Figure 6.

LNGFR silencing decreases nuclear β-catenin levels and decreased EMSC osteogenic differentiation. (a) EMSCs were transfected with siLNGFR or siNC for 24 h, and then LNGFR levels were detected in EMSCs by immunofluorescence assays. Scale bar represents 50 μm. (b–d and f) EMSCs, which had been transfected with siLNGFR or siNC for 24 h, were treated with osteogenic induction solution for another 7 days. On day 3, the osteogenic induction solution was changed. On day 7, the cells were harvested. Then, the mRNA and protein levels of LNGFR were detected by (b) qPCR and (c) Western blot, respectively, (d) the ALP staining depth was observed by optical microscopy, and (f) nuclear β-catenin protein levels were detected by Western blot. Full-length blots of (c) and (f) are presented in Supplementary Figure S4a and b, respectively. (e) EMSCs, which had been transfected with siLNGFR or siNC for 24 h, were treated with osteogenic induction solution for another 21 days. The osteogenic induction solution was changed every three days. Then, the mineralized nodules were photographed after alizarin red staining. GAPDH was used as a total protein control and Lamin B1 was used as a nuclear protein control in Western blot. GAPDH was used as a control in qPCR. Scale bar represents 50 μm in ALP staining and 150 μm in alizarin red staining. ALP staining, alkaline phosphatase staining; Inducer, the osteogenic induction solution; siLNGFR, siRNA for LNGFR; and siNC, negative control siRNA. The results represent the mean ± SD from three independent experiments performed in triplicate. *P < 0.05 and **P < 0.01.