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Figure 3


Fig. 3. RGS10A silencing blocks osteoclast differentiation. (A) TRAP+ MNCs were formed in RANKL-stimulated RAW264.7 cells transfected with mock shRNA or pAVU-scrambled shRNA, but not in the cells transfected with pAVU-R10a or pAVU-R10b. (B) Quantitative analysis of TRAP+ MNCs in (A). TRAP+ MNCs in control groups, mock (*) or pAVU-scrambled (**), are more than 15 times (P<0.05) that of RGS10A-silenced groups (pAVU-R10a or pAVU-R10b). (C) Formation of TRAP+ MNCs in RANKL-stimulated BMMs infected with pLenti-scrambled and the absence of TRAP+ MNCs in the cells infected with pAVU-R10a are shown. (D) Quantitative analysis of TRAP+ MNCs in (C), pLenti-R10a vs the RGS10A-silenced group (*P<0.05). (E) Immunofluorescent staining of cathepsin K and Atp6i in RGS10A-silenced BMMs stimulated with RANKL and M-CSF. RGS10A silencing blocks the expression of cathepsin K and Atp6i as showed in pLenti-R10a groups. (F) Acridine orange staining. Strong orange fluorescence indicates extracellular acidification in the mock or pLenti-scrambled, but not in pLenti-R10a or pLenti-R10b. (G,H) RGS10A silencing-mediated apoptosis in multinucleated osteoclasts with RANKL stimulation. (G) The nuclei of osteoclast cells are condensed and fragmented in the RGS10A-silenced cells as indicated by the arrows, compared with the control cells. (H) Time course of RGS10A silencing-mediated apoptosis. Apoptosis was quantified by counting multinucleated cells with condensed nuclei (each point *P<0.05 vs control).





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