Fig 1: Cyp40 is a secreted factor that enters osteoclasts and inhibits Ras/c-Raf/p-Erk signaling, thereby promoting osteoclastogenesis. a Confocal images of osteoclasts isolated cocultured with SN that were transfected with plasmids containing Cyp40 tagged with EGFP. b ELISAs were performed to analyze the levels of secreted Cyp40 in the cell culture medium from varying numbers of sensory neurons (n = 3). c–e Cyp40 is secreted from sensory nerves and enters osteoclasts via a nonexosomal mechanism. To test this hypothesis, we separated the coculture medium into exosomal (Exo) and nonexosomal supernatants (MS), which were analyzed separately via Western blotting for the presence of Cyp40 (c). d Representative IEM images of the distribution of EGFP-tagged Cyp40 (black granules) in osteoclasts. Osteoclasts on the bone surface were cocultured with SN cells transfected with plasmids containing EGFP-tagged Cyp40. e Representative IEM images of EGFP-tagged Cyp40 (black granules, red arrow) in the brain and membrane vehicles (green arrow). f Western blotting of Ras, c-Raf, Erk, and p-Erk in osteoclasts, which were isolated and cocultured without (Ctrl), or with recombinant Cyp40 (rCyp40), SN (Co-SN), or Cyp40 knockout SN (Co-SN (shCyp40)). g Following the induction of osteoclast differentiation of BMMCs from RTX-treated or Ctrl mice, Western blot analysis was performed to determine the levels of AhR, Ras, c-Raf, and p-Erk proteins in the generated osteoclasts. h, i Western blotting of NFATc1 in osteoclasts, which were isolated and cocultured without (Ctrl), or with recombinant Cyp40 (rCyp40), SN (Co-SN), or Cyp40 knockout SN (Co-SN (shCyp40)). j qPCR analysis of NFATc1 mRNA (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and N.S. means not significant, versus controls, Student’s t test. The results are expressed as the mean ± s.d