recombinant human gdf15 protein  (Bio-Techne corporation)

Journal: Cell Reports Medicine

Article Title: Exercise-induced crosstalk between immune cells and adipocytes in humans: Role of oncostatin-M

doi: 10.1016/j.xcrm.2023.101348

Figure Lengend Snippet:

Article Snippet: GDF15 , Bio-Techne LTD , 9279-GD-050.

Techniques: In Vitro, Recombinant, Plasmid Preparation, Fluorescence, Blocking Assay, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Multiplex Assay, Software, Real-time Polymerase Chain Reaction, Microscopy

Journal: Cell Reports Medicine

Article Title: Exercise-induced crosstalk between immune cells and adipocytes in humans: Role of oncostatin-M

doi: 10.1016/j.xcrm.2023.101348

Figure Lengend Snippet: Oncostatin-M is acutely induced by exercise and increases basal lipolysis in human adipocytes (A–D) Gene expression of candidate secreted factors oncostatin-M, GDF15, SFRP4, and MXRA5 selected for further validation. (E) Human adipocytes were treated with recombinant proteins for 1 h, and kinase activity was assessed using serine-threonine substrate array (n = 5). (F–I) Kinase activity in response to oncostatin-M, GDF15, SFRP4, and MXRA5 was predicted by computational analysis of differentially phosphorylated peptide signatures. The kinase statistic parameter reflects the change in kinase activity, whereas the specificity parameter indicates how specific the peptide signature is to a particular kinase. (J) Protein-protein interaction (PPI) network and reactome pathway analysis of differentially active kinases. Kinase score reflects kinase activation in response to oncostatin-M compared to control cells, and the node degree is a measure of how connected each node is within the network. (K) PPI network and reactome pathway analysis of differentially active kinases. Kinase score reflects kinase activation in response to SFRP4 compared to control cells, and the node degree is a measure of how connected each node is within the network. (L–N) Western-blot quantification and representative image of P-STAT3 Y705, P-ERK1/2 T202/Y204, and P-HSL S563 after 1 h incubation with recombinant proteins in isolated human adipocytes (one-way ANOVA) (n = 5). (O) Lipolysis assay in control, 1 μM, and 10 μM isoprenaline conditions (two-way ANOVA). ∗∗∗∗p < 0,0001; ∗∗∗p < 0.001; ∗∗p < 0,01; ∗p < 0,05. Error bars show SEM.

Article Snippet: The day of the experiments, cells were kept in DMEM/F12 medium without additives for 2 h, then incubated in presence of commercial recombinant human proteins (30–100 nM): Oncostatin-M (295-OM-050, Bio-Techne LTD), GDF15 (9279-GD-050, Bio-Techne LTD), SFRP4 (1827-SF-025/CF, Bio-Techne LTD) and MRXA5 (LS-G26239-10, Nordic Biosite).

Techniques: Expressing, Recombinant, Activity Assay, Activation Assay, Western Blot, Incubation, Isolation

Journal: Cell Reports Medicine

Article Title: Exercise-induced crosstalk between immune cells and adipocytes in humans: Role of oncostatin-M

doi: 10.1016/j.xcrm.2023.101348

Figure Lengend Snippet: OSM expression arises from macrophages in response to exercise-like stimuli (A) Oncostatin-M level in plasma from individuals with either NGT or T2D (n = 15 and 17 per group). # Time effect in both groups (two-way ANOVA test), ### p < 0.001. ¤ Interaction between time and diagnosis group, ¤ p < 0.05. (B) Area under the curve of plasma oncostatin-M after separation between men and women and NGT and T2D (n = 8 per subgroup). § Sex effect (two-way ANOVA on log-transformed values), § p < 0.05. (C and D) Western blot quantification and representative image of P-ERK1/2 and P-HSL in adipose tissue biopsies (n = 5 per group). # Time effect in both groups (two-way ANOVA test), ## p < 0.01. § Condition effect (two-way ANOVA test), § p < 0.05. (E) Schematic of OSM binding to type I and type II receptors in humans. (F–I) Expression of oncostatin-M and oncostatin-M receptor subunits genes OSMR , LIFR , and GPR130 in cells derived from adipose tissue after fractionation. Kruskal-Wallis testing shows significant differences between cell populations for all 4 genes (p < 0.001). (J) Spatial detection of OSM and macrophage markers CD68 mRNAs by fluorescent in situ hybridization in frozen section of adipose tissue of subjects with NGT and T2D (n = 4 and 3). (K and L) Thp1 macrophages were treated with 10 μM forskolin or 100 nM clenbuterol for 3 h, and expression of oncostatin-M and GDF15 was measured. # Treatment effect (two-way ANOVA, log-transformed values), # p < 0.05. n = 3 experiments. (M) Oncostatin M measurement in the conditioned media in response to 10 μM forskolin (two-way ANOVA, log-transformed values). # Treatment effect, ### p < 0.001; ¤polarization effect, ¤¤p < 0.01; ∗Sidak’s post-tests. (N and O) Thp1 macrophages were exposed to conditioned media from 3 h electric-pulse-stimulated (EPS) or 3 h sham EPS human myotubes derived from individuals with either NGT or T2D (n = 5 and 6) for 0, 3, 6, or 24 h, and oncostatin-M and GDF15 gene expressions were measured and presented normalized to expression in response to sham EPS. # Time effect (two-way ANOVA test), ### p < 0.001, # p < 0.05. Error bars show SEM.

Article Snippet: The day of the experiments, cells were kept in DMEM/F12 medium without additives for 2 h, then incubated in presence of commercial recombinant human proteins (30–100 nM): Oncostatin-M (295-OM-050, Bio-Techne LTD), GDF15 (9279-GD-050, Bio-Techne LTD), SFRP4 (1827-SF-025/CF, Bio-Techne LTD) and MRXA5 (LS-G26239-10, Nordic Biosite).

Techniques: Expressing, Transformation Assay, Western Blot, Binding Assay, Derivative Assay, Fractionation, In Situ Hybridization

Journal: Cell Reports Medicine

Article Title: Exercise-induced crosstalk between immune cells and adipocytes in humans: Role of oncostatin-M

doi: 10.1016/j.xcrm.2023.101348

Figure Lengend Snippet:

Article Snippet: The day of the experiments, cells were kept in DMEM/F12 medium without additives for 2 h, then incubated in presence of commercial recombinant human proteins (30–100 nM): Oncostatin-M (295-OM-050, Bio-Techne LTD), GDF15 (9279-GD-050, Bio-Techne LTD), SFRP4 (1827-SF-025/CF, Bio-Techne LTD) and MRXA5 (LS-G26239-10, Nordic Biosite).

Techniques: In Vitro, Recombinant, Plasmid Preparation, Fluorescence, Blocking Assay, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Multiplex Assay, Software, Real-time Polymerase Chain Reaction, Microscopy

Journal: International journal of molecular sciences

Article Title: GDF15 Promotes the Osteogenic Cell Fate of Periodontal Ligament Fibroblasts, thus Affecting Their Mechanobiological Response.

doi: 10.3390/ijms241210011

Figure Lengend Snippet: Figure 1. GDF15 limits cell proliferation of hPdLFs without affecting cell survival in the long term. (a,b) RNA (a) and protein (b) expression of the activin receptor-like kinases (ALK1, 2, and 5) in hPdLFs. (c) Co-immunoprecipitation with ALK1, ALK2, and ALK5-specific antibodies (ALK1/2/5- Ab) to detect GDF15 binding by those receptors in hPdLFs stimulated with 5 ng/mL recombinant human GDF15 (rhGDF15). GDF15 detection in whole cell lysates (WCLs) and GDF15-Ab-precipitated proteins were used as positive controls. (d) Metabolic activity of hPdLFs after stimulation with 5 ng/mL and 20 ng/mL recombinant human GDF15 (rhGDF15) for 12, 24, and 36 days displayed in relation to the 12-day control. (e–g) Ki-67-positive hPdLFs (magenta) after stimulation with rhGDF15 with cell nuclei (blue; (e)). The number of cells per mm2 is displayed in (f), and (g) shows the proportion of Ki-67-positive hPdLFs. (h) The number of trypan blue-positive hPdLFs after rhGDF15 stimulation, indicating the proportion of dead cells. (i,j) TUNEL-positive hPdLFs (green, (i)) after rhGDF15 stimulation with cell nuclei (blue), displayed as a proportion to the cell number (j). * p < 0.05; **/## p < 0.01; ***/### p < 0.001; */**/*** control in relation to 5 ng/mL rhGDF15; ##/### control in relation to 20 ng/mL rhGDF15; one-way ANOVA and Tukey post hoc test. Scale bar: 20 µm in (e) and 25 µm in (i); d, days; dRN, the difference between baseline and measured fluorescence; n.s., not significant.

Article Snippet: For evaluation of GDF15-induced effects, hPdLFs were stimulated for 12, 24, and 36 days with 5 ng/mL or 20 ng/mL recombinant human GDF15 protein (rhGDF15, 9279-GD-050, R&D Systems, Minneapolis, MN, USA) in culture flasks.

Techniques: Expressing, Immunoprecipitation, Binding Assay, Recombinant, Activity Assay, Control, TUNEL Assay

Journal: International journal of molecular sciences

Article Title: GDF15 Promotes the Osteogenic Cell Fate of Periodontal Ligament Fibroblasts, thus Affecting Their Mechanobiological Response.

doi: 10.3390/ijms241210011

Figure Lengend Snippet: Figure 2. GDF15 promote cellular senescence of hPdLFs. (a,b) p21 intensity in hPdLFs stimulated with 5 ng/mL and 20 ng/mL rhGDF15 for 12, 24, and 36 days shown as thermal LUT (a). Grey scattered lines surround the cell nuclei. Mean p21 intensity is shown in relation to the 12-day control in (b). (c,d) β-galactosidase (β-Gal)-positive hPdLFs (magenta) after rhGDF15 stimulation with cell nuclei (blue, (c)). The proportion of β-Gal-positive hPdLFs is displayed in (d). **/## p < 0.01; ***/### p < 0.001; **/*** control in relation to 5 ng/mL rhGDF15; ##/### control in relation to 20 ng/mL rhGDF15; one-way ANOVA and Tukey post hoc test. Scale bar: 10 µm in (a) and 25 µm in (c); d, days.

Article Snippet: For evaluation of GDF15-induced effects, hPdLFs were stimulated for 12, 24, and 36 days with 5 ng/mL or 20 ng/mL recombinant human GDF15 protein (rhGDF15, 9279-GD-050, R&D Systems, Minneapolis, MN, USA) in culture flasks.

Techniques: Control

Journal: International journal of molecular sciences

Article Title: GDF15 Promotes the Osteogenic Cell Fate of Periodontal Ligament Fibroblasts, thus Affecting Their Mechanobiological Response.

doi: 10.3390/ijms241210011

Figure Lengend Snippet: Figure 3. Long-term exposure to GDF15 fosters the osteogenic differentiation of hPdLFs. (a,b) ALPL and RUNX2 expression encoding osteoblast-related markers in hPdLFs stimulated with 5ng/mL and 20 ng/mL recombinant human GDF15 (rhGDF15) for 12, 24, and 36 days displayed in relation to the control at each time point. (c,d) ALP activity (dark blue; (c)) of 36-day rhGDF15-stimulated hPdLFs displayed in relation to the control in (d). (e,f) Alizarin red staining intensity of hPdLFs in wells stimulated for 36 d with rhGDF15 (e) displayed as a percentage (%) in relation to the control in (f). Stimulation with DMS and βGP was used as a positive control for osteogenic differentiation. * p < 0.05; **/##/§§ p < 0.01; ***/###/§§§ p < 0.001; */**/*** in relation to control; ##/### in relation to 5 ng/mL rhGDF15; §§/§§§ in relation to 20 ng/mL rhGDF15; one-way ANOVA and Tukey post hoc test. Scale bar: 25 µm in (c), 5 mm in (e); ARS, alizarin red staining; d, days; n.s., non significant.

Article Snippet: For evaluation of GDF15-induced effects, hPdLFs were stimulated for 12, 24, and 36 days with 5 ng/mL or 20 ng/mL recombinant human GDF15 protein (rhGDF15, 9279-GD-050, R&D Systems, Minneapolis, MN, USA) in culture flasks.

Techniques: Expressing, Recombinant, Control, Activity Assay, Staining, Positive Control

Journal: International journal of molecular sciences

Article Title: GDF15 Promotes the Osteogenic Cell Fate of Periodontal Ligament Fibroblasts, thus Affecting Their Mechanobiological Response.

doi: 10.3390/ijms241210011

Figure Lengend Snippet: Figure 4. Long-term GDF15-exposed hPdLFs show a reduced anti-inflammatory and pro-osteogenic mechanoresponse to tensile forces. (a,b) Quantitative expression levels of IL10 (a) and IL1RN (b) encoding anti-inflammatory markers in hPdLFs stimulated with 5ng/mL and 20 ng/mL recombinant human GDF15 (rhGDF15) for 36 days and stressed with tensile forces for 24 h (+TF) displayed in relation to the untreated control. (c,d) Adherent (activated) THP1 cells (green) on stimulated and stressed hPdLFs (blue, cell nuclei) displayed as the number of THP1 cells per hPdLFs and in relation to the untreated control in (d). (e,f) ALP activity (dark blue) of 36-day rhGDF15-stimulated hPdLFs stressed with TF (c), displayed in relation to the control in (d). (g,h) Alizarin red staining intensity of hPdLFs in wells stimulated for 36 d with rhGDF15 and stressed with TF (g), displayed as a percentage (%) in relation to the control in (h). Stimulation with DMS and βGP was used as a positive control for osteogenic differentiation. Red lines show baseline levels of the respective condition. */#/§ p < 0.05; **/## p < 0.01; ***/###/§§§ p < 0.001; */**/*** in relation to control; #/##/### in relation to control + TF; §§§ baseline (red) in relation to 20 ng/mL rhGDF15 + TF; one-way ANOVA and post hoc test (Tukey). Scale bars: 25 µm in (c,e), 5 mm in (g); ARS, alizarin red staining; d, days.

Article Snippet: For evaluation of GDF15-induced effects, hPdLFs were stimulated for 12, 24, and 36 days with 5 ng/mL or 20 ng/mL recombinant human GDF15 protein (rhGDF15, 9279-GD-050, R&D Systems, Minneapolis, MN, USA) in culture flasks.

Techniques: Expressing, Recombinant, Control, Activity Assay, Staining, Positive Control

Journal: International journal of molecular sciences

Article Title: GDF15 Promotes the Osteogenic Cell Fate of Periodontal Ligament Fibroblasts, thus Affecting Their Mechanobiological Response.

doi: 10.3390/ijms241210011

Figure Lengend Snippet: Figure 5. Long-term GDF15-exposed hPdLFs show an increased pro-inflammatory response to compressive stimuli with limited activation of osteoclasts. (a,b) Quantitative expression levels of IL6 (a) and COX2 (b) encoding pro-inflammatory markers in hPdLFs stimulated with 5 ng/mL and 20 ng/mL recombinant human GDF15 (rhGDF15) for 36 days and stressed with compressive forces for 24 h (+CF), displayed in relation to the untreated control. (c,d) Adherent (activated) THP1 cells (green) on stimulated and stressed hPdLFs (blue, cell nuclei) displayed as the number of THP1 cells per hPdLFs and in relation to the untreated control in (d). (e,f) Quantitative expression levels of OPG (e) and RANKL (f) in stimulated and stressed hPdLFs displayed in relation to the untreated control. (g,h) TRAP-positive THP1 cells (magenta; (g)) indicating the differentiation into osteoclasts, when stimulated with the medium supernatant of 36-day rhGDF15-stimulated hPdLFs additionally stressed with CF. The proportion of TRAP-positive osteoclasts is displayed in (h). Red lines show baseline levels of the respective conditions. */#/§ p < 0.05; **/##/§§ p < 0.01; ***/###/§§§ p < 0.001; */**/*** in relation to control; #/##/### in relation to control + TF; §§/§§§ baseline (red) in relation to 5 ng/mL or 20 ng/mL rhGDF15 + TF; one-way ANOVA and Tukey post hoc test. Scale bars: 25 µm in (c,g); d, days.

Article Snippet: For evaluation of GDF15-induced effects, hPdLFs were stimulated for 12, 24, and 36 days with 5 ng/mL or 20 ng/mL recombinant human GDF15 protein (rhGDF15, 9279-GD-050, R&D Systems, Minneapolis, MN, USA) in culture flasks.

Techniques: Activation Assay, Expressing, Recombinant, Control