Mechanism and signal specificity of the suppressive effect of PTH on apoptosis in cultures of osteoblastic and osteocytic cells. (a) Cultures were maintained for 6 hours in the presence of 10^–7 M dexamethasone (Dex) without or with preincubation for 1 hour with 10^–8 M bPTH(1-34). The pyknotic fragmented nuclei (arrows) typical of apoptotic cells were viewed using HOECHST 33258 fluorescent dye in osteoblastic calvaria cells and by EGFP fluorescence in MLO-Y4 osteocytes. ×400. Insets: percentage of cells undergoing apoptosis, as determined from evaluation of nuclear morphology of 500 cells in randomly selected fields. (b) Cells (10⁴/cm²) were incubated for 1 hour in vehicle or 10^–8 M bPTH(1-34), and then incubated for an additional 6 hours in the absence (basal) or presence of 5 × 10^–5 M etoposide (Etop), 10^–7 M dexamethasone (Dex), or 10^–9 M TNF. (c) Osteoblastic calvaria cells were cultured for 1 hour in vehicle or the indicated log molar concentrations of bPTH(1-34), bPTH(3-34), or dibutyryl cAMP (DB-cAMP), and then for an additional 6 hours in the absence or presence of 10^–7 M dexamethasone. Apoptotic cells in b and c were enumerated by trypan blue staining. Bars represent mean ± SD of triplicate wells. Nearly identical results were obtained in at least 2 additional experiments. Data were analyzed by ANOVA. Etoposide, dexamethasone, and TNF caused a significant (P < 0.05) increase in apoptosis in cultures containing vehicle. *P < 0.05 vs. vehicle (b) or vs. dexamethasone alone (c).