Journal: Biomolecules

Article Title: Anti-Inflammatory and Chondroprotective Effects of Vanillic Acid and Epimedin C in Human Osteoarthritic Chondrocytes

doi: 10.3390/biom10060932

Figure Lengend Snippet: Gene expression assays ( human ) used for real-time PCR.

Article Snippet: GDF-5 , 5′ FAM-3′ NFQ , Hs00167060_m1.

Techniques: Gene Expression

Journal: PLoS ONE

Article Title: Human Chondrocytes Respond Discordantly to the Protein Encoded by the Osteoarthritis Susceptibility Gene GDF5

doi: 10.1371/journal.pone.0086590

Figure Lengend Snippet: The amino acid substitutions of GDF5 variants A and B.

Article Snippet: The cells were stimulated with four different recombinant GDF5 proteins (mouse GDF5, R&D Systems; human wild type GDF5 and two variants A and B, Biopharm GmbH) at 10 ng/ml, 30 ng/ml, 100 ng/ml and 300 ng/ml.

Techniques: Variant Assay

Journal: PLoS ONE

Article Title: Human Chondrocytes Respond Discordantly to the Protein Encoded by the Osteoarthritis Susceptibility Gene GDF5

doi: 10.1371/journal.pone.0086590

Figure Lengend Snippet: Y-axis represents the luciferase activity readings generated in SW1353 cells in response to exogenous growth factor. Cells were stimulated for 6, 12, 24 and 48 hours with wildtype mouse GDF5 (A), wildtype human GDF5 (B), human GDF5 variant A (C) and human GDF5 variant B (D). BMP2 and TGF-β1 stimulations were used as positive controls. Error bars represent the standard error of the mean. GDF5 10, 10 ng/ml; GDF 30, 30 ng/ml; GDF5 100, 100 ng/ml; GDF5 300, 300 ng/ml. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, *****P<0.00001, two-tailed Student’s t-test.

Article Snippet: The cells were stimulated with four different recombinant GDF5 proteins (mouse GDF5, R&D Systems; human wild type GDF5 and two variants A and B, Biopharm GmbH) at 10 ng/ml, 30 ng/ml, 100 ng/ml and 300 ng/ml.

Techniques: Luciferase, Activity Assay, Generated, Variant Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: Human Chondrocytes Respond Discordantly to the Protein Encoded by the Osteoarthritis Susceptibility Gene GDF5

doi: 10.1371/journal.pone.0086590

Figure Lengend Snippet: Chondrocytes were cultured in monolayer and stimulated with each of the four GDF5 proteins for 15 minutes, 30 minutes, 1-β1 stimulation for 1 hour was used as a positive control. Protein was extracted and subjected to western blot analysis using an antibody against Smad 1/5/8 (anti-Smad1/5/8), with anti-β-actin antibody used as a loading control. This data comes from one OA patient. Identical data was obtained for a second OA patient (data not shown).

Article Snippet: The cells were stimulated with four different recombinant GDF5 proteins (mouse GDF5, R&D Systems; human wild type GDF5 and two variants A and B, Biopharm GmbH) at 10 ng/ml, 30 ng/ml, 100 ng/ml and 300 ng/ml.

Techniques: Cell Culture, Positive Control, Western Blot, Control

Journal: PLoS ONE

Article Title: Human Chondrocytes Respond Discordantly to the Protein Encoded by the Osteoarthritis Susceptibility Gene GDF5

doi: 10.1371/journal.pone.0086590

Figure Lengend Snippet: Chondrocytes were stimulated for 6, 12, 24 and 48 hours with wildtype mouse GDF5 (A), wildtype human GDF5 (B), human GDF5 variant A (C), human GDF5 variant B (D) and TGF-β1 (E). Each cross represents a significant (P<0.05, two-tailed Student’s t-test) up/down regulation of gene expression relative to untreated cells in one patient. Twelve patients were studied for each of the GDF5 growth factor treatments and ten patients for the TGF-β1 treatments.

Article Snippet: The cells were stimulated with four different recombinant GDF5 proteins (mouse GDF5, R&D Systems; human wild type GDF5 and two variants A and B, Biopharm GmbH) at 10 ng/ml, 30 ng/ml, 100 ng/ml and 300 ng/ml.

Techniques: Variant Assay, Two Tailed Test, Gene Expression

Journal: PLoS ONE

Article Title: Human Chondrocytes Respond Discordantly to the Protein Encoded by the Osteoarthritis Susceptibility Gene GDF5

doi: 10.1371/journal.pone.0086590

Figure Lengend Snippet: Chondrocytes were stimulated for 5 days with wildtype mouse GDF5 (A), wildtype human GDF5 (B), human GDF5 variant A (C), human GDF5 variant B (D) and TGF-β1 (E). Each cross represents a significant (P<0.05, two-tailed Student’s t-test) up/down regulation of gene expression relative to untreated cells in one patient. Ten patients were studied for each of the growth factor treatments.

Article Snippet: The cells were stimulated with four different recombinant GDF5 proteins (mouse GDF5, R&D Systems; human wild type GDF5 and two variants A and B, Biopharm GmbH) at 10 ng/ml, 30 ng/ml, 100 ng/ml and 300 ng/ml.

Techniques: Variant Assay, Two Tailed Test, Gene Expression

Journal: Stem Cells International

Article Title: Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

doi: 10.1155/2013/326828

Figure Lengend Snippet: Plasmid map of pCMV6-AC-GFP vector containing the true gold ORF of human GDF5 (GenBank accession number NM_000557) with C-terminal TurboGFP.

Article Snippet: The plasmid contains a CMV promoter and a fusion protein of GDF5 including a GFP-tag (OriGene Technologies Inc., Rockville, MD, US) ( ).

Techniques: Plasmid Preparation

Journal: Stem Cells International

Article Title: Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

doi: 10.1155/2013/326828

Figure Lengend Snippet: Primer list used for RT-PCR.

Article Snippet: The plasmid contains a CMV promoter and a fusion protein of GDF5 including a GFP-tag (OriGene Technologies Inc., Rockville, MD, US) ( ).

Techniques:

Journal: Stem Cells International

Article Title: Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

doi: 10.1155/2013/326828

Figure Lengend Snippet: (a) and (b) pmaxGFP-transfected hMSC 48 hours after C-17 (a) or U-23 (b) electroporation using the Amaxa nucleofector. (c) and (d) GDF5 expression of transfected and control hMSC after 14 days of monolayer culture, anti-GDF5 antibody from GeneTex. Blue: cell nucleus stained by DAPI, green: translated GFP, red: intracellular GDF5. (c) Cell population at a resolution of 20x or (d) close-up (68x) hMSC transfected with RG207105, inlet in C = sham control.

Article Snippet: The plasmid contains a CMV promoter and a fusion protein of GDF5 including a GFP-tag (OriGene Technologies Inc., Rockville, MD, US) ( ).

Techniques: Transfection, Electroporation, Expressing, Staining

Journal: Stem Cells International

Article Title: Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

doi: 10.1155/2013/326828

Figure Lengend Snippet: Glycosaminoglycan (GAG)/DNA ratio of outer and inner annulus fibrosus (AF) after 7 days of free-swelling organ culture of bovine intervertebral discs. Please note the difference between untransfected hMSC, control, pmaxGFP, and RG207105 transfected cells. The GDF5 overexpressing cells are pushing partially GAG/DNA ratio back in bovine IVD organ culture.

Article Snippet: The plasmid contains a CMV promoter and a fusion protein of GDF5 including a GFP-tag (OriGene Technologies Inc., Rockville, MD, US) ( ).

Techniques: Organ Culture, Transfection

Journal: Scientific Reports

Article Title: Mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-induced Smad1/5/8 phosphorylation

doi: 10.1038/srep23670

Figure Lengend Snippet: Sections of the molar of E18 mouse were immunostained for GDF5 ( a ), BMPRIA ( b ), BMPRIB ( c ), and BMPRII ( d ). Scale bars: 200 μm. Abbreviations: B, Buccal side; L, Lingual side.

Article Snippet: The rat GDF5 (XM_001066344.2) gene was ligated into the pCMV/hygro-His expression plasmid (RG80131-G-H; Sino Biological Inc., Beijing, China).

Techniques:

Journal: Scientific Reports

Article Title: Mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-induced Smad1/5/8 phosphorylation

doi: 10.1038/srep23670

Figure Lengend Snippet: Expression of GDF5 and various BMPRs, ACVRs was measured in the molar tooth germ cells from embryonic day 13 (E13) to 7 days postnatal (P7) using real-time PCR. Expression of each gene was normalised to that of GAPDH, and the relative expression at each time point was determined based on the expression at E13 (set to 1.0).

Article Snippet: The rat GDF5 (XM_001066344.2) gene was ligated into the pCMV/hygro-His expression plasmid (RG80131-G-H; Sino Biological Inc., Beijing, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-induced Smad1/5/8 phosphorylation

doi: 10.1038/srep23670

Figure Lengend Snippet: SF2 cells were incubated with various combinations of BMP2 (100 ng/ml) and GDF5 (100 or 1000 ng/ml), and the expression of amelogenin (AMEL) and ameloblastin (AMBN) was analysed with real-time PCR.

Article Snippet: The rat GDF5 (XM_001066344.2) gene was ligated into the pCMV/hygro-His expression plasmid (RG80131-G-H; Sino Biological Inc., Beijing, China).

Techniques: Incubation, Expressing, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-induced Smad1/5/8 phosphorylation

doi: 10.1038/srep23670

Figure Lengend Snippet: ( a ) Multiple protein sequence alignment of BMP family. Integrity identity; yellow. High similarity; green. ( b ) The tertiary structure was assembled using the mouse GDF5 sequence alignment. Amino acid 408 is highlighted in red. ( c ) Wild-type and GDF5 Rgsc451 (W408R) mice were evaluated using a micro-CT scanner. Scare bar: 5,000 μm.

Article Snippet: The rat GDF5 (XM_001066344.2) gene was ligated into the pCMV/hygro-His expression plasmid (RG80131-G-H; Sino Biological Inc., Beijing, China).

Techniques: Sequencing, Micro-CT

Journal: Scientific Reports

Article Title: Mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-induced Smad1/5/8 phosphorylation

doi: 10.1038/srep23670

Figure Lengend Snippet: SF2 cells transfected with no plasmid (control), mock expression plasmid, wild-type GDF5, or mutant GDF5 were cultured with BMP2, and the expression of amelogenin (AMEL) and ameloblastin (AMBN) was evaluated by real-time PCR. Expression was normalised to that of GAPDH, and the control sample without BMP2 was considered the baseline and set at 1.0, from which the relative expression for every other condition was compared. *P < 0.01, **P < 0.05.

Article Snippet: The rat GDF5 (XM_001066344.2) gene was ligated into the pCMV/hygro-His expression plasmid (RG80131-G-H; Sino Biological Inc., Beijing, China).

Techniques: Transfection, Plasmid Preparation, Expressing, Mutagenesis, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-induced Smad1/5/8 phosphorylation

doi: 10.1038/srep23670

Figure Lengend Snippet: SF2 cells transfected with no plasmid (control), mock expression plasmid, wild-type GDF5, or mutant GDF5 for 0, 5, 15, 30, or 60 min were analysed by Western blot using anti-Smad5 or anti-phospho Smad1/5/8 antibodies ( a ). Similarly, cells transfected with no plasmid (control), mock expression plasmid, wild type GDF5, or mutant GDF5 were also stimulated for 30 min with BMP2, and the expression of Smad5 and phospho-Smad1/5/8 was evaluated ( b ).

Article Snippet: The rat GDF5 (XM_001066344.2) gene was ligated into the pCMV/hygro-His expression plasmid (RG80131-G-H; Sino Biological Inc., Beijing, China).

Techniques: Transfection, Plasmid Preparation, Expressing, Mutagenesis, Western Blot

Journal: Advanced Science

Article Title: Discovery and Application of Postnatal Nucleus Pulposus Progenitors Essential for Intervertebral Disc Homeostasis and Degeneration

doi: 10.1002/advs.202104888

Figure Lengend Snippet: RegNPs were a metabolically active and mechanically sensitive population, while HomNPs were sensitive to hypoxia with “degenerative” potential. A) t‐SNE plots and representative violin plots showing the expression of Unc5c, Runx3, Bmp7, Wnt4, Tgfb2, CNTFR, Matn3, Grb10, Fgfr3, Epyc, Ptch1, and Pth1r on the t‐SNE map. B) Representation analysis of GO categories showing different functions for RegNPs. C) Heatmap revealing metabolic‐related functions and pathways for RegNPs. D) Representative images of lumbar spine sections from 4‐week‐old wild‐type mice stained for BMP7. Scale bars, 100 µm ( n = 3 mice per group). E) t‐SNE plots and representative violin plots showing the expression of Gdf5, Agt, Eln, Grem1, Mmp3, and Plau on the t‐SNE map. F) Representative analysis of GO categories showing different functions for HomNPs. G) Representative images of lumbar spine sections from 4‐week‐old WT mice stained for GDF5. The lower image shows a high‐magnification view of the indicated area from the upper image. Scale bars, 100 µm ( n = 3 mice per group).

Article Snippet: Dissociated single cells were then stained with AF488 anti‐mouse UTS2R (R&D Systems, FAB9245G‐100UG), APC anti‐mouse GDF5 (ASSAYPRO, 32579‐05161), anti‐mouse CNTFRa (Santa Cruz Biotechnology, SC9993), BB700 anti‐mouse CD146 (BD Biosciences, 742 280), APC anti‐mouse CD44 (BioLegend, 559 250), BV421 anti‐mouse CD73 (BioLegend, 127 217), BV605 anti‐mouse CD90 (BioLegend, 140 317), APC anti‐mouse PDGFa/CD140a (BioLegend, 135 907), FITC anti‐mouse Sca‐1 (BioLegend, 108 106), PerCP/Cy5.5 anti‐CD31 (BioLegend, 102 420), PerCP/Cy5.5 anti‐CD45 (BioLegend, 103 132), PerCP/Cy5.5 anti‐mouse TER‐119 (BioLegend, 116 228), FITC anti‐mouse 6C3/Ly‐51 (BioLegend, 108 305), Brilliant Violet 605 anti‐mouse CD90.2 (BioLegend, 140 317), PE/Cy7 anti‐mouse CD105 (BioLegend, 120 409), APC anti‐mouse CD200 (BioLegend, 123 809), and anti‐mouse Alexa Fluor 488 (Molecular Probes, A21202, 1:1000).

Techniques: Metabolic Labelling, Expressing, Staining

Journal: Advanced Science

Article Title: Discovery and Application of Postnatal Nucleus Pulposus Progenitors Essential for Intervertebral Disc Homeostasis and Degeneration

doi: 10.1002/advs.202104888

Figure Lengend Snippet: Lineage tracing of UTS2R + NP cells. A) Dot plot showing the expression of Uts2r on the t‐SNE map. B) Representative immunofluorescence imaging of UTS2R (green) in postnatal 1‐month‐old WT mice. The right image shows high magnification of the indicated area from the left image ( n = 3 mice per group). Scale bars, 100 µm. C) Construction strategy of Uts2r‐CreER transgenic mice using the CRISPR/Cas9 System. D) Diagram showing postnatal day 1 (P1) Uts2r‐CreER;Ai9 /+ mice administered with one dosage tamoxifen and sacrificed at postnatal day 3 (P3), 1 month (P1M), or 2 months (P2M). E) Representative immunofluorescence imaging of Uts2r‐CreER;Ai9 + cells (red). The right images show high magnification of the indicated area from the left image ( n = 3 mice per group). Scale bars, 100 µm. F,G) Representative immunofluorescence imaging of Uts2r‐CreER;Ai9 + cells (red), BMP7 (green) (F) or GDF5 (green) (G). The right images show high magnification of the indicated area from the left image ( n = 3 mice per group). Scale bars, 100 µm.

Article Snippet: Dissociated single cells were then stained with AF488 anti‐mouse UTS2R (R&D Systems, FAB9245G‐100UG), APC anti‐mouse GDF5 (ASSAYPRO, 32579‐05161), anti‐mouse CNTFRa (Santa Cruz Biotechnology, SC9993), BB700 anti‐mouse CD146 (BD Biosciences, 742 280), APC anti‐mouse CD44 (BioLegend, 559 250), BV421 anti‐mouse CD73 (BioLegend, 127 217), BV605 anti‐mouse CD90 (BioLegend, 140 317), APC anti‐mouse PDGFa/CD140a (BioLegend, 135 907), FITC anti‐mouse Sca‐1 (BioLegend, 108 106), PerCP/Cy5.5 anti‐CD31 (BioLegend, 102 420), PerCP/Cy5.5 anti‐CD45 (BioLegend, 103 132), PerCP/Cy5.5 anti‐mouse TER‐119 (BioLegend, 116 228), FITC anti‐mouse 6C3/Ly‐51 (BioLegend, 108 305), Brilliant Violet 605 anti‐mouse CD90.2 (BioLegend, 140 317), PE/Cy7 anti‐mouse CD105 (BioLegend, 120 409), APC anti‐mouse CD200 (BioLegend, 123 809), and anti‐mouse Alexa Fluor 488 (Molecular Probes, A21202, 1:1000).

Techniques: Expressing, Immunofluorescence, Imaging, Transgenic Assay, CRISPR

Journal: Non-Coding RNA

Article Title: Long Non-Coding RNA Myoparr Regulates GDF5 Expression in Denervated Mouse Skeletal Muscle

doi: 10.3390/ncrna5020033

Figure Lengend Snippet: Knockdown of Myoparr altered global gene expressions in denervated skeletal muscle. The heatmap displayed the changes in expression for 848 genes altered by Myoparr knockdown in denervated TA muscle of C57BL/6J mice (values are z-scores). The black and red arrow indicate the decreased expression of myogenin and increased expression of Gdf5 , respectively.

Article Snippet: The primary antibodies used included the GDF5 antibody A-10 (sc-373744, Santa Cruz Biotechnology, Dallas, TX, USA), the Smad5 antibody YY-6 (sc-101151, Santa Cruz Biotechnology), a phospho-Smad1(Ser463/465)/5(Ser463/465)/8(Ser426/428) antibody (#9511, Cell Signaling Technology, Danvers, MA, USA), and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (#2118, Cell Signaling Technology).

Techniques: Knockdown, Expressing

Journal: Non-Coding RNA

Article Title: Long Non-Coding RNA Myoparr Regulates GDF5 Expression in Denervated Mouse Skeletal Muscle

doi: 10.3390/ncrna5020033

Figure Lengend Snippet: Upregulated genes encoding secretory proteins following Myoparr knockdown in denervated tibialis anterior (TA) muscles of C57BL/6J mice.

Article Snippet: The primary antibodies used included the GDF5 antibody A-10 (sc-373744, Santa Cruz Biotechnology, Dallas, TX, USA), the Smad5 antibody YY-6 (sc-101151, Santa Cruz Biotechnology), a phospho-Smad1(Ser463/465)/5(Ser463/465)/8(Ser426/428) antibody (#9511, Cell Signaling Technology, Danvers, MA, USA), and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (#2118, Cell Signaling Technology).

Techniques: Knockdown, Muscles

Journal: Non-Coding RNA

Article Title: Long Non-Coding RNA Myoparr Regulates GDF5 Expression in Denervated Mouse Skeletal Muscle

doi: 10.3390/ncrna5020033

Figure Lengend Snippet: Myoparr knockdown activated bone morphogenetic protein (BMP) signaling through the expression of growth/differentiation factor 5 (GDF5) in denervated TA muscles of C57BL/6J mice. ( a ) Western blot showing increased expression of GDF5 by Myoparr knockdown in TA muscles 3 d post denervation. The activity of BMP signaling was indicated by the levels of phosphorylated Smad1/5/8 (pSmad1/5/8). GAPDH expression served as an internal control. Three independent experiments were shown. ( b ) Box-and-whisker plots showed relative quantification of GDF5 expression as GDF5/GAPDH ratio ( n = 3 per group). * p < 0.05, unpaired two-tailed Student’s t -test. ( c ) Relative quantification of the activity of BMP signaling was shown as pSmad1/5/8/Smad5 ratio ( n = 3 per group).

Article Snippet: The primary antibodies used included the GDF5 antibody A-10 (sc-373744, Santa Cruz Biotechnology, Dallas, TX, USA), the Smad5 antibody YY-6 (sc-101151, Santa Cruz Biotechnology), a phospho-Smad1(Ser463/465)/5(Ser463/465)/8(Ser426/428) antibody (#9511, Cell Signaling Technology, Danvers, MA, USA), and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (#2118, Cell Signaling Technology).

Techniques: Knockdown, Expressing, Muscles, Western Blot, Activity Assay, Control, Whisker Assay, Quantitative Proteomics, Two Tailed Test