human gdf5  (R&D Systems)

Journal: JCI insight

Article Title: Impaired 1,25-dihydroxyvitamin D3 action underlies enthesopathy development in the Hyp mouse model of X-linked hypophosphatemia.

doi: 10.1172/jci.insight.163259

Figure Lengend Snippet: Figure 8. 1,25D blocks rhGDF5 induced expression of BMP and IHH genes and p-SMAD1/5 and VDR expression is decreased in Hyp entheses by P14. (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 subject- ed 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 ver- sus 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: Expressing, Incubation, Western Blot, 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: We therefore chose to study, in addition to wildtype mouse and human GDF5, a variant form of human GDF5 that was designed by Biopharm to preferentially bind to BMPR-IA.

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: We therefore chose to study, in addition to wildtype mouse and human GDF5, a variant form of human GDF5 that was designed by Biopharm to preferentially bind to BMPR-IA.

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: We therefore chose to study, in addition to wildtype mouse and human GDF5, a variant form of human GDF5 that was designed by Biopharm to preferentially bind to BMPR-IA.

Techniques: Cell Culture, Positive Control, Western Blot

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: We therefore chose to study, in addition to wildtype mouse and human GDF5, a variant form of human GDF5 that was designed by Biopharm to preferentially bind to BMPR-IA.

Techniques: Variant Assay, Two Tailed Test, Expressing

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: We therefore chose to study, in addition to wildtype mouse and human GDF5, a variant form of human GDF5 that was designed by Biopharm to preferentially bind to BMPR-IA.

Techniques: Variant Assay, Two Tailed Test, Expressing

Journal: Biomaterials and Biosystems

Article Title: Growth factor and macromolecular crowding supplementation in human tenocyte culture

doi: 10.1016/j.bbiosy.2021.100009

Figure Lengend Snippet: Fig. 1. SDS-PAGE and densitometric analy- ses of cell layers of human tenocytes treated without ( − )/with ( + ) MMC (carrageenan) and without ( − )/with ( + ) (either in simultaneous or in serial fashion to MMC) IGF1, PDGF 𝛽𝛽, GDF5 and TGF 𝛽3 revealed that MMC supple- mentation, at all timepoints, induced signifi- cantly ( p < 0.05) higher collagen type I depo- sition than the non-MMC groups (without or even with any GF). among the simultaneous GF supplementation to MMC, TGF 𝛽3 induced the highest ( p < 0.05) collagen type I deposition in human tenocyte cultures at all time points. among the serial GF supplementation to MMC, TGF 𝛽3 induced the highest ( p < 0.05) colla- gen type I deposition in human tenocyte cul- tures at all time points. Day 4 ( A ), Day 7 ( B ), Day 10 ( C ), Day 13 ( D ). ∗ indicates significantly ( p < 0.05) higher difference between the simul- taneous and/or serial GF supplementation to MMC and MMC alone groups. Passage 3. N = 3.

Article Snippet: GF and MMC supplementation At passage three, tenocytes were seeded at 25,000 cells/cm 2 in 24- ell plates and allowed to attach for 24 h. The culture media was re- oved and replaced with culture media containing 100 μM L-ascorbic cid phosphate (Sigma Aldrich, Ireland) to induce collagen synthesis nd 100 ng/ml recombinant human IGF1 (R&D Systems, UK), 50 ng/ml ecombinant human PDGF ββ (PeproTech EC, UK), 100 ng/ml recombi- ant human GDF5 (R&D Systems, UK) or 20 ng/ml recombinant human GF β3 (PeproTech EC, UK) with and without 50 μg/ml of carrageenan CR, mixture of κ and lesser amounts of λ CR, C1013, Sigma Aldrich, reland).

Techniques: SDS Page

Journal: Biomaterials and Biosystems

Article Title: Growth factor and macromolecular crowding supplementation in human tenocyte culture

doi: 10.1016/j.bbiosy.2021.100009

Figure Lengend Snippet: Fig. 2. Immunocytochemistry and relative flu- orescence intensity analyses of collagen type I of cell layers of human tenocytes treated with- out ( − )/with ( + ) MMC (carrageenan) and with- out ( − )/with ( + ) (either in simultaneous or in serial fashion to MMC) IGF1, PDGF 𝛽𝛽, GDF5 and TGF 𝛽3 revealed that MMC supplementa- tion, at all timepoints, induced significantly ( p < 0.05) higher collagen type I deposition than the non-MMC groups (without or even with any GF). At day 10 and 13, IGF1 in both simultaneous and serial to MMC supplementa- tion induced significantly higher ( p < 0.05) col- lagen type I deposition than the MMC alone group ( A ). No significant ( p > 0.05) difference was detected in collagen type I deposition be- tween MMC alone and PDGF 𝛽𝛽supplementa- tion in either simultaneous or serial fashion to MMC ( B ). At days 4, 10 and 13, GDF5 sup- plementation in serial fashion to MMC induced significantly ( p < 0.05) higher collagen type I deposition than the MMC alone group ( C ). At day 4, 7 and 13, TGF 𝛽3 supplementation in si- multaneous and serial fashion to MMC induced significantly higher ( p < 0.05) collagen type I deposition than the MMC alone group ( D ). ∗

Article Snippet: GF and MMC supplementation At passage three, tenocytes were seeded at 25,000 cells/cm 2 in 24- ell plates and allowed to attach for 24 h. The culture media was re- oved and replaced with culture media containing 100 μM L-ascorbic cid phosphate (Sigma Aldrich, Ireland) to induce collagen synthesis nd 100 ng/ml recombinant human IGF1 (R&D Systems, UK), 50 ng/ml ecombinant human PDGF ββ (PeproTech EC, UK), 100 ng/ml recombi- ant human GDF5 (R&D Systems, UK) or 20 ng/ml recombinant human GF β3 (PeproTech EC, UK) with and without 50 μg/ml of carrageenan CR, mixture of κ and lesser amounts of λ CR, C1013, Sigma Aldrich, reland).

Techniques: Immunocytochemistry

Journal: Biomaterials and Biosystems

Article Title: Growth factor and macromolecular crowding supplementation in human tenocyte culture

doi: 10.1016/j.bbiosy.2021.100009

Figure Lengend Snippet: Fig. 3. Immunocytochemistry and relative flu- orescence intensity analyses of collagen type III of cell layers of human tenocytes treated with- out ( − )/with ( + ) MMC (carrageenan) and with- out ( − )/with ( + ) (either in simultaneous or in serial fashion to MMC) IGF1, PDGF 𝛽𝛽, GDF5 and TGF 𝛽3 revealed that MMC supplementa- tion, at all timepoints, induced significantly ( p < 0.05) higher collagen type III deposition than the non-MMC groups (without or even with any GF). At day 7 and, IGF1 supplementa- tion in simultaneous and serial fashion to MMC induced significantly higher ( p < 0.05) collagen type III deposition than the MMC alone group ( A ). No significant ( p > 0.05) difference was de- tected in collagen type III deposition between MMC alone and PDGF 𝛽𝛽supplementation in either simultaneous or serial fashion to MMC ( B ). At day 10 and 13, GDF5 supplementation in serial fashion to MMC induced significantly ( p < 0.05) higher collagen type III deposition than the MMC alone group ( C ). At day 4 and 7, TGF 𝛽3 supplementation either simultaneously or in serial fashion to MMC and at day 13 only in serial fashion to MMC induced significantly ( p < 0.05) higher collagen type III deposition than the MMC alone group ( D ). ∗ indicates sig- nificantly (at p < 0.05) higher difference be- tween the simultaneous and/or serial GF sup- plementation to MMC and MMC alone groups. Collagen type III: Green. DAPI: Blue. Scale bars: 100 𝜇m. Passage 3. N = 3.

Article Snippet: GF and MMC supplementation At passage three, tenocytes were seeded at 25,000 cells/cm 2 in 24- ell plates and allowed to attach for 24 h. The culture media was re- oved and replaced with culture media containing 100 μM L-ascorbic cid phosphate (Sigma Aldrich, Ireland) to induce collagen synthesis nd 100 ng/ml recombinant human IGF1 (R&D Systems, UK), 50 ng/ml ecombinant human PDGF ββ (PeproTech EC, UK), 100 ng/ml recombi- ant human GDF5 (R&D Systems, UK) or 20 ng/ml recombinant human GF β3 (PeproTech EC, UK) with and without 50 μg/ml of carrageenan CR, mixture of κ and lesser amounts of λ CR, C1013, Sigma Aldrich, reland).

Techniques: Immunocytochemistry

Journal: Biomaterials and Biosystems

Article Title: Growth factor and macromolecular crowding supplementation in human tenocyte culture

doi: 10.1016/j.bbiosy.2021.100009

Figure Lengend Snippet: Fig. 4. Hierarchal clustering of the fold change (threshold of 3) in gene expression of human tenocytes cultured without ( − )/with ( + ) MMC (carrageenan) and without ( − )/with ( + ) (either in simultaneous or in serial fashion to MMC) IGF1, PDGF 𝛽𝛽, GDF5 and TGF 𝛽3 revealed that at all timepoints TGF 𝛽3 alone and TGF 𝛽3 in either simultaneous or serial supplementation to CR upregulated the most and downregulated the least collagen- and tendon- related genes and upregulated the least and downregulated the most osteo-, chondro-, fibrosis- and adipose- related trans-differentiation genes in comparison to the other GFs without CR and with CR (either in simultaneous or serial fashion to the GFs). IGF1 ( A ), PDGF 𝛽𝛽( B ), GDF5 ( C ), TGF 𝛽3 ( D ). The heatmap was generated by a log transformation of the real-time PCR data presented as ΔCT = (CT miRNA – CT GAPDH) compared to without CR and without GF at each timepoint. Passage 3. Data derived by pooling six wells per sample ( N = 2).

Article Snippet: GF and MMC supplementation At passage three, tenocytes were seeded at 25,000 cells/cm 2 in 24- ell plates and allowed to attach for 24 h. The culture media was re- oved and replaced with culture media containing 100 μM L-ascorbic cid phosphate (Sigma Aldrich, Ireland) to induce collagen synthesis nd 100 ng/ml recombinant human IGF1 (R&D Systems, UK), 50 ng/ml ecombinant human PDGF ββ (PeproTech EC, UK), 100 ng/ml recombi- ant human GDF5 (R&D Systems, UK) or 20 ng/ml recombinant human GF β3 (PeproTech EC, UK) with and without 50 μg/ml of carrageenan CR, mixture of κ and lesser amounts of λ CR, C1013, Sigma Aldrich, reland).

Techniques: Gene Expression, Cell Culture, Comparison, Generated, Transformation Assay, Real-time Polymerase Chain Reaction, Derivative Assay

Journal: Biomaterials and Biosystems

Article Title: Growth factor and macromolecular crowding supplementation in human tenocyte culture ☆

doi: 10.1016/j.bbiosy.2021.100009

Figure Lengend Snippet: SDS-PAGE and densitometric analyses of cell layers of human tenocytes treated without (−)/with (+) MMC (carrageenan) and without (−)/with (+) (either in simultaneous or in serial fashion to MMC) IGF1, PDGF ββ , GDF5 and TGF β 3 revealed that MMC supplementation, at all timepoints, induced significantly ( p < 0.05) higher collagen type I deposition than the non-MMC groups (without or even with any GF). among the simultaneous GF supplementation to MMC, TGF β 3 induced the highest ( p < 0.05) collagen type I deposition in human tenocyte cultures at all time points. among the serial GF supplementation to MMC, TGF β 3 induced the highest ( p < 0.05) collagen type I deposition in human tenocyte cultures at all time points. Day 4 ( A ), Day 7 ( B ), Day 10 ( C ), Day 13 ( D ). * indicates significantly ( p < 0.05) higher difference between the simultaneous and/or serial GF supplementation to MMC and MMC alone groups. Passage 3. N = 3.

Article Snippet: At passage three, tenocytes were seeded at 25,000 cells/cm 2 in 24-well plates and allowed to attach for 24 h. The culture media was removed and replaced with culture media containing 100 μ M L-ascorbic acid phosphate (Sigma Aldrich, Ireland) to induce collagen synthesis and 100 ng/ml recombinant human IGF1 (R&D Systems, UK), 50 ng/ml recombinant human PDGF ββ (PeproTech EC, UK), 100 ng/ml recombinant human GDF5 (R&D Systems, UK) or 20 ng/ml recombinant human TGF β 3 (PeproTech EC, UK) with and without 50 μ g/ml of carrageenan (CR, mixture of κ and lesser amounts of λ CR, C1013, Sigma Aldrich, Ireland).

Techniques: SDS Page

Journal: Biomaterials and Biosystems

Article Title: Growth factor and macromolecular crowding supplementation in human tenocyte culture ☆

doi: 10.1016/j.bbiosy.2021.100009

Figure Lengend Snippet: Immunocytochemistry and relative fluorescence intensity analyses of collagen type I of cell layers of human tenocytes treated without (−)/with (+) MMC (carrageenan) and without (−)/with (+) (either in simultaneous or in serial fashion to MMC) IGF1, PDGF ββ , GDF5 and TGF β 3 revealed that MMC supplementation, at all timepoints, induced significantly ( p < 0.05) higher collagen type I deposition than the non-MMC groups (without or even with any GF). At day 10 and 13, IGF1 in both simultaneous and serial to MMC supplementation induced significantly higher ( p < 0.05) collagen type I deposition than the MMC alone group ( A ). No significant ( p > 0.05) difference was detected in collagen type I deposition between MMC alone and PDGF ββ supplementation in either simultaneous or serial fashion to MMC ( B ). At days 4, 10 and 13, GDF5 supplementation in serial fashion to MMC induced significantly ( p < 0.05) higher collagen type I deposition than the MMC alone group ( C ). At day 4, 7 and 13, TGF β 3 supplementation in simultaneous and serial fashion to MMC induced significantly higher ( p < 0.05) collagen type I deposition than the MMC alone group ( D ). * indicates significantly (at p < 0.05) higher difference between the simultaneous and/or serial GF supplementation to MMC and MMC alone groups. Collagen type I: Green, DAPI: Blue. Scale bars: 100 μm. Passage 3. N = 3.

Article Snippet: At passage three, tenocytes were seeded at 25,000 cells/cm 2 in 24-well plates and allowed to attach for 24 h. The culture media was removed and replaced with culture media containing 100 μ M L-ascorbic acid phosphate (Sigma Aldrich, Ireland) to induce collagen synthesis and 100 ng/ml recombinant human IGF1 (R&D Systems, UK), 50 ng/ml recombinant human PDGF ββ (PeproTech EC, UK), 100 ng/ml recombinant human GDF5 (R&D Systems, UK) or 20 ng/ml recombinant human TGF β 3 (PeproTech EC, UK) with and without 50 μ g/ml of carrageenan (CR, mixture of κ and lesser amounts of λ CR, C1013, Sigma Aldrich, Ireland).

Techniques: Immunocytochemistry, Fluorescence

Journal: Biomaterials and Biosystems

Article Title: Growth factor and macromolecular crowding supplementation in human tenocyte culture ☆

doi: 10.1016/j.bbiosy.2021.100009

Figure Lengend Snippet: Immunocytochemistry and relative fluorescence intensity analyses of collagen type III of cell layers of human tenocytes treated without (−)/with (+) MMC (carrageenan) and without (−)/with (+) (either in simultaneous or in serial fashion to MMC) IGF1, PDGF ββ , GDF5 and TGF β 3 revealed that MMC supplementation, at all timepoints, induced significantly ( p < 0.05) higher collagen type III deposition than the non-MMC groups (without or even with any GF). At day 7 and, IGF1 supplementation in simultaneous and serial fashion to MMC induced significantly higher ( p < 0.05) collagen type III deposition than the MMC alone group ( A ). No significant ( p > 0.05) difference was detected in collagen type III deposition between MMC alone and PDGF ββ supplementation in either simultaneous or serial fashion to MMC ( B ). At day 10 and 13, GDF5 supplementation in serial fashion to MMC induced significantly ( p < 0.05) higher collagen type III deposition than the MMC alone group ( C ). At day 4 and 7, TGF β 3 supplementation either simultaneously or in serial fashion to MMC and at day 13 only in serial fashion to MMC induced significantly ( p < 0.05) higher collagen type III deposition than the MMC alone group ( D ). * indicates significantly (at p < 0.05) higher difference between the simultaneous and/or serial GF supplementation to MMC and MMC alone groups. Collagen type III: Green. DAPI: Blue. Scale bars: 100 μm. Passage 3. N = 3.

Article Snippet: At passage three, tenocytes were seeded at 25,000 cells/cm 2 in 24-well plates and allowed to attach for 24 h. The culture media was removed and replaced with culture media containing 100 μ M L-ascorbic acid phosphate (Sigma Aldrich, Ireland) to induce collagen synthesis and 100 ng/ml recombinant human IGF1 (R&D Systems, UK), 50 ng/ml recombinant human PDGF ββ (PeproTech EC, UK), 100 ng/ml recombinant human GDF5 (R&D Systems, UK) or 20 ng/ml recombinant human TGF β 3 (PeproTech EC, UK) with and without 50 μ g/ml of carrageenan (CR, mixture of κ and lesser amounts of λ CR, C1013, Sigma Aldrich, Ireland).

Techniques: Immunocytochemistry, Fluorescence

Journal: Biomaterials and Biosystems

Article Title: Growth factor and macromolecular crowding supplementation in human tenocyte culture ☆

doi: 10.1016/j.bbiosy.2021.100009

Figure Lengend Snippet: Hierarchal clustering of the fold change (threshold of 3) in gene expression of human tenocytes cultured without (−)/with (+) MMC (carrageenan) and without (−)/with (+) (either in simultaneous or in serial fashion to MMC) IGF1, PDGF ββ , GDF5 and TGF β 3 revealed that at all timepoints TGF β 3 alone and TGF β 3 in either simultaneous or serial supplementation to CR upregulated the most and downregulated the least collagen- and tendon- related genes and upregulated the least and downregulated the most osteo-, chondro-, fibrosis- and adipose- related trans-differentiation genes in comparison to the other GFs without CR and with CR (either in simultaneous or serial fashion to the GFs). IGF1 ( A ), PDGF ββ ( B ), GDF5 ( C ), TGF β 3 ( D ). The heatmap was generated by a log transformation of the real-time PCR data presented as ΔCΤ = (CΤ miRNA – CΤ GAPDH) compared to without CR and without GF at each timepoint. Passage 3. Data derived by pooling six wells per sample ( N = 2).

Article Snippet: At passage three, tenocytes were seeded at 25,000 cells/cm 2 in 24-well plates and allowed to attach for 24 h. The culture media was removed and replaced with culture media containing 100 μ M L-ascorbic acid phosphate (Sigma Aldrich, Ireland) to induce collagen synthesis and 100 ng/ml recombinant human IGF1 (R&D Systems, UK), 50 ng/ml recombinant human PDGF ββ (PeproTech EC, UK), 100 ng/ml recombinant human GDF5 (R&D Systems, UK) or 20 ng/ml recombinant human TGF β 3 (PeproTech EC, UK) with and without 50 μ g/ml of carrageenan (CR, mixture of κ and lesser amounts of λ CR, C1013, Sigma Aldrich, Ireland).

Techniques: Gene Expression, Cell Culture, Comparison, Generated, Transformation Assay, Real-time Polymerase Chain Reaction, Derivative Assay