Journal: American journal of physiology. Endocrinology and metabolism

Article Title: Myostatin propeptide mutation of the hypermuscular Compact mice decreases the formation of myostatin and improves insulin sensitivity.

doi: 10.1152/ajpendo.00216.2016

Figure Lengend Snippet: Fig. 3. Myostatin level in skeletal muscle of Compact, congenic wild-type, and BALB/c mice. M. gastrocnemius protein extracts were subjected to SDS-PAGE and blotted with anti-myostatin or anti-propeptide antibody. Represen- tative images are shown. Note the presence of mature myostatin dimer and myostatin propeptide in Compact sam- ples. Mouse recombinant myostatin was used as a positive control, and muscle homogenates of myostatin knockout (KO) mice served as a negative control. Differences in glycosylation may cause altered electrophoretic mobility. Bar diagrams show the quantification of the results. Data are reported as means SE; n 5 Compact, 5 congenic wild-type, and 6 BALB/c mice. *P 0.05; **P 0.01; ***P 0.001.

Article Snippet: Mouse recombinant myostatin (788-G8; R&D Systems) was used as a positive control.

Techniques: SDS Page, Recombinant, Positive Control, Knock-Out, Negative Control, Glycoproteomics

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: Myostatin propeptide mutation of the hypermuscular Compact mice decreases the formation of myostatin and improves insulin sensitivity.

doi: 10.1152/ajpendo.00216.2016

Figure Lengend Snippet: Fig. 5. Glucose tolerance and insulin sensitivity are improved by Compact myostatin mutation and reduced in congenic wild-type mice. Intraperitoneal (ip) glucose tolerance (A and B) and insulin sensitivity tests (C and D) of 3- to 4-mo-old (A and C) and 10-mo-old animals (B and D). Area under the curve (AUC) values are presented in bar diagrams. Data are reported as means SE. *P 0.05 and **P 0.01; n 3 Compact, 7 congenic wild-type, and 3 BALB/c mice (A), n 7 Compact, 3 congenic wild-type, and 6 BALB/c mice (B), n 3 Compact, 3 congenic wild-type, and 4 BALB/c mice (C), and n 6 Compact, 4 congenic wild-type, and 4 BALB/c mice (D).

Article Snippet: Mouse recombinant myostatin (788-G8; R&D Systems) was used as a positive control.

Techniques: Mutagenesis

Journal: Mediators of Inflammation

Article Title: CRP Stimulates GDF15 Expression in Endothelial Cells through p53

doi: 10.1155/2018/8278039

Figure Lengend Snippet: Women with hsCRP have high levels of GDF15. (a, b) Serum GDF15 levels were quantified by ELISA from HANDLS participants with either low- (<3 mg/L), mid- (>3–20 mg/L), or high hsCRP (>20 mg/L) levels ( n = 39/group). The ELISA assay was performed according to manufacturer's instructions and was repeated in 2 independent experiments. (c) RNA was isolated from PBMCs from HANDLS participants with either low- (<3 mg/L) or high hsCRP (>20 mg/L) levels ( n = 15/group). GDF15 mRNA was quantified by RT-qPCR and normalized to HPRT1 and UBC levels. The histograms represent the mean + SEM from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 by Student's t -test.

Article Snippet: Human GDF15 Quantikine ELISA Kit (R&D Systems) was used according to the manufacturer's directions.

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Quantitative RT-PCR

Journal: Mediators of Inflammation

Article Title: CRP Stimulates GDF15 Expression in Endothelial Cells through p53

doi: 10.1155/2018/8278039

Figure Lengend Snippet: CRP upregulates GDF15 expression. (a) 18 h after CRP treatment with the indicated doses, GDF15 expression in HAECs was analyzed by immunoblotting with anti-GDF15 antibodies. β -Actin was used as a loading control. (b) After CRP treatment for the indicated time points, conditioned media was collected and GDF15 secreted levels were analyzed by ELISA. GDF15 levels were normalized to the 0 h time point for each experiment. The mean of three independent experiments is shown. (c and d) 18 h after CRP (25 μ g/mL) treatment, HAECs were lysed and levels of GDF15 mRNA or protein were quantified by RT-qPCR analysis (c) and western blot analysis (d). (e) HAECs were transfected with pCMV6-control or pCMV6-CRP plasmid for 48 h. Total RNA was isolated, and mRNA levels were quantified using RT-qPCR and normalized to GAPDH . (f) Total cell lysates from the indicated transfected HAECs were analyzed by Western blotting. β -Actin was used as a loading control. The histograms represent the mean + SEM from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 by Student's t -test.

Article Snippet: Human GDF15 Quantikine ELISA Kit (R&D Systems) was used according to the manufacturer's directions.

Techniques: Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Transfection, Plasmid Preparation, Isolation

Journal: Mediators of Inflammation

Article Title: CRP Stimulates GDF15 Expression in Endothelial Cells through p53

doi: 10.1155/2018/8278039

Figure Lengend Snippet: CRP promotes GDF15 transcription. (a) Schematic of GDF15 promotor dual-luciferase constructs. Two p53 binding sites are indicated. (b) The indicated plasmids (1 μ g) were cotransfected with 0.1 μ g of TK-Renilla reporter plasmid in HeLa cells, and 24 h later, the cells were treated with CRP. After 18 h, the promoter activities were measured by luciferase activity. Transfection efficiency for luciferase activity was normalized to the Renilla luciferase activity. The results show the mean + SEM of three independent transfections. ∗∗ p < 0.01 by Student's t -test. (c) Schematic of p53 binding sites and primers used for ChIP assays in the GDF15 promoter. (d) ChIP assays were performed on HAECs transfected for 24 h and treated with or without CRP for 18 h. DNA immunoprecipitated by antibodies to p53 or immunoglobulin G IgG (control) was amplified by qPCR. Each qPCR reaction was performed in triplicate, and the histogram represents the average of three independent ChIP assays + SEM.

Article Snippet: Human GDF15 Quantikine ELISA Kit (R&D Systems) was used according to the manufacturer's directions.

Techniques: Luciferase, Construct, Binding Assay, Plasmid Preparation, Activity Assay, Transfection, Immunoprecipitation, Control, Amplification

Journal: Mediators of Inflammation

Article Title: CRP Stimulates GDF15 Expression in Endothelial Cells through p53

doi: 10.1155/2018/8278039

Figure Lengend Snippet: p53 knockdown inhibits CRP-induced GDF15 expression. HAECs were transfected with either Ctrl siRNA or p53 siRNA for 24 h and treated with or without CRP for 18 h. GDF15 mRNA levels were examined by RT-qPCR (a), and protein levels were analyzed by Western blot analysis (b). The histogram represents the mean + SEM from three independent experiments. ∗ p < 0.05 and ∗∗ p < 0.01 by Student's t -test.

Article Snippet: Human GDF15 Quantikine ELISA Kit (R&D Systems) was used according to the manufacturer's directions.

Techniques: Knockdown, Expressing, Transfection, Quantitative RT-PCR, Western Blot

Journal: Biology

Article Title: Transdifferentiation of Human Fibroblasts into Skeletal Muscle Cells: Optimization and Assembly into Engineered Tissue Constructs through Biological Ligands

doi: 10.3390/biology10060539

Figure Lengend Snippet: Determination of efficacy of C2C12 differentiation in conjunction with the exposure to ligand combinations. C2C12s were differentiated for 7 days and treated with combination ligands of GDF11 (G), TMSB4X (T), IL6 (I), and TNF-α (F) at 10 ng/mL for seven additional days. ( A ) Fusion index was calculated from total myotube nuclei vs. total nuclei ( n = 16, mean + SD). ( B ) Multinucleation of C2C12 myotubes were quantified ( n = 11, mean + SD). ( C ) Nuclear density was evaluated from nuclear count per field of 5x microscopy ( n = 4, mean + SD). ( D ) C2C12 exposed to ligand combinations were stained to express nuclear MYOD1 ( n = 6, mean + SD). ( E ) Cells were stained with Ki67, and where similarly quantified based on average total nuclear count ( n = 6, mean + SD). ( F ) ACTN2 and Ki67 immunostaining of control cells. ( G ) Cells exposed to GTF showed decreases in fusion index and myonucleation levels, although no change in nuclear density and Ki67+ expression was detected. ( H ) GTIF supplementation significantly reduced skeletal muscle differentiation parameters fusion index and multinucleation, in addition to decreasing average nuclear density. ( I ) Control C2C12s expressing nuclear MYOD1. ( J ) GTF treatment greatly reduced nuclear fusion and showed limited differentiation capacity while expressing comparable levels of nuclear MYOD1. ( K ) Exposure of C2C12s to GTIF combination significantly inhibited skeletal muscle differentiation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Proteins utilized included recombinant human Follistatin (Fs, 669-FO-025), recombinant human Myostatin (GDF8, 788-G8-010) or Growth Differentiation factor (GDF8), recombinant human basic Fibroblast Growth Factor 2 (FGF2, 233-FB-025), recombinant human GDF11 (1958-GD-010), recombinant human GDF15 (957-GD-025/CF), recombinant human Bone Morphogenetic Protein 4 (BMP4, 314-BP-010/CF), recombinant human BMP7 (354-BP-010), recombinant human Growth Hormone (hGH, 1067-GH-025), recombinant human Interleukin 6 (IL6, 206-IL-010), recombinant human Tumor Necrosis Factor Alpha (TNF-α, 210-TA-005) (All R&D Systems), and Thymosin β (TOCRIS, 3390).

Techniques: Microscopy, Staining, Immunostaining, Control, Expressing

Journal: Biology

Article Title: Transdifferentiation of Human Fibroblasts into Skeletal Muscle Cells: Optimization and Assembly into Engineered Tissue Constructs through Biological Ligands

doi: 10.3390/biology10060539

Figure Lengend Snippet: Effect of ligand combination exposure on differentiation of skeletal muscle cells derived from tHFs. Cells were transduced with MYOD1 fragments and induced to express the skeletal muscle phenotype via the induction of doxycycline and SB431542 over a 7-day period. Ligand combinations of GDF11 (G), TMSB4X (T), IL6 (I), and TNF-α (F) at 10 ng/mL were introduced for an additional week, and SB and Dox administration was discontinued. Skeletal muscle cells were fixed and stained on day 14 and characterized by various differentiation and proliferation parameters from 5× microscopy. ( A ) Fusion index of tHFs was evaluated by determining the ratio of myotube nuclei vs total nuclear count ( n = 16, mean + SD). ( B ) Cellular multinucleation was quantified to assess tHF development of differentiation ( n = 22, mean + SD). ( C ) Nuclear density was similarly assessed by quantifying nuclear count per field ( n = 4, mean + SD). ( D ) Nuclear MYOD1 was quantified ( n = 6, mean + SD). ( E ) Ki67 nuclei were also assessed with a nuclear count ( n = 6, mean + SD). ( F ) Control tHF myotubes were immunostained with ACTN2 and Ki67. ( G ) IL6 and TNF-α combination demonstrated significant decrease in differentiation parameters fusion index, multinucleation, myotube length, and diameter , although Ki67+ expression had increased. ( H ) Exposure of tHFs to combined GDF11, TMSB4X, IL6, and TNF-α showed similar results, however nuclear Ki67 expression was unchanged. ( I ) Untreated tHFs with ACTN2 and MYOD1 nuclear stains. ( J ) Cells treated with IF showed a decrease in MYOD1 nuclear expression. ( K ) Additionally, GDF11, TMSB4X, and IL6 exposure yielded similar results with respect to MYOD1+. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Proteins utilized included recombinant human Follistatin (Fs, 669-FO-025), recombinant human Myostatin (GDF8, 788-G8-010) or Growth Differentiation factor (GDF8), recombinant human basic Fibroblast Growth Factor 2 (FGF2, 233-FB-025), recombinant human GDF11 (1958-GD-010), recombinant human GDF15 (957-GD-025/CF), recombinant human Bone Morphogenetic Protein 4 (BMP4, 314-BP-010/CF), recombinant human BMP7 (354-BP-010), recombinant human Growth Hormone (hGH, 1067-GH-025), recombinant human Interleukin 6 (IL6, 206-IL-010), recombinant human Tumor Necrosis Factor Alpha (TNF-α, 210-TA-005) (All R&D Systems), and Thymosin β (TOCRIS, 3390).

Techniques: Derivative Assay, Transduction, Staining, Microscopy, Control, Expressing

Journal: Biology

Article Title: Transdifferentiation of Human Fibroblasts into Skeletal Muscle Cells: Optimization and Assembly into Engineered Tissue Constructs through Biological Ligands

doi: 10.3390/biology10060539

Figure Lengend Snippet: Skeletal muscle tissues were engineered from a composite fibrin/Matrigel hydrogel mixture with mouse skeletal myoblasts C2C12s, and subject to 10 ng/mL biological ligands. C2C12s were encapsulated and differentiated in a fibrin-based hybrid hydrogel over a 7-day period, 10 ng/mL biological ligands GDF11, TMSB4X, IL6 or TNF-α were administered after a week of tissue plating. ( A ) Immunohistochemical staining of C2C12 skeletal muscle constructs with ACTN2 and DAPI, demonstrated high cellular density. ( B ) Skeletal myotubes increased compactness and alignment towards central pillar regions where tensile force is maximal ( C ) Structural organization of C2C12s at pillar regions appeared disrupted due to gel contraction. ( D ) Cross-striated, multinucleated skeletal muscle form condensed tissues as demonstrated with high magnification 60× confocal microscopy. ( E ) Myotube diameter (µm) was not affected by one-week exposure to 10 ng/mL ligands. ( n > 32, mean + SD). ( F ) Nuclear density of skeletal muscle C2C12s within tissue were not impacted with ligand administration. ( n = 6, mean + SD).

Article Snippet: Proteins utilized included recombinant human Follistatin (Fs, 669-FO-025), recombinant human Myostatin (GDF8, 788-G8-010) or Growth Differentiation factor (GDF8), recombinant human basic Fibroblast Growth Factor 2 (FGF2, 233-FB-025), recombinant human GDF11 (1958-GD-010), recombinant human GDF15 (957-GD-025/CF), recombinant human Bone Morphogenetic Protein 4 (BMP4, 314-BP-010/CF), recombinant human BMP7 (354-BP-010), recombinant human Growth Hormone (hGH, 1067-GH-025), recombinant human Interleukin 6 (IL6, 206-IL-010), recombinant human Tumor Necrosis Factor Alpha (TNF-α, 210-TA-005) (All R&D Systems), and Thymosin β (TOCRIS, 3390).

Techniques: Immunohistochemical staining, Staining, Construct, Confocal Microscopy

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: Endocrinology

Article Title: TGFβ Superfamily Members Mediate Androgen Deprivation Therapy-Induced Obese Frailty in Male Mice.

doi: 10.1210/en.2016-1580

Figure Lengend Snippet: Figure 7. Castration induces myostatin protein levels in skeletal muscle. A, Representative immunoblots of myostatin and -actin (reprobing of the myostatin blot) expression in GAS muscle. B, Quantification of mean myostatin levels in GAS muscles from 4 mice at each time, assessed by 3 independent measurements. C, Representative immunoblot of myostatin and - actin (reprobing of the myostatin blot) expression level in TRI muscle. Lanes of immunoblots marked (C) contain identical control sample for interblot comparison. D, Quantification of mean myostatin levels in TRI muscles from 4 mice at each time, assessed by 3 independent measurements. The values shown in B and D are relative to the sham-castrated (0-wk castrate) animals. Sham-castrated (blue) and castrated groups (red) were compared by one-way ANOVA with Dunnett’s testing. Bars are SEM; *, P .05; **, P .01; ***, P .001 vs sham-castrated group.

Article Snippet: Target Antigen Sequence (if Known) Name of Antibody Manufacturer, Catalog Number, and/or Name of Individual Providing the Antibody Species Raised in; Monoclonal or Polyclonal Dilution Used Myostatin Mouse GDF8 Asn25Ser376 MAb (clone 84214) R&D Systems, MAB788 Monoclonal; rat IgG2B 1:1000 Phospho-Smad2 Smad3S423/5 MAb (clone EP823Y) Abcam, 52903 Monoclonal; rabbit 1:2000 Smad2 Amino terminus hSmad2 L16D3 Cell Signaling, 3103 Monoclonal; mouse 1:2000 Eukaryotic EF2 Complete protein G270 Nastiuk (Ref. 63) Rabbit 1:2000 -Actin -Actin aa 1–14 AC15 Sigma, A5441 Monoclonal; mouse 1:5000 Rat IgG (H L) Rabbit antirat unconjugated Pierce, 31218 Rabbit 1:20 000 Rabbit IgG (H L) Goat antirabbit HRP- conjugated secondary Pierce, 31460 Goat 1:20 000 Mouse IgG (H L) Goat antimouse HRPconjugated secondary Pierce, 31430 Goat 1:20 000 D ow nloaded from https://academ ic.oup.com /endo/article/157/11/4461/2758425 by G ITAM (D eem ed to be U niversity) user on 01 January 2025 tion are an acutely sensitive biological measure of circulating androgen elimination (34, 35).

Techniques: Western Blot, Expressing, Muscles, Control, Comparison