Fig. 5: Darapladib suppresses osteoclast formation via Alox12/12-HETE signaling axis.

A–D The expression of enzymes associated with the arachidonic acid metabolism pathway in osteoclasts treated with the inhibitor or ablation of Pla2g7 according to immunoblotting and relative RNA analysis. n = 3 biologically independent wells per group associated with cyclooxygenase (COX) and cytochrome P450 (CYP) pathways and n = 4 per group associated with lipoxygenase (LOX) pathways. E, F The immunofluorescence images of Alox12 in osteoclasts treated with Darapladib and quantification of fluorescence intensity. n = 3 biologically independent wells per group (scale bar, 10 μm). G, H The expression of Alox12 and Nfatc1 in mice treated with drug and statistical analysis of fluorescence intensity. n = 6 biologically independent mice per group (scale bar, 100 μm). I TRAP staining indicating Alox12 inhibitor treatment on Pla2g7-induced osteoclasts differentiation. (scale bar, 200 μm). J–L ML355 treatments (200 nM) on Pla2g7-induced osteoclasts in immunoblotting and RT-qPCR. n = 3 per group. n = 3 biologically independent wells per group. M The concentration of 12-HETE in RANKL or RANKL and Darapladib treatments were assessed following a 3-day induction period, coinciding with osteoclast formation. n = 4 biologically independent mice per group. N, O Darapladib or 12-HETE treatments (100 ng/mL) on RANKL-induced BMMs in TRAP staining and quantification of TRAP positive MNCs and average osteoclast size in each group. n = 6 biologically independent wells per group. P, Q Relative mRNA and protein expression in osteoclasts treated with Darapladib or 12-HETE, and the densitometric analysis of Western blot bands. For RNA analysis, n = 3 biologically independent wells per group. For Western blot analysis, n = 3 biologically independent cell samples per group. R Immunoblotting showing these markers in osteoclasts incubated with or without 12-HETE. Data were showed as mean ± SD. Two-sided Student’s t test (B, D, F, H, M) was employed to assess the difference between the two groups. Two-way ANOVA (K, O–Q) was utilized for multiple comparisons test. Significance: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, ns non-significance.