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