Fig. 1

Enhanced SLIT3-ROBO1 signaling induced by LPS in OCPs regulates osteoclast differentiation by switching the C/EBP-β isoform. A, B OCPs were incubated with LPS for 4 h and the Slit3 (A) and Robo1 (B) mRNA levels were determined by qPCR. C OCPs were incubated with PBS or 10 ng/mL LPS for 6 h and immunofluorescence analysis was performed to detect SLIT3 and ROBO1. Scale bar, 20 μm. D OCPs were incubated with or without LPS for 24 h and the SLIT3 level was measured by ELISA. E, F. LPS-dependent SLIT3 upregulation was analyzed by qPCR (E) and ELISA (F) in OCPs isolated from WT or Tlr4^−/− mice in the presence of 10 ng/mL LPS. G-I OCPs isolated from WT or Slit3^−/− mice were cultured in a medium containing RANKL and LPS. After TRAP staining (G), the number of TRAP⁺ MNCs in the panel was quantified (H) and the increase of OC numbers in Slit3^−/− (Slit3^−/−/WT) was calculated with LPS stimulation or not (I). Scale bar, 200 μm. J The mRNA expression of Pu.1, Nfatc1, and Ctsk were measured in pOCs from WT or Slit3^−/− mice, which had been incubated with or without LPS in a medium including M-CSF and RANKL for 24 h. K The NFATc1 protein level in cell lysates of WT or Slit3^−/− mature OCs, treated with or without LPS, was determined by immunoblotting. L The C/EBP- β protein isoforms LAP, LIP, and MafB, in addition to SLIT3 and β-actin, were analyzed. M, N OCPs were cultured with M-CSF in the presence of rSLIT3. The mRNA level of C/ebp-β was determined by qPCR (M), and the protein level of the LAP, LIP isoforms, and actin were analyzed by immunoblotting (N). Data are shown as mean ± s.d., * P < 0.05, **P < 0.005, ***P < 0.0001. All representative data from three independent experiments are shown