Fig. 1

Silencing of parkin enhanced bone-resorbing activity. A–C Parkin mRNA and protein levels were determined by A qPCR and C (upper) western blot analysis, respectively, during differentiation of OBs in OM at the indicated times. BMMs were cultured with M-CSF (M) and RANKL (R) to induce differentiation into mOCs. Parkin transcripts and protein levels were determined by B qPCR and C (lower) western blot analysis. D Overexpression of parkin in primary mouse calvarial osteoblastic precursor cells generated by a virus infection system was detected by western blot analysis (upper). After 1 week, the cells were stained with ALP and AR to assess the degree of OB differentiation (lower). E Overexpression of parkin in OCPs using a virus infection system was confirmed by western blot analysis (left, upper). The infected cells were stained with TRAP solution (left, lower), and TRAP-positive MNCs were counted under a light microscope (×100) (right). F BMMs or OCPs primed with M-CSF (M) and RANKL (R) were transfected with control (Con) or parkin-specific siRNA #1 and #2 and incubated with fresh medium or osteoclastogenic medium for 24 h. The cell lysates were collected and analyzed by western blot analysis to quantify the levels of parkin protein expression. G Transfected cells were additionally cultured for 3 days in osteoclastogenic medium and stained with TRAP solution (upper). TRAP-positive MNCs (≥ 3 nuclei) were counted (lower) under a light microscope (×100). n.s. indicates not significant. H RANKL-primed OCPs on dentin were transfected with Con or parkin-specific siRNA. Resorption pits were visualized by staining with toluidine blue (upper), and the resorption pit area was quantified (lower). Original magnification, ×200. Scale bar, 100 μm. *P < 0.05, **P < 0.005, and ***P < 0.001 between the indicated groups. Data are represented as means ± SD from three independent experiments; P-values were calculated by Kruskal–Wallis or Tukey post hoc comparison tests