Figure 1.
Figure 1. PMCA expression was increased during RANKL-induced osteoclast differentiation. (A) Human PBMCs were cultured in the presence of 30 ng/ml M-CSF and 100 ng/ml RANKL. After 3 and 7 d of culture (preosteoclast and mature osteoclast stages, respectively), total RNAs were extracted and subjected to DNA microarray analysis. The relative signal intensity of ATP2B4 (the gene encoding PMCA4) mRNA detected in a DNA microarray analysis is shown. Data are means ± SD (**, P < 0.01). (B) Mouse BMMs were incubated with 30 ng/ml M-CSF or M-CSF plus 100 ng/ml RANKL for 36 h. Biotinylated cell surface proteins were prepared as described in Materials and methods and subjected to LC/MS/MS analysis. The elution profile of peptides from LC is shown. Arrows indicate PMCA1 peptides identified by subsequent mass spectrometry analyses. (C and D) Mouse BMMs were stimulated with 100 ng/ml RANKL in the presence of 30 ng/ml M-CSF for the indicated times. Total RNA was subjected to RT-PCR analysis to examine the expression levels of Atp2b and Slc8a families (the genes encoding NCXs). The mRNA expression in mouse brain was used as a positive control. Ctsk (cathepsin K) is a marker gene for osteoclast differentiation. (E) Quantitative real-time PCR analysis of Atp2b1 and Atp2b4 transcripts was performed with BMMs treated with 100 ng/ml RANKL plus 30 ng/ml M-CSF for the indicated times. The mRNA expression levels were normalized against those of Hprt1. The error bars show the mean ± SD of three independent experiments. (F) Cell lysates from BMMs treated with 100 ng/ml RANKL plus 30 ng/ml M-CSF for the indicated times were subjected to Western blotting. The expression levels of total PMCA and NFATc1 were examined with pan-PMCA and NFATc1 antibodies. (G) PMCA immunostaining (Cy3) was performed with mature mouse osteoclasts cultured on dentin discs. Plasma membrane ganglioside GM1 was costained using FITC-conjugated cholera toxin B. Cross-sectional images were reconstructed through the z-axis by confocal microscopy (A, apical; B, basolateral). Bars, 5 µm. (H) BMMs were infected with viruses harboring empty vector (pMSCV) or constitutively active NFATc1 (pMSCV-NFATc1-CA). 2 d after infection, PMCA and NFATc1 expression was examined by Western blotting. (I) BMMs were treated with 100 ng/ml RANKL plus 30 ng/ml M-CSF for 2 d. ChIP was performed using an NFATc1 antobody followed by a PCR amplification of the promoter regions of Atp2b1 or Atp2b4 gene. Input DNA (1% of total) was also amplified using the same primer sets.

PMCA expression was increased during RANKL-induced osteoclast differentiation. (A) Human PBMCs were cultured in the presence of 30 ng/ml M-CSF and 100 ng/ml RANKL. After 3 and 7 d of culture (preosteoclast and mature osteoclast stages, respectively), total RNAs were extracted and subjected to DNA microarray analysis. The relative signal intensity of ATP2B4 (the gene encoding PMCA4) mRNA detected in a DNA microarray analysis is shown. Data are means ± SD (**, P < 0.01). (B) Mouse BMMs were incubated with 30 ng/ml M-CSF or M-CSF plus 100 ng/ml RANKL for 36 h. Biotinylated cell surface proteins were prepared as described in Materials and methods and subjected to LC/MS/MS analysis. The elution profile of peptides from LC is shown. Arrows indicate PMCA1 peptides identified by subsequent mass spectrometry analyses. (C and D) Mouse BMMs were stimulated with 100 ng/ml RANKL in the presence of 30 ng/ml M-CSF for the indicated times. Total RNA was subjected to RT-PCR analysis to examine the expression levels of Atp2b and Slc8a families (the genes encoding NCXs). The mRNA expression in mouse brain was used as a positive control. Ctsk (cathepsin K) is a marker gene for osteoclast differentiation. (E) Quantitative real-time PCR analysis of Atp2b1 and Atp2b4 transcripts was performed with BMMs treated with 100 ng/ml RANKL plus 30 ng/ml M-CSF for the indicated times. The mRNA expression levels were normalized against those of Hprt1. The error bars show the mean ± SD of three independent experiments. (F) Cell lysates from BMMs treated with 100 ng/ml RANKL plus 30 ng/ml M-CSF for the indicated times were subjected to Western blotting. The expression levels of total PMCA and NFATc1 were examined with pan-PMCA and NFATc1 antibodies. (G) PMCA immunostaining (Cy3) was performed with mature mouse osteoclasts cultured on dentin discs. Plasma membrane ganglioside GM1 was costained using FITC-conjugated cholera toxin B. Cross-sectional images were reconstructed through the z-axis by confocal microscopy (A, apical; B, basolateral). Bars, 5 µm. (H) BMMs were infected with viruses harboring empty vector (pMSCV) or constitutively active NFATc1 (pMSCV-NFATc1-CA). 2 d after infection, PMCA and NFATc1 expression was examined by Western blotting. (I) BMMs were treated with 100 ng/ml RANKL plus 30 ng/ml M-CSF for 2 d. ChIP was performed using an NFATc1 antobody followed by a PCR amplification of the promoter regions of Atp2b1 or Atp2b4 gene. Input DNA (1% of total) was also amplified using the same primer sets.

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