Fig. 8: Osteopontin regulates bone marrow-derived macrophages through the lactate/ATP6V0d2 axis.

a Relative expression of different V-ATPase subunits in BMDMs treated with or without (ctrl) rOPN (0.5 μg/ml) under osteoclastogenic induction for 3 days in vitro (n = 3 biologically independent samples). b Representative western blot images (left) and quantification (right, n = 3 biologically independent samples) of ATP6V0d2 in BMDMs, Oc and rOPN-treated Oc for 4 days in vitro. c Total intracellular ATP concentration in BMDMs (ctrl), rOPN (0.5 μg/ml)-treated BMDMs, and BMDMs treated with rOPN (0.5 μg/ml) plus antagonist 27 (100 nM) under osteoclastogenic induction for 0.5 h (n = 4 biologically independent samples). d Intracellular lactate concentration in BMDMs (ctrl), rOPN (0.5 μg/ml)-treated BMDMs, and BMDMs treated with rOPN (0.5 μg/ml) plus antagonist 27 (100 nM) under osteoclastogenic induction for 10 h (n = 4 biologically independent samples). e Relative expression of the indicated V-ATPase subunits in BMDMs stimulated with a gradient concentration of lactate for 24 h under osteoclastogenic induction. ^#represents significantly decreased (P < 0.0001) expression of Atp6v0d2 in 50 mM lactate-treated BMDMs compared with untreated BMDMs (n = 3 biologically independent samples). f, g Representative images of TRAP staining (f) and counting of Trap+ osteoclasts (n = 6 biologically independent samples) (g) after osteoclastogenic induction in the presence or absence (ctrl) of rOPN (0.5 μg/ml), lactate (50 mM), and rOPN with siRNA Cd61 for 5 days. Scale bar: 50 μm. Images are representative of 3 independent experiments. All data are presented as mean ± SD. Two-way ANOVA with Tukey’s post-hoc test (a, e) and one-way ANOVA with Tukey’s post-hoc test (b–d, g) were used. Source data are provided as a Source Data file.