Fig. 3.

Osteocytes acidify their microenvironment in vitro in response to PTHrP and this is mediated by the vacuolar ATPase. Cells were treated for 20 min with PTHrP in the presence of SNARF; (A) NIH3T3 fibroblasts (8 experiments, n=3 each) did not respond to PTHrP, while (B) SaOs2 osteosarcoma cells (5 experiments, n=3-4 each) and (C) primary osteocytes (4 experiments, n=2-3 each) demonstrated significant increase in SNARF fluorescence upon PTHrP treatment (mean and SEM, unpaired two-tailed t-test compared to vehicle). (D) PTHrP-induced acidification upon osteocyte differentiation in IDG-SW3 cells (mean and SEM, 3 experiments, n=3 each, one-way; ANOVA and Bonferroni posthoc test compared to vehicle group on each day). (E) Bafilomycin blocked PTHrP-induced acidification in primary osteocytes (5 experiments, n=2-3 each, mean and SEM, one-way ANOVA and Tukey posthoc test comparing all groups).