Fig 1: 7,8-DHF positively regulates OPG expression via activating CREB.a MC3T3 cells cultured in OIM were treated with 7,8-DHF in different time points. Western blotting showed that 7,8-DHF inhibited C/EBPβ, increased Akt (S473), MAPK (p38), C-Jun, CREB phosphorylation. b Relative protein level of C/EBPβ, p- C/EBPβ, AEP, phosphorylated C-Jun, CREB, Akt, MAPK, and TrkB in MC3T3 cells treated with 7,8-DHF in different time points. Data are shown as mean ± SEM of three independent experiments, one-way ANOVA. c Western blotting showed that knockdown of CREB blunted 7,8-DHF-induced OPG expression. d Relative protein levels of RANKL, OPG, and RANKL/OPG ratio. Data are shown as mean ± SEM of three independent experiments, one-way ANOVA. e qPCR results showed that knockdown of CREB inhibited OPG mRNA expression induced by 7,8-DHF. Data are shown as mean ± SEM of three independent experiments, one-way ANOVA.
Fig 2: 7,8-DHF promotes MC3T3-E1 cells differentiation, mineralization and OPG secretion.a Representative images of ALP staining in MC3T3-E1 cells treated with BDNF or 7,8-DHF combined with or without K252a for 14 days (n = 3 independent experiments) (Scale bar, 5 mm (upper panel), 200 μm (lower panel)). b Representative images of Alizarin Red S mediated calcium staining in MC3T3-E1 cells treated with BDNF or 7,8-DHF combined with or without K252a for 21 days showed that 7,8-DHF promoted MC3T3 cells mineralization (n = 3 independent experiments) (Scale bar, 5 mm (upper panel), 200 μm (lower panel)). c MC3T3 cells were cultured in complete medium or osteogenic induction medium (OIM) with BDNF or 7,8 DHF combined with or without K252a for 4 days. Western blotting results showed 7,8-DHF inhibited C/EBPβ/AEP pathway and increased OPG expression, and K252 inhibited the effect of 7,8-DHF. d Relative protein levels of C/EBPβ, p-C/EBPβ, AEP, RANKL, OPG, Osterix, p-TrkB/TrkB, and p-Akt/Akt in MC3T3 cells cultured in the complete medium or OIM with BDNF or 7,8-DHF combined with or without K252a for 4 days. Data are shown as mean ± SEM of three independent experiments, one-way ANOVA; e AEP enzymatic activity assay. BDNF and 7,8-DHF inhibit AEP activities, and K252a abolish BDNF and 7,8-DHF’s effects. Data are shown as mean ± SEM of three independent experiments, one-way ANOVA. f qPCR results show that OPG mRNA expression increases in MC3T3 cells after 7,8-DHF treatment for 4 days. Data are shown as mean ± SEM of three independent experiments, one-way ANOVA. g 7,8-DHF increases OPG level and decreases the RANK-L/OPG ratio. Levels of OPG and RANK-L secreted into the medium were measured by ELISA. Data are shown as mean ± SEM of 3 independent experiments, one-way ANOVA.
Fig 3: The effects of osteoblast lineage‐specific deletion of GPR39 on bone structure and metabolism in female mice. Sixteen‐week‐old female mice with a conditional deletion of GPR39 in the osteoblast lineage (Gpr39Ob−/Ob−) were compared to mice harboring the floxed allele (Gpr39Fl/Fl) and osterix cre expressing mice (Osx cre). (A) Body length; (B) body weight; (C–F) μCT analysis of vertebral trabecular bone structural parameters. Femural (G) cortical bone area (Ct B. Ar); (H) cortical thickness (Ct.Th); (I) cortical bone area to total area (Ct.Ar/Tt.Ar); (J) cortical mean mineral content (Ct. TMD); (K–O) histomorphometric analyses of vertebral bone sections, and (P) the ratio between the serum concentrations of RANKL and OPG. Data presented as means ± SD (one‐way ANOVA). *p < 0.05, **p < 0.01, and ***p < 0.001.
Supplier Page from Abcam for Mouse RANKL ELISA Kit (TRANCE/TNFSF11)