Priority Research Paper Volume 13, Issue 24 pp 25607—25642

Figure 6. PTH protects osteocytes from oxidative stress-induced cell death and senescence. (A) Representative images of immunohistochemistry for 4-HNE on the L3/4 vertebrae from male animals are shown. The frequency of 4-HNE⁺ osteocytes per image field was analyzed. Mean ± SEM is shown. (B) Expression of p16^Ink4a in the tibiae of male control and Dmp1-PPR^KO mice was analyzed by qPCR. N = 4–6 per group. Mean ± SEM is shown. (C–E) Control and 12H-PPR^KO osteocytic cell line was pretreated with either 10 nM hPTH(1–34) or 10 μM forskolin (FSK) for 18-22 hrs prior to H₂O₂ exposure. (C) After H₂O₂ exposure (1 mM, overnight), cell viability was measured by resazurin-based assays. (D) After H₂O₂ exposure (1 mM, 4 h) intracellular ROS levels were measured using a fluorescent probe (DCFDA). Data are presented as relative fluorescence unit (RFU). (E) After continuous exposure to H₂O₂ (150 μM, 14 d), cells were stained for SA β-gal. Representative SA β-gal staining images and the quantification of SA β-gal⁺ cells (red arrowheads) are shown. Bar = 100 μm. (F) Representative TRAP staining images are shown of BMMCs isolated from 3–4-month-old male control mice under osteoclastic differentiation in the presence of conditioned medium from H₂O₂-treated control osteocytic cell line (–H₂O₂ or +H₂O₂, 150 μM for 7 days). Bar = 400 μm. N = 18 per group. (G) After continuous exposure to H₂O₂ (100 or 150 μM, 7 or 14 d), cells were harvested for RNA isolation. mRNA expression of p16^Ink4a, p21, Sost, and RANKL were analyzed by qPCR. N = 3 per group. Kruskal-Wallis test with Dunn’s post hoc test, two-way ANOVA with Tukey’s post hoc test, one-way ANOVA with Sidak’s post hoc test or Mann-Whitney test were performed. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Same letter indicates n.s. Data are presented as mean ± SD.