Fig. 1: The inhibitory effect of Cldn11 overexpression on OC differentiation and bone resorption was not associated with the expression of OPG and RANKL in OBs.

a Primary OBs were transfected with pMX or pMX-Cldn11 and cultured in a medium containing IL-1 (10 ng/mL) or PGE₂ (10⁻⁶ M) and VitD₃ (10⁻⁸ M) for 1 day. Quantitative real time RT-PCR was performed to determine the mRNA expression of OPG and RANKL. b OBs and BMCs transfected with pMX or pMX-Cldn11 were co-cultured for 7 days in a medium containing IL-1 (10 ng/mL) or PGE₂ (10⁻⁶ M) and VitD₃ (10⁻⁸ M). After culturing, cells were fixed, and the number of TRAP-positive MNCs (nuclei >5) was counted. c The mRNA level of Cldn11 was assessed by quantitative real time RT-PCR during osteoclastogenesis in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL). d BMMs transfected with pMX or pMX-Cldn11 were cultured in the presence of the indicated concentrations of RANKL. After 4 days, GFP expression to confirm the efficiency of retrovirus was visualized under a fluorescence microscope, and cultured cells were stained for TRAP. e The number of TRAP-positive MNCs (nuclei >5) per well was counted. f Mature OCs transfected with pMX or pMX-Cldn11 from the co-culture system were seeded in a 48-well plate for 48 h, in a hydroxyapatite-coated plate for 24 h, or in dentin slices for 48 h. The cells attached to the 48-well plate were stained with TRAP solution (left), and those attached to the hydroxyapatite-coated plate (middle) and dentin slices (right) were removed. Then, the plates were photographed under a light microscope. g The number of TRAP-positive MNCs (nuclei >5) was counted (left), and relative resorption areas in the hydroxyapatite-coated plate and dentin slices were quantified using Image-Pro Plus (Ver 4.5) software (right). Data are presented as the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. (not significant) vs. the pMX (control)