Journal: International Journal of Oncology

Article Title: Effects of non-steroidal anti-inflammatory drug-activated gene-1 on Ganoderma lucidum polysaccharides-induced apoptosis of human prostate cancer PC-3 cells

doi: 10.3892/ijo.2018.4578

Figure Lengend Snippet: Expression levels of NAG-1, and its transcription factor EGR-1, are induced in PC-3 cells upon GLP treatment. (A) Quantification of the mRNA expression levels of NAG-1 and EGR-1 in a time-dependent manner (0-24 h) following treatment of PC-3 cells with 5 mg/ml GLP. Data are presented as the means ± standard error from three independent experiments. Two-way ANOVA with post hoc Bonferroni's correction for multiple comparison was used to determine statistical significance. * indicates time-dependent effects; # indicates effects of GLP treatment. * P<0.05 and ** P<0.01 compared with the 0 h group for each treatment. # P<0.05 and ## P<0.01 compared with the untreated control group at each time-point. (B) Quantification of the mRNA expression levels of NAG-1 and EGR-1 in a dose-dependent manner following treatment of PC-3 cells with GLP (0-10 mg/ml) for 24 h. Induction of EGR-1 and NAG-1 protein expression upon GLP treatment in a (C) time-dependent and (D) dose-dependent manner, as determined by western blotting. β-actin was used as an internal control. (E) GLP induced NAG-1 promoter activity, as determined by luciferase assay. Fold-changes were normalized to pRL-null expressing Renilla luciferase protein. (F) ELISA of the concentration of NAG-1 protein in the cell culture medium following treatment with 0-10 mg/ml GLP for 48 h. Data presented were normalized to the concentration of protein in lysates from each sample. All data are presented as the means ± standard error of three independent experiments. * P<0.05, ** P<0.01 compared with the control group (one-way ANOVA with Dunnett's correction). ANOVA, analysis of variance; EGR-1, early growth response-1; GLP, Ganoderma lucidum polysaccharides; NAG-1, non-steroidal anti-inflammatory drug-activated gene-1; RLU, relative light units.

Article Snippet: The Quantikine Human GDF15/NAG-1 ELISA kit (cat. no. DY957) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing, Comparison, Western Blot, Activity Assay, Luciferase, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture

Journal: International Journal of Oncology

Article Title: Effects of non-steroidal anti-inflammatory drug-activated gene-1 on Ganoderma lucidum polysaccharides-induced apoptosis of human prostate cancer PC-3 cells

doi: 10.3892/ijo.2018.4578

Figure Lengend Snippet: GLP-induced apoptosis of PC-3 cells is mediated through NAG-1 induction. (A) NAG-1 siRNA successfully knocked down NAG-1 expression in PC-3 cells, as determined by western blotting. (B) NAG-1 siRNA inhibited GLP-induced NAG-1 expression, as determined by western blotting. (C) NAG-1 siRNA inhibited GLP-induced apoptosis, as determined by flow cytometry. Percentage of early and late apoptotic of PC-3 cells induced by GLP is presented in the lower panel. (D) NAG-1 siRNA inhibited GLP-induced PARP cleavage and the suppression of pro-caspase-3, -6 and -9 protein expression. β-actin was used as an internal control. Data are presented as the means ± standard error. * P<0.05, ** P<0.01 compared with the control group (one-way analysis of variance with Dunnett's correction). ## P<0.01, compared with GLP-treated cells. GLP, Ganoderma lucidum polysaccharides; NAG-1, non-steroidal anti-inflammatory drug-activated gene-1; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; siRNA, small interfering RNA.

Article Snippet: The Quantikine Human GDF15/NAG-1 ELISA kit (cat. no. DY957) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Flow Cytometry, Small Interfering RNA

Journal: International Journal of Oncology

Article Title: Effects of non-steroidal anti-inflammatory drug-activated gene-1 on Ganoderma lucidum polysaccharides-induced apoptosis of human prostate cancer PC-3 cells

doi: 10.3892/ijo.2018.4578

Figure Lengend Snippet: Working model of the molecular mechanisms by which GLP exerts its anticancer activity in prostate cancer PC-3 cells. GLP-induced apoptosis is mediated by NAG-1 induction, which may serve a pivotal role in GLP-induced cell death in prostate cancer cells.

Article Snippet: The Quantikine Human GDF15/NAG-1 ELISA kit (cat. no. DY957) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Activity Assay

Journal: Cancer Science

Article Title: Growth differentiation factor 15 induces cisplatin resistance through upregulation of xCT expression and glutathione synthesis in gastric cancer

doi: 10.1111/cas.15869

Figure Lengend Snippet: Growth differentiation factor 15 (GDF15) expression is elevated in gastric cancer and high serum GDF15 level is a poor prognostic factor. (A) GDF15 gene expression level in gastric cancer patients illustrated with boxplots by the Gene Expression Profiling Interactive Analysis (GEPIA) online database. (B) The Kaplan–Meier plotter online database was used to analyze the clinical effect of GDF15 gene expression in gastric cancer patients ( http://kmplot.com/analysis/ ). (C) Clinical effect of GDF15 serum levels (≥upper quartile 1066.79 ng/mL vs.

Article Snippet: The GDF15 neutralizing Ab (MAB957) and recombinant human GDF15 ( Escherichia coli ‐expressed) protein (#9279‐GD‐050) were obtained from R&D Systems.

Techniques: Expressing, Gene Expression

Journal: Cancer Science

Article Title: Growth differentiation factor 15 induces cisplatin resistance through upregulation of xCT expression and glutathione synthesis in gastric cancer

doi: 10.1111/cas.15869

Figure Lengend Snippet: Clinical characteristics of patients with gastric cancer with different growth differentiation factor 15 (GDF15) expression of tumor tissues

Article Snippet: The GDF15 neutralizing Ab (MAB957) and recombinant human GDF15 ( Escherichia coli ‐expressed) protein (#9279‐GD‐050) were obtained from R&D Systems.

Techniques: Expressing

Journal: Cancer Science

Article Title: Growth differentiation factor 15 induces cisplatin resistance through upregulation of xCT expression and glutathione synthesis in gastric cancer

doi: 10.1111/cas.15869

Figure Lengend Snippet: Clinical characteristics of patients with gastric cancer with low or high serum growth differentiation factor 15 (GDF15) levels

Article Snippet: The GDF15 neutralizing Ab (MAB957) and recombinant human GDF15 ( Escherichia coli ‐expressed) protein (#9279‐GD‐050) were obtained from R&D Systems.

Techniques:

Journal: Cancer Science

Article Title: Growth differentiation factor 15 induces cisplatin resistance through upregulation of xCT expression and glutathione synthesis in gastric cancer

doi: 10.1111/cas.15869

Figure Lengend Snippet: Growth differentiation factor 15 (GDF15) expression is essential for cell proliferation and migration of gastric cancer cells. (A, C) siGDF15 (180 pmol for 3 × 10 5 cells in a 6‐cm dish for 48 h) or (B, D) pcDNA‐GDF15 (pGDF15, 6 μg for 3 × 10 5 cells in a 6‐cm dish for 12 h, followed by replacement of fresh medium for a total of 48 h) were used to knockdown or overexpress GDF15. Efficiencies of knockdown and overexpression were analyzed by quantitative real‐time PCR. (A, B) After transfection with siGDF15 or pGDF15, cells were reseeded with a density of 3000 cells per well in a 96‐well plate. Cell proliferation was analyzed with sulforhodamine B (SRB) assay. (C, D) After transfection with siGDF15 or pGDF15, cells were reseeded with a density of 1 × 10 5 cells per Transwell insert. Cell migration was determined by Transwell migration assay (siGDF15: AGS, NUGC‐3, and TSGH9201 for 12, 16, and 24 h migration, respectively; magnification, 200×) (pGDF15: AGS, NUGC‐3, and TSGH9201 for 8, 12, and 24 h migration, respectively; magnification, 100×). Graph is presented as mean ± SEM ( n ≥ 3). *Significant vs. individual control.

Article Snippet: The GDF15 neutralizing Ab (MAB957) and recombinant human GDF15 ( Escherichia coli ‐expressed) protein (#9279‐GD‐050) were obtained from R&D Systems.

Techniques: Expressing, Migration, Knockdown, Over Expression, Real-time Polymerase Chain Reaction, Transfection, Sulforhodamine B Assay, Transwell Migration Assay, Control

Journal: Cancer Science

Article Title: Growth differentiation factor 15 induces cisplatin resistance through upregulation of xCT expression and glutathione synthesis in gastric cancer

doi: 10.1111/cas.15869

Figure Lengend Snippet: Growth differentiation factor 15 (GDF15) contributes to cisplatin resistance in human gastric cancer cells. (A–C) GDF15 (A) gene and (B) protein expressions and (C) released GDF15 level between parental (P) and cisplatin‐resistant (CisR) gastric cancer cells were analyzed with quantitative real‐time PCR, western blotting, and ELISA assays, respectively. (D, E) Cisplatin sensitivity (48 h) of the gastric cancer cells was assessed using (D) sulforhodamine B (SRB) assay and (E) propidium iodide (PI) exclusion assay. (F–H) Effects of (F) GDF15 neutralizing Ab (GDF15 NAb), (G) recombinant human GDF15 (rhGDF15), and (H) GDF15 overexpression on sensitivity of cisplatin were evaluated with SRB assay. G, GDF15 plasmid; V, empty vector. Quantitative real‐time PCR and western blotting were used to validate the efficiencies of GDF15 knockdown or overexpression, respectively. Graph is presented by mean ± SEM ( n ≥ 3). *Significant vs. individual control. ** , ***Significant, rhGDF15 (20 and 50 ng/mL) vs. individual control.

Article Snippet: The GDF15 neutralizing Ab (MAB957) and recombinant human GDF15 ( Escherichia coli ‐expressed) protein (#9279‐GD‐050) were obtained from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Sulforhodamine B Assay, Exclusion Assay, Recombinant, Over Expression, Plasmid Preparation, Knockdown, Control

Journal: Cancer Science

Article Title: Growth differentiation factor 15 induces cisplatin resistance through upregulation of xCT expression and glutathione synthesis in gastric cancer

doi: 10.1111/cas.15869

Figure Lengend Snippet: Growth differentiation factor 15 (GDF15)‐upregulated xCT expression through the eukaryotic initiation factor 2α (eIF2α)‐activating transcription factor 4 (ATF4) pathway enhances intracellular glutathione (GSH) levels in cisplatin‐resistant gastric cancer cells. (A) Knockdown efficiency was validated using western blotting. (B) Effects of siGDF15 and glial cell‐derived neurotrophic factor family receptor a‐like siRNA (siGFRAL) on GSH levels were evaluated using the GSH detection kit. (C, D) After treatment of GDF15‐knockdown cisplatin‐resistant (CisR) cells with cisplatin (24 h), intracellular and mitochondrial reactive oxygen species were evaluated with (C) dichlorodihydro‐fluorescein (DCF) and (D) MitoSox Red using flow cytometry. (E) GDF15 and xCT gene expressions were evaluated using quantitative real‐time PCR. (F) Protein expression of GDF15 and the eIF2α‐xCT pathway were evaluated using western blotting. (G) After transfections with different xCT promoters (WT, antioxidant‐responsive element [ARE]‐mutant, and amino acid response element [AARE]‐mutant), the cells were further transfected with siGDF15. Graph is presented as mean ± SEM ( n ≥ 3). *Significant vs. individual control. **Significant vs. WT/ARE‐mutant‐xCT promoters.

Article Snippet: The GDF15 neutralizing Ab (MAB957) and recombinant human GDF15 ( Escherichia coli ‐expressed) protein (#9279‐GD‐050) were obtained from R&D Systems.

Techniques: Expressing, Knockdown, Western Blot, Derivative Assay, Flow Cytometry, Real-time Polymerase Chain Reaction, Transfection, Mutagenesis, Control

Journal: Cancer Science

Article Title: Growth differentiation factor 15 induces cisplatin resistance through upregulation of xCT expression and glutathione synthesis in gastric cancer

doi: 10.1111/cas.15869

Figure Lengend Snippet: Eukaryotic initiation factor 2α (eIF2α)‐activating transcription factor 4 (ATF4)‐xCT‐elevated glutathione (GSH) contributes to growth differentiation factor 15 (GDF15)‐mediated cisplatin resistance in gastric cancer cells. (A, B, D) Cells were transfected with pcDNA‐GDF15. G, GDF15 plasmid; V, empty vector. (A, D) Gene expressions of GDF15 and xCT were evaluated using quantitative real‐time PCR. (B) The eIF2α‐ATF4‐xCT pathway was evaluated using western blotting. (C) After transfection with different types of xCT promoters, HEK293T cells were further transfected with pcDNA‐GDF15. (D) After overexpression of GDF15, the effects of sulfasalazine (SSA, 350 μM) and buthionine sulfoximine (BSO, 0.5 mM) on cisplatin sensitivity (48 h) were evaluated with propidium iodide (PI) exclusion assay. Graph is presented as mean ± SEM ( n ≥ 3). *Significant vs. individual control. **Significant vs. WT/antioxidant‐responsive element (ARE)‐mutant xCT promoters. # Significant vs. pcDNA. ## Significant vs. cisplatin treatment. AARE, amino acid response element; Con, control.

Article Snippet: The GDF15 neutralizing Ab (MAB957) and recombinant human GDF15 ( Escherichia coli ‐expressed) protein (#9279‐GD‐050) were obtained from R&D Systems.

Techniques: Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Over Expression, Exclusion Assay, Control, Mutagenesis

Journal: Cancer Science

Article Title: Growth differentiation factor 15 induces cisplatin resistance through upregulation of xCT expression and glutathione synthesis in gastric cancer

doi: 10.1111/cas.15869

Figure Lengend Snippet: Growth differentiation factor 15 (GDF15)/ glial cell‐derived neurotrophic factor family receptor a‐like (GFRAL)‐mediated signaling in cisplatin‐resistant gastric cancer cells could be through general control nonderepressible 2 (GCN2). (A, B) siGDF15 and siGFRAL were transfected into (A) AGS cisplatin‐resistant (CisR) and (B) NUGC‐3CisR cells. (C) AGSCisR and (D) NUGC‐3CisR cells were treated with SPP86 (5 μΜ) for 24 h. Upstream regulators of the eukaryotic initiation factor 2α (eIF2α) and eIF2α‐activating transcription factor 4 (ATF4)‐xCT pathways were analyzed using western blotting. Graph is presented as mean ± SEM ( n ≥ 3). *Significant vs. individual control (Con). PERK, PKR‐like endoplasmic reticulum kinase; PKR, protein kinase R.

Article Snippet: The GDF15 neutralizing Ab (MAB957) and recombinant human GDF15 ( Escherichia coli ‐expressed) protein (#9279‐GD‐050) were obtained from R&D Systems.

Techniques: Derivative Assay, Control, Transfection, Western Blot

Journal: Cancer Science

Article Title: Growth differentiation factor 15 induces cisplatin resistance through upregulation of xCT expression and glutathione synthesis in gastric cancer

doi: 10.1111/cas.15869

Figure Lengend Snippet: General control nonderepressible 2 (GCN2) is responsible for growth differentiation factor 15 (GDF15)‐mediated glial cell‐derived neurotrophic factor family receptor a‐like (GFRAL)‐eukaryotic initiation factor 2α (eIF2α)‐activating transcription factor 4 (ATF4)‐xCT signaling and cisplatin resistance. (A, B, D) After GDF15 overexpression by pcDNA‐GDF15 (G, pcDNA‐GDF15; V, pcDNA alone), cells were treated with siRNAs against (A) protein kinase R (PKR), (B) heme‐regulated eIF2α kinase (HRI), and (D) GCN2 for 48 h. The effect of siRNAs against PKR, HRI, and GCN2 on GDF15‐mediated eIF2α‐ATF4‐xCT regulation was assessed using western blotting. (C) Effects of siHRI and siPKR on cisplatin resistance in cisplatin‐resistant (CisR) cells was evaluated with sulforhodamine B (SRB) assay. Graph is presented as mean ± SEM ( n ≥ 3). *Significant vs. individual control. **Significant vs. siScr with GDF15 overexpression.

Article Snippet: The GDF15 neutralizing Ab (MAB957) and recombinant human GDF15 ( Escherichia coli ‐expressed) protein (#9279‐GD‐050) were obtained from R&D Systems.

Techniques: Control, Derivative Assay, Over Expression, Western Blot, Sulforhodamine B Assay

Journal: Cancer Science

Article Title: Growth differentiation factor 15 induces cisplatin resistance through upregulation of xCT expression and glutathione synthesis in gastric cancer

doi: 10.1111/cas.15869

Figure Lengend Snippet: Proposed mechanism of growth differentiation factor 15 (GDF15)‐mediated cisplatin resistance. In the present study, we found that GDF15‐elevated glutathione (GSH) through the glial cell‐derived neurotrophic factor family receptor a‐like (GFRAL)‐general control nonderepressible 2 (GCN2)‐eukaryotic initiation factor 2α (eIF2α)‐activating transcription factor 4 (ATF4)‐xCT pathway enhances cisplatin resistance for gastric cancer. Figure was created by Servier Medical Art. PKR, protein kinase R; RET, rearranged during transfection; ROS, reactive oxygen species.

Article Snippet: The GDF15 neutralizing Ab (MAB957) and recombinant human GDF15 ( Escherichia coli ‐expressed) protein (#9279‐GD‐050) were obtained from R&D Systems.

Techniques: Derivative Assay, Control, Transfection

Journal: Cell reports

Article Title: Exploring the In Vivo Role of the Mitochondrial Calcium Uniporter in Brown Fat Bioenergetics

doi: 10.1016/j.celrep.2019.04.013

Figure Lengend Snippet:

Article Snippet: FGF21 and GDF15 were measured from plasma samples using R&D Systems Quantikine ELISA Kits MF2100 and MGD150, respectively, according to the manufacturer’s protocols.

Techniques: Synthesized, Recombinant, Saline, Protease Inhibitor, Lysis, Modification, Gene Expression, Enzyme-linked Immunosorbent Assay, Generated, Software

Journal: Journal of Tissue Engineering

Article Title: GDF15 promotes osteogenic differentiation of human dental pulp stem cells by activating the TGF-β/SMAD signaling pathway

doi: 10.1177/20417314251357752

Figure Lengend Snippet: GDF15 secretion was significantly upregulated during the osteogenic differentiation of hDPSCs. (a) Morphology of primary hDPSCs. Scale bars (white): 100 μm. Morphology of passage 3 hDPSCs at 24 h of culture. Scale bars (black): 250 μm. P0 = Passage 0; P3 = Passage three. (b) Colony-forming assay to assess the self-renewal ability of hDPSCs. Scale bars: 250 μm. (c) The CCK-8 assay was employed to assess the proliferation of hDPSCs. (d) Proliferation of hDPSCs detected by crystal violet staining. Scale bars: 250 μm. (e) ALP staining of hDPSCs grown in OM for a week. Scale bars: 250 μm. (f) ARS staining of 21-day-cultured hDPSCs in OM and quantitative analysis of mineralized nodule deposition. Scale bars: 250 μm. (g) Flow cytometry demonstrated that the hDPSCs highly expressed CD105, CD90, and CD73; and lowly expressed CD45, CD19, and CD14. (h) During induction, intracellular mRNA levels of Gdf15 and osteogenic-specific genes ( Alp, Runx2, Osx, Ocn , and D spp ) were elevated. (i) During induction, osteogenic-specific proteins (ALP, RUNX2, OPN, DMP1, and DSPP) were upregulated and intracellular GDF15 protein was reduced in hDPSCs. Relative quantitative analysis of gray scale values of protein bands. The internal control was GAPDH. (j) Increased GDF15 secretion was detected by ELISA after osteogenic induction. Data were displayed as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00001).

Article Snippet: The GDF15 antibody (Santa Cruz Biotechnology, Dallas, USA; sc-377195) was conjugated to protein A/G magnetic beads (MedChemExpress, New Jersey, USA) using a rotary mixer (Bio-Rad, Hercules, CA, USA) under sequential conditions: 30 min at room temperature and 2 h at 4°C.

Techniques: CCK-8 Assay, Staining, Cell Culture, Flow Cytometry, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of Tissue Engineering

Article Title: GDF15 promotes osteogenic differentiation of human dental pulp stem cells by activating the TGF-β/SMAD signaling pathway

doi: 10.1177/20417314251357752

Figure Lengend Snippet: G df15 overexpression promoted osteogenic differentiation of hDPSCs in vitro , while Gdf15 knockdown suppressed it. (a) Images of GFP-positive hDPSCs were observed using fluorescence microscopy. Scale bars: 200 μm. GFP, Green Fluorescent Protein. (b) Relative mRNA expression of Gdf15 in the NC and G df15 overexpression ( Gdf15 ) groups. (c) Protein levels of G DF15 in the NC and Gdf15 groups. (d) Relative mRNA expression of G df15 in Gdf15 overexpressing hDPSCs after 7, 14, and 21 days of incubation in the OM. (e) ALP staining of the G df15 and NC groups on day 7 of osteogenic differentiation. (f) ARS staining and quantitative analysis of overexpressed hDPSCs were performed on days 7, 14, and 21 of osteogenic differentiation. (g) Osteogenic-specific genes ( Alp, Col1a1, Runx2, Osx , and Dmp1 ) and their relative mRNA levels in hDPSCs was examined by qPCR on days 7, 14, and 21 of the induced differentiation. (h) Relative mRNA expression of Gdf15 in the siNC and G df15 knockdown ( siGdf15 ) groups. (i) GDF15 protein levels in siNC and siGdf15 groups. (j) Relative mRNA expression of Gdf15 in siRNA-transfected hDPSCs after 7, 14, and 21 days of incubation in the OM. (k) ALP staining in the siNC and siGDF15 groups on day 7 of induced differentiation. (l) ARS staining and quantitative analysis were performed on hDPSCs transfected with siRNA on days 7, 14, and 21 of induced differentiation. (m) Relative mRNA levels of Alp, Col1a1, Runx2, Osx , and Dmp1 in siRNA-transfected hDPSCs on days 7, 14, and 21 of induced differentiation. Scale bars: 250 μm (ALP and ARS staining); Mean ± SD was employed to express all data (* p < 0.05, ** p < 0.01, *** p < 0.0001, **** p < 0.00001).

Article Snippet: The GDF15 antibody (Santa Cruz Biotechnology, Dallas, USA; sc-377195) was conjugated to protein A/G magnetic beads (MedChemExpress, New Jersey, USA) using a rotary mixer (Bio-Rad, Hercules, CA, USA) under sequential conditions: 30 min at room temperature and 2 h at 4°C.

Techniques: Over Expression, In Vitro, Knockdown, Fluorescence, Microscopy, Expressing, Incubation, Staining, Transfection

Journal: Journal of Tissue Engineering

Article Title: GDF15 promotes osteogenic differentiation of human dental pulp stem cells by activating the TGF-β/SMAD signaling pathway

doi: 10.1177/20417314251357752

Figure Lengend Snippet: rhGDF15 activated TGF-β/SMAD signaling pathway in hDPSCs. (a and b) Tgf-βr2 mRNA levels in hDPSCs overexpressing or knockdown of Gdf15 were measured after culturing in OM for 7, 14, and 21 days. (c) Tgf-βr2 mRNA levels in hDPSCs treated with OM containing rhGDF15 were measured after 7, 14, and 21 days of culture. (d) Structural modeling for molecular docking analysis of GDF15 and TGF-βR2. GDF15 and TGF-βR2 are depicted in blue-purple and orange-yellow, respectively. Hydrogen bonds are indicated in yellow. (e) Co-IP assay showing representative protein bands of GDF15 and TGFβ-R2 in hDPSCs. (f) Levels of TGF-β/SMAD signaling pathway-specific proteins after 20 ng/mL rhGDF15 treatment of hDPSCs for the indicated times were tested utilizing WB and grayscale analysis of protein bands. (g) Levels of TGF-β/SMAD signaling pathway-specific proteins after rhGDF15 (0–100 ng/mL) treatment of hDPSCs for 30 min were detected by WB and grayscale analysis of protein bands. (h) Expression of total, plasma, and nuclear proteins of p-SMAD2 and p-SMAD3 in hDPSCs after stimulation with rhGDF15 (20 ng/mL) for 30 min, and grayscale analysis of protein bands. (i) Immunofluorescence co-localization of TGF-β/SMAD signaling proteins in implants in the nude mouse subcutaneous transplantation model. Scale bars: 100 μm. The area of new bone formation is delineated by yellow dashed lines. The white dashed box demarcates the regions selected for high-magnification demonstration of osteogenic areas. Scale bars: 100 μm (enlarged view). Data were displayed as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: The GDF15 antibody (Santa Cruz Biotechnology, Dallas, USA; sc-377195) was conjugated to protein A/G magnetic beads (MedChemExpress, New Jersey, USA) using a rotary mixer (Bio-Rad, Hercules, CA, USA) under sequential conditions: 30 min at room temperature and 2 h at 4°C.

Techniques: Knockdown, Co-Immunoprecipitation Assay, Expressing, Clinical Proteomics, Immunofluorescence, Transplantation Assay

Journal: Journal of Tissue Engineering

Article Title: GDF15 promotes osteogenic differentiation of human dental pulp stem cells by activating the TGF-β/SMAD signaling pathway

doi: 10.1177/20417314251357752

Figure Lengend Snippet: Activation of the TGF-β/SMAD signaling pathway by GDF15 is partially reversed by the inhibitors. (a) The concentration of the inhibitor was selected based on the analysis of the protein level ratios of phosphorylated to total SMAD2 or SMAD3. (b and c) The protein expression levels and quantitative analysis of p-SMAD2/SMAD2 and p-SMAD3/SMAD3 were assessed in hDPSCs pretreated with inhibitor for 1 h followed by rhGDF15 stimulation for 30 min. (d) Representative images and quantitative analysis of p-SMAD2 or p-SMAD3 immunofluorescence staining. Scale bars: 100 μm. (e and f) The osteogenic effects of inhibitor-treated hDPSCs were analyzed by qPCR. (g) ALP staining following 7 days of treatment. Scale bars: 250 μm. (h) ARS staining following 21 days of treatment and relative quantitative analysis. Scale bars: 250 μm. (i and j) The osteogenic effects of hDPSCs treated with inhibitors for 7 days were evaluated by WB, and the protein bands were analyzed in gray. Mean ± SD was employed to express all data (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: The GDF15 antibody (Santa Cruz Biotechnology, Dallas, USA; sc-377195) was conjugated to protein A/G magnetic beads (MedChemExpress, New Jersey, USA) using a rotary mixer (Bio-Rad, Hercules, CA, USA) under sequential conditions: 30 min at room temperature and 2 h at 4°C.

Techniques: Activation Assay, Concentration Assay, Expressing, Immunofluorescence, Staining