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: JCI Insight

Article Title: Impaired 1,25-dihydroxyvitamin D 3 action underlies enthesopathy development in the Hyp mouse model of X-linked hypophosphatemia

doi: 10.1172/jci.insight.163259

Figure Lengend Snippet: ( A ) Primary murine chondrocytes were pretreated for 0.5, 1, 4, or 18 hours with or without (–) 1 × 10 –8 M 1,25D prior to incubation with (+) or without (–) 100 ng/mL rhGDF5 (for 30 minutes) and subjected to Western blot analyses for p-SMAD1/5/9, SMAD1, and β-actin. Data are representative of those obtained from 3 independent experiments. ( B ) Primary murine chondrocytes were pretreated for 18 hours with 1 × 10 –8 M 1,25D followed by treatment with 200 ng/mL rhGDF5 (for 4 hours). Gene expression analyses were performed for BMP and IHH target genes. Data are representative of those obtained from 5–7 independent experiments. One-way ANOVA followed by Fisher’s least significant difference test was used to analyze significance between all genotype groups. * P < 0.05 versus WT; # P < 0.05 versus rhGDF5; a indicates P < 0.05 versus rhGDF5 plus 1,25D. ( C ) IHC for VDR was performed on P7, P14, P30, and P60 entheses from WT, Hyp , and C –/– mice. In all representative pictures, the enthesis region is outlined with a black box. Scale bar: 20 μm. Data are representative of 6 mice per age or genotype group.

Article Snippet: For Western blot analyses, chondrocytes were pretreated for 0.5, 1, 4, or 18 hours with 1 × 10 –8 M 1,25D prior to exposure to recombinant human GDF5 (rhGDF5) (100 ng/mL; R&D Systems) for 30 minutes.

Techniques: Incubation, Western Blot, Expressing

Journal: Heliyon

Article Title: GDF5 induces TBX3 in a concentration dependent manner - on a gold nanoparticle gradient

doi: 10.1016/j.heliyon.2020.e04133

Figure Lengend Snippet: (A) GDF5 gradient surface, 18 × 18 mm, seeded with c-iPSCs visualized using high-throughput confocal IN CELL Analyzer 6000. The bar on the left upper side indicates the extent of the gradient, where the continuous density increase is shown with a marker on the left-hand side, with low GDF5 density at the bottom of the image and high at the top (the damaged lower left corner does not affect the gradient result). (B) Focusing on the budding cell clusters identified at a density range 500–1,500 particles/μm 2 . Two white lines indicate the budding zone. (C–D) Close-up of buds identified in the budding zone. Scale bars 10 μm.

Article Snippet: Recombinant human GDF5 (R&D Systems, Bio-Techne, MN, USA), TGFβ-1 (R&D Systems, Bio-Techne, MN, USA), and TGFβ-3 (R&D Systems, Bio-Techne, MN, USA) were biotinylated to facilitate binding to streptavidin.

Techniques: High Throughput Screening Assay, Marker

Journal: Heliyon

Article Title: GDF5 induces TBX3 in a concentration dependent manner - on a gold nanoparticle gradient

doi: 10.1016/j.heliyon.2020.e04133

Figure Lengend Snippet: GDF5, TGFβ-1, and TGFβ-3 h.d. surfaces, 18 × 18 mm, visualized using high-throughput confocal IN CELL Analyzer 6000. (A) GDF5 surfaces with a density of 400 particles/μm 2 (control). No buds are identified. (B) GDF5 surface with a density in the range of the budding zone, 900 particles/μm 2 . Buds are clearly identified as brighter colored clusters. (C) TGFβ-1 surfaces with a density of 600 particles/μm 2 . Cell-free cavities were formed, a response comparable to that of cells on laminin 521 gradient surfaces (Fig. S2). (D) Evenly distributed cells on TGFβ-1 surfaces with a density of 2,000 particles/μm 2 , (E) TGFβ-3 surfaces with a density of 600 particles/μm 2 , and (F) a density of 1,900 particles/μm 2 .

Article Snippet: Recombinant human GDF5 (R&D Systems, Bio-Techne, MN, USA), TGFβ-1 (R&D Systems, Bio-Techne, MN, USA), and TGFβ-3 (R&D Systems, Bio-Techne, MN, USA) were biotinylated to facilitate binding to streptavidin.

Techniques: High Throughput Screening Assay, Control

Journal: Heliyon

Article Title: GDF5 induces TBX3 in a concentration dependent manner - on a gold nanoparticle gradient

doi: 10.1016/j.heliyon.2020.e04133

Figure Lengend Snippet: Immunostaining for SOX9 in c-iPSCs after five days of differentiation on a GDF5 gradient. (A1–A3) Images from the budding zone were acquired using fluorescence microscopy, 20X objective. Scale bars denote 100 μm. (B1–B3) Images from the budding zone were acquired using a confocal microscope, 40X objective. Scale bars denote 10 μm.

Article Snippet: Recombinant human GDF5 (R&D Systems, Bio-Techne, MN, USA), TGFβ-1 (R&D Systems, Bio-Techne, MN, USA), and TGFβ-3 (R&D Systems, Bio-Techne, MN, USA) were biotinylated to facilitate binding to streptavidin.

Techniques: Immunostaining, Fluorescence, Microscopy

Journal: Heliyon

Article Title: GDF5 induces TBX3 in a concentration dependent manner - on a gold nanoparticle gradient

doi: 10.1016/j.heliyon.2020.e04133

Figure Lengend Snippet: TBX3 are localized in the nuclei and in vesicles in the budding zone. TBX3 expression is visualized in c-iPSCs after five days of differentiation on a GDF5 gradient. (A) Budding zone on a GDF5 gradient. Scale bar 100 μm B1–B3 and C1–C3 show two different budding clusters on the GDF5 gradient. (D1-D3) Close-up of a budding zone. (B–C) Images were acquired with a confocal microscope, 40X objective. Scale bars 20 μm. (D) Images were acquired using a 60X objective. Scale bars 10 μm.

Article Snippet: Recombinant human GDF5 (R&D Systems, Bio-Techne, MN, USA), TGFβ-1 (R&D Systems, Bio-Techne, MN, USA), and TGFβ-3 (R&D Systems, Bio-Techne, MN, USA) were biotinylated to facilitate binding to streptavidin.

Techniques: Expressing, Microscopy

Journal: Heliyon

Article Title: GDF5 induces TBX3 in a concentration dependent manner - on a gold nanoparticle gradient

doi: 10.1016/j.heliyon.2020.e04133

Figure Lengend Snippet: iPSCs on h.d. surfaces immunostained with TBX3 monoclonal (Table S2) antibody; images acquired using confocal microscopy. TBX3 expression is clearly upregulated on the GDF5 h.d. surface with a particle density in the range of the budding zone, as compared with on all other surfaces. (A) GDF5 surface with low particle density. (B) GDF5 budding zone surface. (C) TGFβ-3 surface with the same particle density as (B). (D) TGFβ-3 surface with the same particle density as (B), but with GDF5 (10 ng/ml) added to the differentiation medium. (E) Surface without biomolecules, coated with Coat-1 (DEF-CS 500 Coat-1, Cellartis, Sweden).

Article Snippet: Recombinant human GDF5 (R&D Systems, Bio-Techne, MN, USA), TGFβ-1 (R&D Systems, Bio-Techne, MN, USA), and TGFβ-3 (R&D Systems, Bio-Techne, MN, USA) were biotinylated to facilitate binding to streptavidin.

Techniques: Confocal Microscopy, Expressing

Journal: Heliyon

Article Title: GDF5 induces TBX3 in a concentration dependent manner - on a gold nanoparticle gradient

doi: 10.1016/j.heliyon.2020.e04133

Figure Lengend Snippet: Histological sections of pellets generated from c-iPSCs (A–C) and chondrocytes (D–E). A1–A2 to E1–E2 were stained with Alcian Blue van Gieson, demonstrating the presence of proteoglycans (blue) and collagen (red/purple). A3–A6 & B3–C3 were immunostained for TBX3, which was clearly upregulated in the areas with higher amounts of GAG. (A1–A6) c-iPSCs pre-differentiated on a GDF5 h.d. surface with low particle density. (B1–B3) c-iPSCs pre-differentiated on a GDF5 h.d. surface with budding zone particle density. (C1–C3) c-iPSCs pre-differentiated on a GDF5 gradient surface. (D1–D2) Chondrocyte pellet pre-differentiated on a GDF5 gradient surface. (E1–E2) Chondrocyte pellet with 5 % human serum in the medium. (A1–E1, A3–C3, A5–A6) 100 μm scale bars. (A2–E2) 50 μm scale bars. (A4) 20 μm scale bars.

Article Snippet: Recombinant human GDF5 (R&D Systems, Bio-Techne, MN, USA), TGFβ-1 (R&D Systems, Bio-Techne, MN, USA), and TGFβ-3 (R&D Systems, Bio-Techne, MN, USA) were biotinylated to facilitate binding to streptavidin.

Techniques: Generated, Staining