Fig 2: The role played by mitophagy in osteoclast fission and apoptosis(A) Representative Annexin-V/PI staining fluorescence images showing fused osteoclasts, osteoclasts undergoing fission, and apoptosis osteoclasts (Annexin-V, green; PI, red) (n = 5).(B) The relative mRNA levels of Fbxo7 genes in fused osteoclasts, osteoclasts undergoing fission, and apoptosis osteoclasts (n = 3).(C) Western blot analysis of the protein levels of TOM20, BNIP3, PINK1, Parkin, and GAPDH in fused osteoclasts, osteoclasts undergoing fission, and apoptosis osteoclasts (n = 3).(D) Quantification of TOM20, BNIP3, PINK1, and Parkin by immunoblotting.(E) Representative transmission electron microscope images showing fused osteoclasts, osteoclasts undergoing fission, and apoptosis osteoclasts (n = 3). The red arrowheads indicate the condensation of nuclear chromatin. The red dotted circles indicate mitochondria fracture and decreased density. Yellow arrowheads indicate autolysosomes.(F) Quantification of mitochondria and autolysosomes by transmission electron microscope.*p < 0.05 by one-way ANOVA (B, D and F).

Fig 3: Generation and re-fusion of osteomorphs(A) Representative images of immunofluorescence staining (Fbxo7, green; DAPI, blue) in BMMs, fused osteoclasts, and osteoclasts undergoing fission (n = 5). Yellow dotted circles indicate large osteoclasts. The red arrowhead indicates osteoclasts undergoing fission.(B) Representative images showing osteoclast division and re-fusion. Red arrowhead indicates osteoclasts undergoing fission. Purple arrowhead indicates osteoclasts undergoing re-fusion. Yellow arrowheads indicate the re-fused osteoclasts generated after treatment with RANKL for 12 h.

Fig 4: Osteoclast-osteomorph recycling in vitro(A) The timeline of the experiment.(B) Representative images of osteoclast fission. Red arrowheads indicate daughter cells.(C) Screenshot of the key frame of Video S1. Red arrowheads indicate cell bridged process. Yellow arrowhead indicates daughter cell. Yellow dotted box indicates the cell outline.(D) Screenshot of the key frame of Video S2. Red arrowheads indicate cell bridged process. Yellow arrowhead indicates daughter cell.(E) Proportion of osteoclasts with different fates.(F) The relative mRNA levels of DC-STAMP, TRAP, CTSK, and NFATc1 genes in bone marrow macrophages (BMMs), fused osteoclasts, and osteoclasts undergoing fission (n = 3).(G) Western blot analysis of the protein levels of DC-STAMP, TRAP, CTSK, NFATc1, and GAPDH in BMMs, fused osteoclasts, and osteoclasts undergoing fission.(H) The relative mRNA levels of the Fbxo7 and Bpgm genes in fused osteoclasts and osteoclasts undergoing fission (n = 3).(I) Western blot analysis of the protein levels of Fbxo7, Bpgm, and GAPDH in fused and osteoclasts undergoing fission (n = 3).(J) Quantification of Fbxo7 and Bpgm by immunoblotting.*p < 0.05 by Student’s t test (H and J) or by one-way ANOVA (F).