Fig 1: P2X7 expression levels and intracellular Ca2+ concentrations are increased during osteoclast differentiation. Bone marrow mononuclear macrophages were cultured in the presence of M-CSF (30 ng/ml) and RANKL (60 ng/ml), and differentiation into mature osteoclasts was evaluated. Control BMMs were cultured in the presence of M-CSF (30 ng/ml). (A) Western blot analysis was performed to examine the protein expression levels of P2X7, TRAP, NFATc1 and c-fos. (B) Reverse transcription-quantitative polymerase chain reaction was performed to detect the mRNA expression levels of P2X7, TRAP, NFATc1 and c-fos. (C) TRAP-positive multinucleated cells were counted as osteoclasts. (D) Fluo3-AM was used to observe intracellular Ca2+ fluorescence intensity. Data are presented as the mean ± SD from three independent experiments. **P<0.01 vs. control. M-CSF, macrophage colony-stimulating factor; NFATc1, nuclear factor of activated T cells c1; RANKL, receptor activator of NF-?B ligand; TRAP, tartrate-resistant acid phosphatase.
Fig 2: Effects of P2X7 overexpression on osteoclast differentiation, Ca2+/calcineurin/NFATc1 signaling and autophagy. Bone marrow mononuclear macrophages were transduced with sh-P2X7 or transduced with HBLV-m-P2Rx7-3×fag-2s Green-Puro in the presence of M-CSF with or without RANKL. (A and B) Western blot analysis of the protein expression levels of P2X7, TRAP, CK, CAII, MMP-9, NFATc1, c-fos and calcineurin. (C and D) TRAP-positive multinucleated cells were counted as osteoclasts and scanning electron microscopy was used to observe bone resorption lacuna. (E and F) Western blot analysis of the protein expression levels of Beclin-1 and LC3II, and immunofluorescence analysis of LC3 fluorescent puncta. (G) Fluo3-AM staining was performed to detect intracellular Ca2+ concentration. Data are presented as the mean ± SD from three independent experiments. *P<0.05, **P<0.01 vs. NC group or as indicated. CAII, carbonic anhydrase II; CK, cathepsin K; M-CSF, macrophage colony-stimulating factor; MMP-9, matrix metalloproteinase-9; NC, negative control; NFATc1, nuclear factor of activated T cells c1; OE, overexpression; RANKL, receptor activator of NF-?B ligand; sh, short hairpin; TRAP, tartrate-resistant acid phosphatase.
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