Fig. 6: Darapladib and Gpr31 mediate osteoclast differentiation via regulating p38 MAPK and mitochondrial energy metabolism.

A, B Knockdown efficiency of Gpr31 with siRNA from the level of gene and protein. n = 3 biologically independent wells per group. C TRAP staining to demonstrated the knockdown efficiency of Gpr31. D Immunoblotting showing deceased osteoclast markers expression in Gpr31 siRNA knockdown compared to control group during osteoclast formation. E KEGG analysis according to the differentially expressed genes of RNA-seq. F GSEA of oxidative phosphorylation and MAPK signaling pathway between RANKL and RANKL with Darapladib treatment. G, H The expression of MAPK signaling pathway in RANKL-induce osteoclast formation treated with drug or Gpr31 siRNA knockdown in Western blot. I–L OCR (left) and ECAR (right) of RANKL-induced BMMs with or without Darapladib, and statistical analysis of related parameters including Maximal respiration, ATP-linked respiration and Maximal ECAR. n = 3 biologically independent wells per group. M–P OCR (left) and ECAR (right) of BMMs incubated with Gpr31 siRNA compared with control group, and quantification of parameters Maximal respiration and Maximal ECAR. n = 3 biologically independent wells per group. (Q, R) The expression of mitochondrial respiratory chain, electron transport chain and osteoclast markers in WT BMMs treated with Darapladib or Gpr31 siRNA compared with control group. S The densitometric analysis of Western blot bands. n = 3 biologically independent cell samples per group. T, U Immunoblotting showing the expression of mitochondrial dynamics and osteoclast markers in WT BMMs treated with Darapladib or Gpr31 siRNA compared with control group. V The densitometric analysis of Western blot bands. n = 3 biologically independent cell samples per group. W–Z The flow diagram of MitoSOX and quantification of fluorescence intensity of probe. n = 3 biologically independent wells per group. Data were showed as mean ± SD. Two-sided Student’s t test (S, V, Z) was employed to assess the difference between the two groups. One-way ANOVA (A, X) and two-way ANOVA (J, L, N, P) were utilized for multiple comparisons test. Significance: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, ns non-significance.