **P = 0.004 5. r, s Representative images of immunostaining for p-H3 and ZsGreen⁺ cells in the proximal tibial growth plate in the control (r) and DR (s) groups. Arrow heads, ZsGreen⁺ cells expressing p-H3 (r, s). t Percentage of p-H3-positive cells among ZsGreen⁺ cells in the top 50 µm. Control group (n = 10), DR group (n = 12). **P = 0.003 5. Ad lib ad libitum, DR dietary restriction, SOC secondary ossification center, p-H3 phospho histone H3. The white dashed lines demarcate the growth plate from the surrounding tissues. Scale bars: 200 μm (b [left-most], c [left-most], f [left], g [left], i, j, l, m), 50 μm (b [right two], c [right two], f [right], g [right]), 20 μm (o, p, r, s). All data are presented as the mean ± SD. Statistical significance was determined by unpaired two-tailed t test