Fig 1: FMT treatment promoted osteogenesis. (A) OPN (B) RUNX2, and (C) OPG stained the sections of distal femur. Scale bars represented 50 ?µm; (D) No. OPN+ cells (E) No. RUNX2+ cells; (F) No. OPG+ cells (G) The serum concentration of OPG. Data were expressed as the mean ?± ?SD (n ?= ?5). One-way ANOVA procedure followed by the Tukey test and Pearson's correlation were used to assess statistical significance. The different letters represented significant differences between different groups, *P ?< ?0.05, **P ?< ?0.01, and ***P ?< ?0.001 compared with corresponding group.
Fig 2: Effects of BMP2 overexpression on Dex-induced protein alterations in MC3T3-E1 cells. MC3T3-E1 cells were infected with pLVX-BMP2 prior to 1 µM Dex treatment for 48 h, in the absence or presence of 60 µM mangiferin pretreatment for 3 h. (A) Cell lysates were subjected to western blot analysis using BMP2, p-Smad-1 and Smad-1-specific antibodies. (B) Intensity of the protein bands of a typical experiment was semi-quantified using ImageJ 1.41o software. (C) Cell lysates were subjected to western blot analysis using OSX, OCN, OPG, RANK, RANKL, Bcl-2, and Bax-specific antibodies. (D and E) Intensity of the protein bands of a typical experiment was semi-quantified using ImageJ 1.41o software. +++P<0.001 compared with the control group; #P<0.05, ##P<0.01 and ###P<0.001 compared with the Dex treatment group; &P<0.01 and &&P<0.01 compared with the Dex + pLVX-BMP2 treatment group. Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; BMP2, bone morphogenetic protein 2; Dex, dexamethasone; OCN, osteocalcin; OPG, osteoprotegerin; OSX, osterix; p-Smad-1, phosphorylated-Smad-1; t-Smad-1, total Smad-1; RANK, receptor activator of nuclear factor-?B; RANKL, RANK ligand; Smad-1, SMAD family member 1.
Fig 3: LncRNA SNHG1 enhances Opg methylation to suppress the expression of Opg. (A) The mRNA expression of Opg by qPCR in BMSCs treated with adipogenic differentiation inducer for 14 days; (B) MSP analysis to detect the methylation status of Opg; (C) The mRNA expression of Opg by qPCR in BMSCs transfected with pcDNA-SNHG1/empty vector or sh-SNHG1/sh-NC; (D) The protein level of Opg by western blot in BMSCs transfected with pcDNA-SNHG1/empty vector or sh-SNHG1/sh-NC; (E, F) The mRNA expression and protein level of Opg in pcDNA-SNHG1 or empty vector transfected BMSCs after 5-Aza treatment for 96 h; (G) MSP analysis to detect the methylation status of Opg in pcDNA-SNHG1 or empty vector transfected MSCs after 5-Aza treatment for 96 h. NM, normal growth medium; AM, adipogenic induced medium. Comparisons were conducted using Student's t-test or one-way ANOVA with post-hoc analysis where appropriate. *p < 0.05; **p < 0.01
Fig 4: Illustration of this study’s results and a working model. BMP signaling enhances the Rankl/Opg expression ratio in cells of the osteoblastic lineage to promote osteoclastic bone resorption in bone remodeling. Moreover, the BMP-Smad pathway directly induces Atoh8 in immature osteoblasts. Atoh8 interacts with Runx2 and inhibits Runx2 transcriptional activity to suppress autoregulation of Runx2 and subsequent osteoblastic differentiation, as well as the Rankl/Opg expression ratio, thereby regulating the osteoclast number negatively to prevent excessive BMP-mediated bone resorption (left panel). The enhanced Runx2 activity in Atoh8-KO mice likely limited osteoblast maturation to maintain bone formation, while Runx2 increased the Rankl/Opg expression ratio to promote bone resorption and loss (right panel)
Fig 5: Silencing lncRNA SNHG1 suppresses BMSC adipogenic differentiation but promotes BMSC osteogenic differentiation. BMSCs were transfected with sh-SNHG1 or sh-NC. (A) LncRNA SNHG1 expression of BMSCs transfected with sh-SNHG1 or sh-NC; (B) Calculated adipocyte density after ORO staining with represented images; (C) The mRNA expressions of Opg, Runx2, Ppar-?, Fabp4, ALP, Bmp4, Bglap and Cebpa by qPCR; (D) Immunofluorescence staining for Ppar-?; (E) The protein levels of Opg, Runx2, Ppar-? and Fabp4 by western blot; (F) ARS absorbance after ARS staining with represented images; (G) ALP absorbance after ALP staining with represented images. Comparisons were conducted using Student's t-test. *p < 0.05; **p < 0.01
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