Fig. 2: LRF expression is associated with osteoclast survival.

A Diagram of experimental setup. BMMs were incubated with M-CSF (10 ng/ml) and RANKL (50 ng/ml) for 48 h. Cells were detached, re-seeded in culture dishes or dentin slices, and cultured for another 48 h. B Immunofluorescence staining of osteoclasts formed on culture dishes or dentin slices with LRF and cathepsin K antibodies. Bar = 20 μm. After culturing, cells were fixed, and stained for LRF (green) and cathepsin K (red). C RT-qPCR analysis of Lrf, Ctsk, or Bcl-xl expression in the cells formed on culture dishes or dentin slices. D 10 μM LY294002 was added to osteoclasts for 12 h. After culturing, cells were fixed, and stained for LRF (green). Nuclei were stained with To-Pro-3 iodide (blue). E After 48 h of BMMs stimulated with RANKL, alendronate was added for 24 h. RT-qPCR analysis of Lrf expression in osteoclasts was performed. In RT-qPCR analysis, expression was normalized with Gapdh (n = 3). Data are presented as mean ± SD. Student’s t-test was used for statistical analysis in C. *P < 0.05, **P < 0.01 compared to osteoclasts formed on culture dishes (C). The differences between means and effects of treatments were determined by one-way ANOVA followed by Tukey’s post hoc test in (E). *P < 0.05, compared to osteoclasts cultured without alendronate (E).