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. 2023 Apr 17;133(8):e162519. doi: 10.1172/JCI162519

Figure 1. Sn osteocytes accumulate in bone with aging and are cleared by AP treatment in old DMP1-Cre+/– p16-LOX-ATTAC mice.

Figure 1

(A) Changes in female (pink bars) and male (blue bars) murine p16Ink4a mRNA expression throughout life in osteocyte-enriched bone from C57BL/6 WT mice, relative to young adult (6-month-old) mice. ***P < 0.001, by ANOVA with Tukey’s post hoc test. (B) Representative images of a non-Sn osteocyte (original magnification, ×63 oil) in a young (6-month-old) mouse versus a Sn osteocyte in an old (24-month-old) mouse according to the TAF (white arrows) assay (n = 8 females/group). Scale bars: 20 μm and 2 μm (enlarged insets). (C–E) Quantification of (C) the mean TAF/osteocyte and (D) the mean percentage of TAF+ osteocytes/mouse based on: the percentage of osteocytes with 1 or more TAF, the percentage of osteocytes with 2 or more TAF, and the percentage of osteocytes with 3 or more TAF, respectively; and quantification of (E) the mean percentage of 3 or more TAF+ osteocytes/mouse (n = 8 females/group). (F) Schematic of the unrecombined p16-LOX-ATTAC construct and cross with DMP1-Cre mice. (G) Study design for local clearance of Sn osteocytes in old (20 months) p16-LOX-ATTAC x DMP1-Cre mouse cohorts, males and females combined, randomized to Veh (gray) or AP (teal) treatment for 4 months. (H) RT-qPCR analysis of p16Ink4a mRNA expression across tissues in mice (males and females combined, n = 12–18 per tissue) treated with Veh (gray) or AP (teal). (I and J) Quantification of (I) the mean percentage of TAF+ osteocytes/mouse and (J) the mean percentage of TAF+ osteoblasts per mouse (OBs/mouse) based on the percentage of cells with 1 or more TAF, the percentage of cells with 2 or more TAF, and the percentage of cells with 3 or more TAF, respectively. (K) Representative images of perigonadal adipose tissue staining for SA–β-Gal+ cells in Veh- and AP-treated mice. Scale bars: 100 μm. (L) Quantification of SA–β-Gal+ adipocytes in mice treated with Veh (gray, n = 9: n = 5 females, n = 4 males) or AP (teal, n = 11: n = 6 females, n = 5 males). Data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by independent samples Student’s t test or Wilcoxon rank-sum test, as appropriate. OCY, osteocytes.