Fig. 4: Overexpression of Cldn11 enhances OB differentiation and its intracellular signaling.

a–e Primary OB precursor cells were cultured in osteogenic medium containing AA (50 μg/mL) and β-gly (10 mM). a Cells were cultured for the indicated times, and mRNA levels of Cldn11 were analyzed by quantitative real-time RT-PCR. b OBs transfected with pMX or pMX-Cldn11 were cultured for 7 days in osteoblastogenic medium. ALP-positive cells were monitored by ALP staining, and ALP activity was expressed a function of released p-nitrophenol. c Calcium deposits for matrix mineralization were measured by ARS staining after 21 days, and the intensity of staining was quantified with CPC. d Total cell lysates from OBs transfected with pMX or pMX-Cldn11 were analyzed by performing western blotting with the indicated antibodies. β-actin was used as an internal control. e Cells were cultured for the indicated times, and the mRNA expression of the indicated genes was analyzed by performing quantitative real time RT-PCR. Data are presented as the mean ± SD of three independent experiments. **P < 0.01; ***P < 0.001 vs. pMX (control) of the indicated time point, respectively