Figure 5. Bone-resorbing function of osteoclasts is impaired in Becn1 cKO mice.
(a) qRT-PCR for Cre gene expression during osteoclast differentiation in BMMs isolated from Becn1 WT or cKO mice. (b) Western blotting for Beclin1 expression level during osteoclast differentiation. (c) TRAP staining following RANKL/M-CSF treatment on BMMs for 4 and 5 days. Bar = 100 μm. (d) Quantification of TRAP+ cells. (e) Dentin slice assay following RANKL/M-CSF treatment on BMMs isolated from Becn1 WT or cKO mice. Cells were removed with cotton swabs, and stained with FITC-wheat germ agglutinin (WGA). The resorbed pit depth on dentin slice was visualized in z stack by confocal microscopy. The image was reconstructed by Amira software. (f) Normalized resorbed pit depth excavated by Becn1 WT and cKO osteoclasts. Three different experiments (cell number = 100) were normalized to the average depth. (g) TRAP activity measurement from cell culture supernatant from dentin slices. (h) Western blotting for Beclin1 expression level from osteoclasts harvested from the dentin slices. (i) Western blotting against Beclin1, LC3B and β-actin during RANKL-induced osteoclast differentiation in BMMs from Becn1+/+ (WT) or Becn1+/− (Het) mice.