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Fig. 7. Detection of nuclear factor of activated T cells (NFAT) nuclear accumulation in osteoclasts. (A) Phase contrast image of freshly isolated multinucleated rat osteoclast. (B–F) NFATc1 localization in fixed rat osteoclasts was assessed by immunofluorescence and confocal microscopy. (B) Minimal staining was observed in control preparations where primary antibody was omitted. (C) Example of osteoclast exhibiting cytoplasmic localization of NFATc1. (D) Example of osteoclast exhibiting similar levels of NFATc1 in cytoplasm and nuclei. (E) Example of osteoclast in which NFATc1 levels are higher in the nuclei than in the cytoplasm. (F) Example of osteoclast in which virtually all NFATc1 is localized in the nuclei. Osteoclasts exhibiting NFATc1 distributions similar to those shown in E and F were enumerated as having nuclear accumulation of NFATc1. (Calibration bar, 10 mm.)

Fig. 8. Representative images showing effects of RANKL on nuclear accumulation of NFATc1 in rat osteoclasts. Osteoclasts were stimulated with vehicle or RANKL (1 mg/ml) at pH 7.6 for 45 min at 37°C and then fixed. NFATc1 localization was assessed by immunofluorescence (green) and nuclei were stained by using TOTO-3 (red). (A) In most osteoclasts treated with vehicle, NFATc1 was localized predominantly in the cytoplasm. (B) RANKL induced nuclear accumulation of NFATc1, most often in all nuclei of single osteoclast. Yellow on superimposed image indicates nuclear localization of NFATc1. (Calibration bar, 20 mm.)

Fig. 9. Effects of RANKL and pH on nuclear accumulation of NF-kB in rat osteoclasts. Localization of the p65 subunit of NFkB was assessed by using immunofluorescence. (A) RANKL, but not extracellular acidification, induced nuclear translocation of NF-kB. (Left) NF-kB is localized in the nuclei of an osteoclast treated with RANKL (1 m g/ml, pH 7.4) for 30 min. (Right) Cytoplasmic localization of NF-kB in an osteoclast exposed to pH 7.0 for 30 min. (Calibration bar, 10 m m.) (B) Time course of the effect of RANKL and acidification on nuclear accumulation of NF-kB. Osteoclasts were incubated in media of pH 7.4 (Control, white circles), RANKL (1 mg/ml) in media of pH 7.4 (red circles), or media of pH 7.0 (green circles) and then fixed at the indicated times. The number of osteoclasts exhibiting nuclear accumulation of NF-kB was expressed as a percentage of the total number of osteoclasts. Data are means ± SEM (three independent experiments). *, Significant difference compared with control for the same time points (P < 0.01) assessed by one-way ANOVA and a Bonferroni test.

Fig. 10. Inhibition of phospholipase C (PLC) or depletion of intracellular Ca²⁺ stores blocks acid-induced elevation of [Ca²⁺]_i in rat osteoclasts. (A–C) We used ATP as a positive control to confirm the effectiveness of U73122. Osteoclasts were superfused with Ca²⁺-containing buffer (pH 7.4) and stimulated with ATP (100 m M), applied for 10 s by pressure ejection from a micropipette where indicated by arrowheads. Where indicated by shaded regions, samples were treated with (A) vehicle (0.1% DMSO, Control), (B) U73122 (1 mM) or (C) the inactive control compound U73343 (1 m M) and then stimulated again with ATP. Acidification from pH 7.4 to 7.0 was induced where indicated by the bars below the Ca²⁺ traces. Responses are from three separate osteoclasts, representative of five for vehicle, seven for U73122, and six for U73343. (D) To deplete intracellular Ca²⁺ stores, osteoclasts were pretreated with the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (5 mM) for 30 min. Acidification from pH 7.4 to 7.0 was induced where indicated by the bars below the Ca²⁺ traces. Responses from a control osteoclast (Left) and an osteoclast treated with thapsigargin (Right) are shown. Data are representative of six osteoclasts for each condition.

Fig. 11. Identification of ovarian cancer G protein-coupled receptor 1 (OGR1) in osteoclast-like cells and osteoclasts. (A–B) Detection of transcripts encoding OGR1 by conventional RT-PCR. (A) Transcripts for OGR1 (Gpr68) were detected in RNA isolated from in vitro derived osteoclast-like cells (OCL, lane 2) and murine lung (lane 8), but were barely detectable in murine liver (lane 6). Amplicons were of the expected size (533 bp), and their identity was confirmed by sequencing. (B) Expression of mouse glyceraldehyde-3-phosphate dehydrogenase mRNA. There were no amplification products in samples that did not undergo reverse transcription (–, lanes 3, 7, and 9) or did not contain template (W, lane 4). Lanes 1 and 5 show 100-bp ladders (M). Data are representative of results from four independent experiments for osteoclast-like cells and three independent preparations for murine liver and lung. (C–F) Detection of OGR1 protein by using immunofluorescence. Monochrome images show confocal sections immediately above the substrate. (C) Minimal staining was observed in control preparations where primary antibody was omitted. (D) OGR1 in undifferentiated RAW 264.7 cells. (E) Staining was more intense in osteoclast-like cells differentiated from RAW 264.7 cells in the presence of RANKL. Arrowheads indicate presence of OGR1 at the plasma membrane. (F) OGR1 staining in authentic rat osteoclast. (Calibration bar, 20 mm.)

Fig. 12. Effects of calcitonin and cyclosporin A on acid-stimulated osteoclastic resorption. Rabbit osteoclasts were plated on dentin slices and pretreated with calcitonin (10 mM) or cyclosporin A (CsA, 1 mM) for 30 min at pH 7.6. Where indicated, the pH was altered to 7.0 by increasing the partial pressure of CO₂ (respiratory acidosis). Experiments were stopped at 24 h and the number and planar area of the resorption pits were quantified. (A) Number of pits per slice. (B) Average pit area. Data are means ± SEM, n = 3 independent experiments each performed in triplicate. Differences were assessed using Student’s t test. * indicates significant difference for the effect of acidosis. # indicates significant difference for the effects of calcitonin or cyclosporin A compared to untreated samples at the same pH.