Fig. 6: Cldn11 negatively modulates osteoclastogenesis via interaction with the EphrinB2 ligand.

a The expression of Cldn11, EphrinB2, and EphB4 and the co-localization of these proteins in osteoclast precursor cells were observed with a confocal imaging assay. b The mRNA expression of EphrinB2 and EphB4 was evaluated during preOC differentiation into mature OCs by quantitative RT-PCR. c BMMs were cultured in the presence of M-CSF (10 ng/mL) for 1 day prior to cell lysis. Lysates were immunoprecipitated with EphrinB2 or EphB4 antibody and subsequently immunoblotted with Cldn11 antibody. d BMMs transfected with siControl or siCldn11 were cultured and lysed in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 24 and 48 h. Lysates were immunoprecipitated with EphrinB2 antibody and subsequently immunoblotted with Cldn11 antibody. c, d Co-immunoprecipitated samples (top) or total cell extracts (bottom) were subjected to western blotting to detect Cldn11, EphrinB2, EphB4, and β-actin. e BMMs were transfected with control siRNA or siCldn11, and one of the siCldn11 groups was treated with EphB4-Fc. Subsequently, the cells were cultured in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 4 days. After culturing, cells were fixed and stained (left), and the number of TRAP-positive MNCs was counted (right). (f) Mature OCs transfected with siControl or siCldn11 from the co-culture system were seeded on dentin slices, and one of the siCldn11 groups was treated with EphB4-Fc, and then mature OCs were cultured for 48 h. Attached cells on the plates were removed and photographed under a light microscope (left). Pit areas were quantified using Image-Pro Plus (Ver 4.5) software (right). *P < 0.05; ***P < 0.001 vs. the siControl, ^#P < 0.05; ^###P < 0.001 vs. the siCldn11