Fig. 3: Knockdown of Cldn11 promotes OC differentiation.

a BMMs were transfected with non-target control siRNA (siControl) or siRNA targeting Cldn11 (siCldn11), and the level of Cldn11 protein expression was analyzed by western blotting to confirm the knockdown efficiency of siCldn11. b, c BMMs transduced with siControl or siCldn11 were cultured in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 4 days. b Cultured cells were stained for TRAP. c The number of TRAP-positive MNCs (nuclei>5) was counted. d, e OBs and BMCs transfected with siControl or siCldn11 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). d After culturing, cells were fixed, and stained for TRAP. e The number of TRAP-positive MNCs (nuclei>5) was determined. f Mature OCs transfected with siControl or siCldn11 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). h, i BMMs were transduced with siControl or siCldn11 and h then cultured in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) for the indicated time. i Infected BMMs were serum starved for 6 h and stimulated with RANKL (100 ng/mL) for the indicated time points. Total cell lysates were analyzed by performing western blotting with the indicated antibodies. β-actin was used as an internal control. Data are presented as the mean ± SD of three independent experiments. *P < 0.05; **P < 00.01; n.s. (not significant) vs. the siControl