Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of fibroblast growth factor receptor 2 in bone marrow mesenchymal stem cells enhances osteogenesis and promotes critical cranial bone defect regeneration

doi: 10.3389/fcell.2023.1208239

Figure Lengend Snippet: The therapeutic effects of the Fgfr2 overexpression BMSCs carried by GelMA on mouse cranial bone defects. (A) Diagram of skull bone defect modeling and GelMA implantation with 405 nm light induction. (B) The skull bone defects in visual after 6 weeks of repair (n = 6). (C–E) The micro-CT images, bone defect area quantification, and bone volume analysis of the skulls repaired after 6 weeks (n = 6). (F) H&E staining, Masson staining, and OCN staining of bone defect areas after 6 weeks of repair. Graphs show the mean value ±SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For immunofluorescence staining, the sections were incubated with anti-OCN (1:200, A6205, Abclonal) and then incubated with secondary antibodies for inmmunohistochemistry.

Techniques: Over Expression, Micro-CT, Staining

Journal: Journal of Orthopaedic Surgery and Research

Article Title: T. gondii excretory proteins promote the osteogenic differentiation of human bone mesenchymal stem cells via the BMP/Smad signaling pathway

doi: 10.1186/s13018-024-04839-0

Figure Lengend Snippet: TgEP promoted the osteogenic differentiation of hBMSCs. ( A ) Cytotoxic effect of TgEP on hBMSCs detected using a cytotoxicity detection kit. ( B ) Effect of TgEP on cell proliferation assessed using a CCK-8 kit. ( C ) ALP staining was performed to evaluate ALP expression in the cells. Scale bar = 500 μm. ( D ) Quantification of ALP staining results using ImageJ software and statistical analysis. ( E ) Alizarin red staining was performed to measure the content of mineralized nodules in the cells. Scale bar = 500 μm. ( F ) Quantification of alizarin red staining results using ImageJ software and statistical analysis. ( G ) mRNA transcription levels of osteogenic genes were assessed using qRT‒PCR after the cells were treated for 3 or 7 days. ( H , J ) Western blotting analysis of the osteogenic proteins ALP, Runx2, Osx, and OCN in hBMSCs treated for 5 days ( H ) or 7 days ( J ). The expression levels were quantified using β-actin as a loading control ( I , K ). ( L ) Immunofluorescence staining was performed to detect the expression of Runx2 in the cells. Scale bar = 30 μm. (Data are presented as the mean ± SD; n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001, ns P >0.05 compared to the control group)

Article Snippet: For immunohistochemistry analysis, tissue sections were stained according to standard protocols using anti-osteocalcin (OCN) antibodies (Cat.#: 614,487) (1:100, ZEN-BIO, China).

Techniques: CCK-8 Assay, Staining, Expressing, Software, Western Blot, Control, Immunofluorescence

Journal: Journal of Orthopaedic Surgery and Research

Article Title: T. gondii excretory proteins promote the osteogenic differentiation of human bone mesenchymal stem cells via the BMP/Smad signaling pathway

doi: 10.1186/s13018-024-04839-0

Figure Lengend Snippet: TgEP promotes the repair of bone defects in vivo. ( A ) Histological analysis of bone tissue sections at 6 weeks, including H&E staining, Masson’s trichrome staining, and osteocalcin immunohistochemical staining. Scale bar = 500 μm. ( B ) Histological analysis of bone tissue sections at 8 weeks, including H&E staining, Masson’s trichrome staining, and osteocalcin immunohistochemical staining. Scale bar = 500 μm.

Article Snippet: For immunohistochemistry analysis, tissue sections were stained according to standard protocols using anti-osteocalcin (OCN) antibodies (Cat.#: 614,487) (1:100, ZEN-BIO, China).

Techniques: In Vivo, Staining, Immunohistochemical staining

Journal: PLoS ONE

Article Title: Murine GPRC6A Mediates Cellular Responses to L-Amino Acids, but Not Osteocalcin Variants

doi: 10.1371/journal.pone.0146846

Figure Lengend Snippet: Osteocalcin (OCN) variants used in this study.

Article Snippet: Purified bovine OCN was purchased from Meridian Life Science (Memphis, TN, USA).

Techniques: Sequencing, Purification, Recombinant, Produced

Journal: PLoS ONE

Article Title: Murine GPRC6A Mediates Cellular Responses to L-Amino Acids, but Not Osteocalcin Variants

doi: 10.1371/journal.pone.0146846

Figure Lengend Snippet: (A) Variants of the putative GPRC6A peptide ligand osteocalcin (OCN) fail to induce inositol phosphate accumulation in FlpIn-TREx-HEK293-mGPRC6A cells co-expressing Gα qG66D . (B) OCN variants (40 ng/ml) do not modulate L-ornithine-stimulated inositol phosphate accumulation at mGPRC6A (OCN variants as described in ).

Article Snippet: Purified bovine OCN was purchased from Meridian Life Science (Memphis, TN, USA).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Murine GPRC6A Mediates Cellular Responses to L-Amino Acids, but Not Osteocalcin Variants

doi: 10.1371/journal.pone.0146846

Figure Lengend Snippet: (A) OCN variants do not stimulate cAMP accumulation in FlpIn-TREx-HEK293 cells stably expressing mGPRC6A (open circles). Neither L-ornithine nor OCN variants inhibit forksolin (3 μM)-stimulated cAMP accumulation in the same cell line (filled circles; OCN variants as described in ). (B) Neither L-ornithine (1 mM) nor OCN variants (40 ng/ml) stimulate ERK1/2 phosphorylation in FlpIn-TREx-HEK293-mGPRC6A cells; (C) a similar lack of activity was observed in cells co-expressing Gα qG66D .

Article Snippet: Purified bovine OCN was purchased from Meridian Life Science (Memphis, TN, USA).

Techniques: Stable Transfection, Expressing, Phospho-proteomics, Activity Assay

Journal: PLoS ONE

Article Title: Murine GPRC6A Mediates Cellular Responses to L-Amino Acids, but Not Osteocalcin Variants

doi: 10.1371/journal.pone.0146846

Figure Lengend Snippet: (A) L-ornithine (20 mM) significantly increases GLP-1 release from GLUTag cells (** P < 0.01 vs Vehicle, one-way ANOVA followed by Sidak’s multiple comparisons test). This effect is significantly reversed by NPS-2143 (^ P < 0.05 vs. L-ornithine alone). Human synthetic OCN (0.01–10 ng/ml) did not significantly modulate GLP-1 release with or without cell washing. (B) High [glucose] significantly enhanced insulin secretion by MIN6 cells ( P < 0.0001, two-way ANOVA). L-ornithine (20 mM), but not mouse synthetic OCN (acid form; 0.03–100 ng/mL), significantly increased glucose-sensitive insulin secretion (** P < 0.01 vs. Vehicle, one-way ANOVA for high [glucose] group followed by Sidak’s multiple comparison test. ^^ P < 0.01 vs. L-ornithine alone). Open bars, no glucose; filled bars, 16.7mM glucose. Data normalised to Insulin release in the presence of 16.7mM glucose.

Article Snippet: Purified bovine OCN was purchased from Meridian Life Science (Memphis, TN, USA).

Techniques: Comparison

Journal: PLoS ONE

Article Title: Murine GPRC6A Mediates Cellular Responses to L-Amino Acids, but Not Osteocalcin Variants

doi: 10.1371/journal.pone.0146846

Figure Lengend Snippet: Concentration-dependent increases in cell index (a measure of impedance) in INS-1(832) cells (that do not express Gprc6a ) with synthetic human, purified bovine and synthetic mouse OCN. Statistical analysis performed by two-way ANOVA ([OCN] and treatment); there was a significant effect of both [OCN] and treatment for all studies; * P < 0.05, ** P < 0.01 vs. Vehicle by Sidak’s multiple comparison test.

Article Snippet: Purified bovine OCN was purchased from Meridian Life Science (Memphis, TN, USA).

Techniques: Concentration Assay, Purification, Comparison

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: The levels of osteocalcin (OCN) in the serum or cerebrospinal fluid (CSF) of control and 6-hydroxydopamine (6-OHDA)-induced Parkinson’s disease (PD) rat models. The CSF OCN levels in PD rats dramatically decreased compared to control rats ( A , n = 17). No difference in the serum OCN levels was found between control and PD rats ( B , n = 19). Expression of GPR158 and GPRC6A in various brain-derived tissues of 8-week-old rats ( C , n = 3). Coimmunoprecipitation of 8-week-old mice striata and proteins were then subjected to western blot analysis using anti-Gpr158 and anti-GPRC6a antibodies ( D , n = 3). Values were obtained from three independent experiments and results are given as the mean ± SEM. * P < 0.05.

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Control, Expressing, Derivative Assay, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: Effect of intraperitoneal (i.p.) injection of OCN on distance traveled (A) and rearing responses (B) in the open-field test (OFT). The cylinder test (C) and elevated body swing test (EBST; D ) were used to evaluate locomotor asymmetry. Compared with the sham groups, 6-OHDA injection decreased the distance traveled by rats in the OFT, and reduced the use of contralateral (left) forelimb in the cylinder test. The injection of 4 μg/kg OCN significantly improved the movement distance of 6-OHDA-lesioned rats in the 4th week ( P < 0.001), while 40 μg/kg OCN increased the number of rearing responses in the 3rd week ( P < 0.05). The representative traces of the OFT are shown in the lower panel of (A,B) . In the cylinder test, 4 μg/kg OCN improved the use of the contralateral forelimb ( P < 0.05). No obvious differences were observed in the EBST. n = 8–10, * P < 0.05 (compared with the 6-OHDA group).

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Injection

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: Effect of intra-striatum injection of OCN on distance traveled (A) , rearing responses (B) in OFT. The cylinder test (C) and EBST (D) were used to evaluate locomotor asymmetry. Compared with the sham groups, 6-OHDA injection decreased the distance traveled by rats, while the injection of OCN (4 μg) or nerve growth factor (NGF) significantly improved movement distance (A) , but not rearing impairment (B) of 6-OHDA-lesioned rats in OFT. The representative traces of the OFT are shown in the lower panel of (B) . No obvious differences were observed in the cylinder test (C) or the EBST (D) . n = 9–11, * P < 0.05 (compared with the 6-OHDA group).

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Injection

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: The protective effect of OCN on tyrosine hydroxylase (TH + ) neurons and fibers in the nigrostriatal system in the unilateral 6-OHDA-lesioned PD rat model. 6-OHDA induced ~90% dopaminergic neuronal loss on the lesioned side compared to the intact side of the substantia nigra (SN), which was improved by i.p. OCN injection (A) . Similarly, the decrease in TH-positive cell number in the SN of the lesioned side was also significantly alleviated by OCN injection (B) . The results from western blots measuring TH were consistent with immunohistochemistry in the striatum (C) . In the intra-striatum injection OCN group, 6-OHDA induced ~50% dopaminergic neuronal loss in the lesioned side compared to the intact side of the SN, which was improved by OCN injection (D) . Similarly, the decrease in TH-positive cell number in the SN of lesioned side was also significantly alleviated by intra-striatum OCN injection (E) . The results from western blots measuring TH were consistent with immunohistochemistry in the striatum (F) . n = 16, * P < 0.05 (compared with the 6-OHDA group).

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Injection, Western Blot, Immunohistochemistry

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: The effect of OCN on the proliferation of astrocytes and microglia. 6-OHDA significantly increased the number of GFAP-positive astrocytes in the lesioned side compared to the intact side both in the SN (A) and striatum (B) , which was inhibited by OCN injection. The same function of OCN on microglial proliferation was also observed, which was stained with Iba1 (C,D) . n = 16. Green = TH, Red = GFAP or Iba1, Blue = Nucleus.

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Injection, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: Neuroprotective effects of OCN on 6-OHDA-induced neurotoxicity. PC12 cells were incubated with different concentrations of 6-OHDA for 24 h, and cell viability was measured by cell counting kit-8 (CCK-8; A ). Pretreatment of PC12 cells with various concentrations of OCN (1–100 ng/ml) was performed 2 h before exposure to 100 μM 6-OHDA, and the cell viability was measured at 24 h (B) . The effects of OCN on the auto-oxidation of 6-OHDA were measured spectrophotometrically at 490 nm using a microplate reader (C) . The results from western blots showed that treatment with 100 μM 6-OHDA increased the expression of caspase-3 and decreased the expression of bcl-2, and both changes were reversed by pre-incubation with OCN (D) . 6-OHDA decreased the expression of phosphorylated AKT (Ser473) and phosphorylated GSK3β (Ser9) at 24 h (E) , and both reductions were reversed by pretreatment with OCN (F) . Similarly, 6-OHDA injection into the right striatum also resulted in decreased expression of p-AKT and p-GSK3β, and both reductions were inhibited by direct injection of OCN into the striatum (G) . Values were obtained from three independent experiments. * P < 0.05 (compared with the control group), # P < 0.05 (compared with the 6-OHDA group).

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Incubation, Cell Counting, CCK-8 Assay, Western Blot, Expressing, Injection, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: OCN protects PC12 cells from 6-OHDA-induced neurotoxicity. Apoptotic cells were measured by flow cytometry (A–E) . The number of apoptotic cells was counted in the R2 (AV + /7-amino-actinomycin, 7-AAD + , the late phase apoptotic cells) and R4 (AV + /7-AAD − , the early phase apoptotic cells) regions, and the apoptotic rate was calculated (F) . The CCK-8 test showed that OCN could attenuate the neuronal toxicity induced by 6-OHDA through activating AKT/GSK3β, while LY294002 reversed the neuroprotective effect of OCN on PC12 cells (G) through inhibiting the phosphorylation of AKT and GSK3β (H) . Values were obtained from three independent experiments. * P < 0.05 (compared with the control group), # P < 0.05 (compared with the 6-OHDA group).

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Flow Cytometry, CCK-8 Assay, Phospho-proteomics, Control