Fig 1: Irisin facilitated osteogenesis in a mouse model of OI (A) Representative images of TRAP staining and HE staining of tibial sections from all groups after injecting Irisin (100 ?µg/kg/week) or vehicle for 8 weeks. Red-stained regions around the trabecular bone in TRAP-staining tibial sections were considered as TRAP-positive osteoclasts. Blue-stained regions around the subchondral trabecular bone in HE-staining tibial sections were considered mature osteoblasts. Scale bar represents 100 ?µm (B-C) Quantitative measurements of the number of osteoclasts/field of bone tissue (N.Oc/BS) in TRAP-staining sections and the number of osteoblasts/field of bone tissue (N.Ob/BS) in HE-staining sections were analyzed for each group (D) Representative immunohistochemical pictures of ALP, OCN, Runx2, and Collagen I in tibial sections. Scale bar represents 50 ?µm (E–H) Quantification of ALP, OCN, Runx2, and Collagen I expression in the immunohistochemical staining were analyzed by OsteoMetrics software. Wt/wt ?+ ?veh group (n ?= ?10), oim/oim ?+ ?veh group (n ?= ?7) and oim/oim ?+ ?Irisin group (n ?= ?8). Data are shown as mean ?± ?SD. *p ?< ?0.05, **p ?< ?0.01, ***p ?< ?0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig 2: Irisin improved bone mass and increased bone formation in a mouse model of OI (A) Representative 3D micro-CT images of trabecular (Red area) and cortical (Green area) regions in femora from each group after treatment with Irisin (100 ?µg/kg/week) or vehicle for 8 weeks (B–I) Tb. BMD (g/cm3), Tb.Th (µm), Tb.N (1/µm), Tb.BV/TV (%), Tb. Sp (µm), Ct. Th (µm), Ct. Ar (mm2), and Marrow area (mm2) in the corresponding region of femora in wt/wt ?+ ?veh group (n ?= ?10), oim/oim ?+ ?veh group (n ?= ?6) and oim/oim ?+ ?Irisin group (n ?= ?8) (J) Dynamic histomorphometry of tibia was performed through injecting calcein (3 days) and xylenol orange (10 days) before euthanasia. Representative images (green, calcein label; red, xylenol orange label) at the mid-diaphysis were radiographed by fluorescence light microscopy. Scale bars represent 200 ?µm and 50 ?µm (K–P) Bone formation parameters, including MAR, MS/BS, and BFR of endosteum and periosteum, were quantified in available hard tissue sections (n ?= ?6). Data are shown as mean ?± ?SD. *p ?< ?0.05, **p ?< ?0.01, ***p ?< ?0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig 3: Irisin alleviated TGF-ß1 mediated inhibition of Runx2 function at the osteocalcin promoter through promoting HADC4/5 degeneration via inducing sumoylation (A) Representative immunofluorescence photos of osteocalcin in oim/oim osteoblasts treated with TGF-ß1 (10 ?ng/mL), Irisin (100 ?nM), and RGDS peptide (20 ?µM) for 10 days. Blue indicated DAPI staining of nuclei. Scale bar represents 100 ?µm (B) Quantitative analysis of osteocalcin-positive areas in immunofluorescence-stained images (n ?= ?3) (C) MC3T3-E1 cells were seeded on 24-well plate and co-transfected with Runx2 overexpression plasmid, firefly reporter constructs containing 6OSE-promoter, and Renilla-expressing plasmid for 24 ?h. Firefly/renilla luciferase activities were determined 6 ?h after osteoblastic induction with TGF-ß1 (10 ?ng/mL), Irisin (100 ?nM), and RGDS peptide (20 ?µM) using Dual-Luciferase Reporter Gene Assay Kit (n ?= ?3) (D) MC3T3-E1 cells were treated with TGF-ß1 (10 ?ng/mL) and Irisin (100 ?nM) for 6 ?h, ChIP assays were performed using the antibody against Acetyl-Histone H4 and semi-quantitative PCR analysis was conducted with osteocalcin-specific primers (E–F) qRT-PCR and western blot analysis of HDAC4 and HDAC5 were performed for MC3T3-E1 cells after incubation with TGF-ß1 (10 ?ng/mL) and Irisin (100 ?nM) for 6 ?h (n ?= ?3) (G–H) Co-IP assay was used to detect the sumoylation of HDAC4 and HDAC5 by anti-Sumo-1 antibody in MC3T3-E1 cells after Irisin (100 ?nM) treatment for 6 ?h (I) MC3T3-E1 cells were seeded on 24-well plate and co-transfected with Runx2 overexpression plasmid, firefly reporter constructs containing 6OSE-promoter, and Renilla-expressing plasmid for 24 ?h. Firefly/renilla luciferase activities were determined 6 ?h after osteoblastic induction with TGF-ß1 (10 ?ng/mL), Irisin (100 ?nM), and COH000 (2 ?µM) using Dual-Luciferase Reporter Gene Assay Kit (n ?= ?3). R: RGDS peptide. Data are shown as mean ?± ?SD. *p ?< ?0.05, **p ?< ?0.01, ***p ?< ?0.001 versus Ctrl; #p ?< ?0.05, ##p ?< ?0.01, ###p ?< ?0.001 versus TGF-ß1; &p ?< ?0.05, &&p ?< ?0.01, &&&p ?< ?0.001 versus TGF-ß1+Irisin. (J) Schematic diagram illustrating the mechanism of Irisin-mediated osteogenic differentiation through promoting osteogenesis and antagonizing TGF-ß1-induced suppression of osteoblast differentiation. Path 1: TGF-ß1 bound to its receptor TßRII, then recruited TßRI and activated the phosphorylation of Smad2/3, resulting in inhibition of Runx2 function. Path 2: Irisin competitively antagonized TGF-ß/Smad signaling and co-induced ERK/p38 signaling, promoted HDAC4/5 degradation via inducing sumoylation, leading to attenuation of TGF-ß1-induced depression of osteogenesis. Path 3: Irisin bound to integrin receptors to activate ERK/p38 signaling and promoted osteoblast differentiation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig 4: Irisin antagonized TGF-ß1-induced suppression of osteogenic differentiation through integrin receptor-mediated and integrin receptor-independent mechanisms (A) Western blot analysis showing integrin receptors counteracted TGF-ß1-induced Smad2/3 phosphorylation and co-enhanced ERK/p38 signaling with RGDS peptide (20 ?µM) pretreatment for 30 ?min, followed by co-incubation with TGF-ß1 (10 ?ng/mL) for 1 ?h along with adding Irisin (100 ?nM), U0126 (10 ?µM), and SB203585 (10 ?µM) in oim/oim osteoblasts (B) Quantitative analysis of protein expression of Smad signaling and MAPK signaling (n ?= ?3) (C) Western blot analysis showing integrin receptors attenuated TGF-ß1-induced Smad2/3 phosphorylation in cytoplasm and nucleus with RGDS peptide (20 ?µM) pretreatment for 30 ?min, followed by co-incubation with TGF-ß1 (10 ?ng/mL) and Irisin (100 ?nM) for 1 ?h in oim/oim osteoblasts (D) Quantitative analysis of protein expression of Smad signaling in cytoplasm and nucleus (n ?= ?3) (E) Representative images of ALP and ARS staining of oim/oim osteoblasts after inducing osteoblastic differentiation with Irisin (100 ?nM) in the presence of TGF-ß1 (10 ?ng) and supplemented with RGDS peptide (20 ?µM), U0126 (10 ?µM), and SB203585 (10 ?µM) for 14 days and 21 days. Scale bar represents 200 ?µm (F) Quantification analysis of Cfu-ALP, ALP activity, Cfu-ARS, and ARS activity (n ?= ?3). Data are shown as mean ?± ?SD. *p ?< ?0.05, **p ?< ?0.01, ***p ?< ?0.001 versus Ctrl; #p ?< ?0.05, ##p ?< ?0.01, ###p ?< ?0.001 versus TGF-ß1; &p ?< ?0.05, &&p ?< ?0.01, &&&p ?< ?0.001 versus TGF-ß1+Irisin.
Fig 5: FNDC5/Irisin was decreased in a mouse model of OI (A) Quantification analysis of serum Irisin with ELISA in 3-month-old oim/oim and wt/wt mice for both male and female mice (n ?= ?8) (B) Representative images of HE & FNDC5 staining of TA muscles from wt/wt mice, unfractured and fractured side of oim/oim mice (n ?= ?8). Scale bar represents 50 ?µm (C–D) Quantification analysis of FNDC5 expression in HE staining of TA muscles from wt/wt mice, unfractured and fractured side of oim/oim mice (n ?= ?8) (E) Representative immunohistochemistry images characterizing the expression of FNDC5 in tibia from wt/wt mice, unfractured and fractured side of oim/oim mice (n ?= ?8). Scale bar represents 100 ?µm (F–K) Quantification analysis of FNDC5 expression in tibial growth plate, tibial cortical bone, and tibial trabecular bone from wt/wt mice, unfractured and fractured side of oim/oim mice (n ?= ?8). Data are shown as mean ?± ?SD. *p ?< ?0.05, **p ?< ?0.01, ***p ?< ?0.001.
Supplier Page from Novus Biologicals, a Bio-Techne Brand for Mouse Irisin/FNDC5 ELISA Kit (Colorimetric)