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Fig. 5. Overexpression of RGS10A significantly increases the sensitivity to RANKL signaling during osteoclast differentiation. BMMs were infected with pLenti-LacZ or pLenti-R10A. (A) Western blotting. Without RANKL stimulation, the signals of RGS10A were only detectable in cells infected with pLenti-R10A but not in the pLenti-LacZ-transfected cells (control). (B) Immunostaining. 80-90% of the cells express RGS10A in pLenti-R10A; however, there are no stained cells in pLenti-LacZ-infected cells. (C) TRAP+ staining. The cells were treated with 0, 5 and 10 ng/ml of RANKL in the presence of 10 ng/ml of M-CSF for 4 days. Without RANKL stimulation, 8% of precursor cells differentiated into mononuclear TRAP+ cells in pLenti-R10A (left lower panel). In pLenti-R10A cells, there are more TRAP+ cells and mature multinuclear cells compared with the control group in the presence of 5 or 10 ng/ml RANKL together with 10 ng/ml M-CSF (middle and right lower panels). (D) Quantitative analysis of TRAP+ MNCs in (C). The number of TRAP+ MNCs in pLenti-R10A is 1.5 times higher than that of the control group (P<0.05 vs pLenti-LacZ). (E) Immunostaining. With 5 ng/ml RANKL stimulation, pLenti-R10A-infected cells expressed Atp6i, cathepsin K, and NFAT2 at higher levels than the control cells. (F) The effect of FK506 on osteoclastogenesis induced by RGS10A overexpression in RGS10A-silenced RAW264.7 or control cells. FK506 (1 µg/ml) inhibited osteoclast differentiation from RANKL-stimulated RAW264.7 cells infected by pLenti-LacZ (panel 2) as compared with the culture without FK506 (panel 1). FK506 inhibited the rescue effect of RGS10A reintroduction as shown in panel 3 in RGS10A-silenced RAW264.7 cells infected with pLenti-RGS10a (panel 4).
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