Journal: Cancer Cell International

Article Title: Clinical usefulness and acceleratory effect of macrophage inhibitory cytokine-1 on biliary tract cancer: an experimental biomarker analysis

doi: 10.1186/s12935-022-02668-x

Figure Lengend Snippet: Histological immunostaining, TMA, western blot and RT–PCR analyses of MIC-1 expression. MIC-1 immunostaining was performed on tumour specimens from two patients with BTC (magnification: A × 20, B × 200, D × 20, E × 100). MIC-1 expression was observed in specimens from both patients. C MIC-1 expression was also observed in normal bile duct epithelial cells from the first patient (magnification: × 100). F The intensity of TMA immunostaining is shown (0: none, 1: weakly positive, 2: moderately positive, and 3: strongly positive) (magnification × 200). G MIC-1 expression was higher in BTC tissues than in normal tissues in the TMA. H MIC-1 was more expressed at higher levels in tumour cell lines (HuCCT-1 and TFK-1) than in a normal bile duct epithelial cell line (MMNK-1), as determined using western blotting. I MIC-1 expression was also more frequently detected in tumour cell lines using RT–PCR

Article Snippet: An anti-GDF-15/MIC-1 polyclonal antibody (Bioss Antibodies Inc., Massachusetts, USA) was used for MIC-1 immunostaining according to the manufacturer’s protocol.

Techniques: Immunostaining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Cancer Cell International

Article Title: Clinical usefulness and acceleratory effect of macrophage inhibitory cytokine-1 on biliary tract cancer: an experimental biomarker analysis

doi: 10.1186/s12935-022-02668-x

Figure Lengend Snippet: Results of the cell culture experiments (n = 3). A Cell proliferation assay. The proliferation of both BTC cell lines was significantly accelerated by MIC-1. At three days, the minimum effective concentration of MIC-1 was 50 ng/ml in HuCCT-1 cells and 6.25 ng/ml in TFK-1 cells. These concentrations were applied in the other cell culture experiments. B Cell invasion assay. BTC cell invasion was accelerated by MIC-1. C Apoptosis assay. A higher level of apoptosis was observed in cell lines not treated with MIC-1 than in those treated with MIC-1. D Anticancer drug sensitivity assay. The effective concentration of GEM was 100 nM in both tumour cell lines (left two figures). MIC-1 inhibited the anticancer effect of GEM (right two figures). * P < 0.05 and ** P < 0.01

Article Snippet: An anti-GDF-15/MIC-1 polyclonal antibody (Bioss Antibodies Inc., Massachusetts, USA) was used for MIC-1 immunostaining according to the manufacturer’s protocol.

Techniques: Cell Culture, Proliferation Assay, Concentration Assay, Invasion Assay, Apoptosis Assay, Sensitive Assay

Journal: Cancer Cell International

Article Title: Clinical usefulness and acceleratory effect of macrophage inhibitory cytokine-1 on biliary tract cancer: an experimental biomarker analysis

doi: 10.1186/s12935-022-02668-x

Figure Lengend Snippet: Correlation between serum MIC-1 levels and several clinical parameters in patients with BTC. A Serum MIC-1 levels were significantly higher in patients with stage IV BTC than in patients with stage I/II/III BTC (526.6 (231.1–788.4) vs. 288.1 (42.7–720.2) × 10 –2 ng/ml, P < 0.01). B Serum MIC-1 levels and UICC stage showed a significant positive correlation. C Serum MIC-1 and ALT levels did not show a significant correlation. D Serum M30 levels were significantly higher in patients with stage III/IV BTC than in patients with stage I/II BTC (558.0 (105.4–1128.2) vs. 277.0 (100.6–1110.6) U/L, p = 0.015). E Serum M30 levels and the UICC stage showed a significant positive correlation. F Serum M30 and MIC-1 levels showed a significant positive correlation. * P < 0.05 and ** P < 0.01

Article Snippet: An anti-GDF-15/MIC-1 polyclonal antibody (Bioss Antibodies Inc., Massachusetts, USA) was used for MIC-1 immunostaining according to the manufacturer’s protocol.

Techniques:

Journal: Cancer Cell International

Article Title: Clinical usefulness and acceleratory effect of macrophage inhibitory cytokine-1 on biliary tract cancer: an experimental biomarker analysis

doi: 10.1186/s12935-022-02668-x

Figure Lengend Snippet: Ability to diagnose BTC using serum markers. A The AUCs of M30 and MIC-1 were higher than that of CA19-9. The AUC of the combination of CA19-9 and M30 was significantly higher than that of CA19-9. Furthermore, the combination of MIC-1 and M30 resulted in the highest AUC. B The ability to diagnose BTC was significantly greater using a combination of MIC-1 and M30 than using bile cytology or biliary brush cytology. * P < 0.05, ** P < 0.01

Article Snippet: An anti-GDF-15/MIC-1 polyclonal antibody (Bioss Antibodies Inc., Massachusetts, USA) was used for MIC-1 immunostaining according to the manufacturer’s protocol.

Techniques:

Journal: Cancer Cell International

Article Title: Clinical usefulness and acceleratory effect of macrophage inhibitory cytokine-1 on biliary tract cancer: an experimental biomarker analysis

doi: 10.1186/s12935-022-02668-x

Figure Lengend Snippet: Ability to diagnose early BTC using serum markers. A The AUCs of MIC-1 and M30 were higher than that of CA19-9. The AUC was highest for the combination of MIC-1 and M30, indicating a greater ability to diagnose early BTC. The AUC of the combination of MIC-1 and M30 was significantly higher than that of CA19-9 ( P value < 0.05). B Although the difference was not significant, the ability to diagnose BTC was improved using the combination of MIC-1 and M30 levels compared with biliary cytology or brush cytology. * P < 0.05

Article Snippet: An anti-GDF-15/MIC-1 polyclonal antibody (Bioss Antibodies Inc., Massachusetts, USA) was used for MIC-1 immunostaining according to the manufacturer’s protocol.

Techniques:

Journal: Cancer Cell International

Article Title: Clinical usefulness and acceleratory effect of macrophage inhibitory cytokine-1 on biliary tract cancer: an experimental biomarker analysis

doi: 10.1186/s12935-022-02668-x

Figure Lengend Snippet: DFS, OS of BTC patients evaluated based on M30 or MIC-1. A The DFS was not significantly different between the patients with serum M30 ≥ the median and the patients with serum M30 < the median. B The DFS was not significantly different between the patients with serum MIC-1 ≥ the median and the patients with serum MIC-1 < the median. C The OS was not significantly different between the patients with serum M30 ≥ the median and the patients with serum M30 < the median. D The OS was significantly longer in the patients with serum MIC-1 < the median than in the patients with serum MIC-1 ≥ the median

Article Snippet: An anti-GDF-15/MIC-1 polyclonal antibody (Bioss Antibodies Inc., Massachusetts, USA) was used for MIC-1 immunostaining according to the manufacturer’s protocol.

Techniques:

Journal: Nature immunology

Article Title: A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma

doi: 10.1038/s41590-020-0777-3

Figure Lengend Snippet: a , flow cytometric analysis and frequencies of IL13 + ILC2 (Lineage – T1/ST2 + cells) in mice of respective genotypes treated as indicated (n=5). b , c , In vitro suppression assays using ILC2 from OVA+UFP-treated Foxp3 YFPCre mice and lung T reg cells of the respective genotypes, treated as indicated (n=4) . d , GDF15 transcripts in T reg cells of Foxp3 YFPCre , Foxp3 YFPCre Notch4 Δ/Δ and Foxp3 YFPCre Ctnnb1 Δ/Δ (n=5). e , flow cytometric analysis and frequencies of GDF15 + lung T reg cells in the respective mouse genotypes treated as indicated (n=5). f , flow cytometric analysis and frequencies of IL-13 induced in naive ILC2 stimulated with IL-33, GDF15 or both (n=3). g , IL-13 expression in naive ILC2 incubated with Notch4 hi T reg cells from OVA+UFP treated mice without or with blocking GDF15 peptide (n=6). h , In vitro suppression assays using lung T reg cells and ILC2 isolated from OVA+UFP-treated Foxp3 YFPCre mice and incubated without or with GDF15 blocking peptide (n=4). Each symbol represents an independent sample. Numbers in flow plots indicate percentages. Error bars indicate SEM. Statistical tests: two-way ANOVA with Sidak’s post hoc analysis ( a - e , h ); One-way ANOVA with Dunnett’s post hoc analysis ( f,g ). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data representative of two or three independent experiments.

Article Snippet: Polyclonal rabbit anti-GDF15(catalogue no: 32572–05171, 1:200, Assaypro), anti-CD16/CD32 (clone: 93, Catalogue no: 101319, 1:1000, Biolegend), Alexa Fluor 647 goat anti-rabbit IgG Ab (clone PA5–39741, catalogue no: A32733, 1:1000, Thermofischer), p-Mob1 Ab (T35; clone D2F10, Catalogue no: 8699S, 1:300, CST), rabbit anti-mouse p-Lats1 Ab (T35; clone D57D3, 1:300, CST), rabbit anti-mouse p-Lats1/2 Ab (S909/872; clone PA5–39741, catalogue no: PA5–105895, 1:300, Thermofischer), Rat anti-mouse IL-6 mAb (Clone: MP5–20F3, Catalogue no: BE0046, 1:500, Bioxcell), anti-CD28 (Clone: 37.51, Catalogue no: 122004, ,1.1000, Biolegend) , anti-IL-4 (Catalogue no: 500-P54, 1:1000, Peprotech).

Techniques: In Vitro, Expressing, Incubation, Blocking Assay, Isolation

Journal: Nature immunology

Article Title: A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma

doi: 10.1038/s41590-020-0777-3

Figure Lengend Snippet: a , d , Representative PAS-stained sections of lung tissues isolated from Foxp3 YFPCre and Foxp3 YFPCre Notch4 Δ/Δ with either PBS or OVA+UFP, the latter either alone or supplemented with GDF15 or GDF15 blocking peptide, as indicated (200X magnification), Inflammation score for the respective mouse groups (n=10). b , e , AHR in Foxp3 YFPCre and Foxp3 YFPCre Notch4 Δ/Δ treated as indicated (n=10). c , f , Frequencies and absolute numbers of ILC2, eosinophils, IL-4, and IL-13, expression in lung Foxp3 – CD4 + T eff cells in the respective groups (n=10) g, AHR in Rora Cre and Rora Cre Il4/Il13 Δ/Δ treated as indicated (n=5). h, Frequencies and absolute numbers of eosinophils, ILC2, IL-4, and IL-13, expression in lung Foxp3 – CD4 + T eff cells in the respective groups (n=5). Error bars indicate SEM. Statistical tests. One-way ANOVA with Dunnett’s post hoc analysis. ( a,c,d,f ), two-way ANOVA with Sidak’s post hoc analysis ( b , e , g , h ); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data representative of two or three independent experiments.

Article Snippet: Polyclonal rabbit anti-GDF15(catalogue no: 32572–05171, 1:200, Assaypro), anti-CD16/CD32 (clone: 93, Catalogue no: 101319, 1:1000, Biolegend), Alexa Fluor 647 goat anti-rabbit IgG Ab (clone PA5–39741, catalogue no: A32733, 1:1000, Thermofischer), p-Mob1 Ab (T35; clone D2F10, Catalogue no: 8699S, 1:300, CST), rabbit anti-mouse p-Lats1 Ab (T35; clone D57D3, 1:300, CST), rabbit anti-mouse p-Lats1/2 Ab (S909/872; clone PA5–39741, catalogue no: PA5–105895, 1:300, Thermofischer), Rat anti-mouse IL-6 mAb (Clone: MP5–20F3, Catalogue no: BE0046, 1:500, Bioxcell), anti-CD28 (Clone: 37.51, Catalogue no: 122004, ,1.1000, Biolegend) , anti-IL-4 (Catalogue no: 500-P54, 1:1000, Peprotech).

Techniques: Staining, Isolation, Blocking Assay, Expressing

Journal: Nature immunology

Article Title: A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma

doi: 10.1038/s41590-020-0777-3

Figure Lengend Snippet: a , b , Flow cytometric analysis, cell frequencies and MFI of Notch4 expression on circulating T reg cells ( a ) and T eff cells ( b ) of control and asthmatic subjects, the latter segregated for asthma severity (control: n=39; mild n=31; moderate: n=27; severe: n=11). c , flow cytometric analysis, cell frequencies and MFI of Notch4 expression on Helios + versus Helios – circulating T reg cells of control and asthmatic subjects (control: n=13; mild n=9, moderate n=14; severe: n=11). d , e , Flow cytometric analysis, cell frequencies and MFI of Yap ( d ) and β-catenin ( e ) expression on circulating T reg cells of control and severe asthmatic subjects (control n=24; mild n=15; moderate n=15; severe: n=11). f , Serum GDF15 concentrations in moderate and severe asthmatic subjects plotted as a function of Notch4 expression on circulating T reg cells (n=21). g , In vitro suppression third party CD4 + T cells (T eff ) by the Notch4 hi versus Notch4 lo T reg cells from severe asthmatics compared to T reg cells of control subjects (n=2 subjects, 3 replicates per dilution per subject). h , In vitro suppression assays of ILC2 activation using circulating Notch4 hi T reg cells of asthmatics subjects and control T reg cells of healthy controls, incubated at the indicated T reg cell:ILC2 ratios without or with GDF15 blocking peptide (n=5). i , Flow cytometric analysis of Notch4 expression in T reg cells of a healthy control and a severe asthmatic before and after treatment with anti-IL-6R mAb (n=1). Error bars indicate SEM. Statistical tests: One-way ANOVA with Dunnett’s post hoc analysis ( a - e ); simple linear regression analysis ( f ); two-way ANOVA with Sidak’s post hoc analysis ( g,h ); ***P<0.001, ****P<0.0001. Data representative of two or three independent experiments.

Article Snippet: Polyclonal rabbit anti-GDF15(catalogue no: 32572–05171, 1:200, Assaypro), anti-CD16/CD32 (clone: 93, Catalogue no: 101319, 1:1000, Biolegend), Alexa Fluor 647 goat anti-rabbit IgG Ab (clone PA5–39741, catalogue no: A32733, 1:1000, Thermofischer), p-Mob1 Ab (T35; clone D2F10, Catalogue no: 8699S, 1:300, CST), rabbit anti-mouse p-Lats1 Ab (T35; clone D57D3, 1:300, CST), rabbit anti-mouse p-Lats1/2 Ab (S909/872; clone PA5–39741, catalogue no: PA5–105895, 1:300, Thermofischer), Rat anti-mouse IL-6 mAb (Clone: MP5–20F3, Catalogue no: BE0046, 1:500, Bioxcell), anti-CD28 (Clone: 37.51, Catalogue no: 122004, ,1.1000, Biolegend) , anti-IL-4 (Catalogue no: 500-P54, 1:1000, Peprotech).

Techniques: Expressing, In Vitro, Activation Assay, Incubation, Blocking Assay

Journal: Nature immunology

Article Title: A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma

doi: 10.1038/s41590-020-0777-3

Figure Lengend Snippet: a , b , Flow cytometric analysis, cell frequencies and mean fluorescence intensity (MFI) of Notch1, 2 and 3 expression in peripheral blood T reg cells ( a ) and T eff cells ( b ) of control and asthmatic subjects, the latter segregated for asthma severity (control n=22, M.P n= 15, Mod n= 16. S.P n=11). c , Flow cytometric analysis and cell frequencies of Notch4 peripheral blood T reg cells of healthy control, food allergy (FA), eczema and FA+eczema (Control n=37, FA n= 28, Eczema n=10 and FA+Eczema n=20) d , Serum GDF15 concentrations in asthmatic subjects plotted as a function of Notch4 expression on circulating T reg cells (n=73) e , Cell frequencies of Notch4 expression in peripheral blood T reg cells in healthy subjects, allergic and non-allergic asthmatics (control = 56, non-allergic n=21, allergic n=85). Error bars indicate SEM. Statistical tests: One-way ANOVA with Dunnett’s post hoc analysis. ( a - c,e ); simple regression analysis ( d ). ***P<0.001, ****P<0.0001. Data representative of two or three independent experiments.

Article Snippet: Polyclonal rabbit anti-GDF15(catalogue no: 32572–05171, 1:200, Assaypro), anti-CD16/CD32 (clone: 93, Catalogue no: 101319, 1:1000, Biolegend), Alexa Fluor 647 goat anti-rabbit IgG Ab (clone PA5–39741, catalogue no: A32733, 1:1000, Thermofischer), p-Mob1 Ab (T35; clone D2F10, Catalogue no: 8699S, 1:300, CST), rabbit anti-mouse p-Lats1 Ab (T35; clone D57D3, 1:300, CST), rabbit anti-mouse p-Lats1/2 Ab (S909/872; clone PA5–39741, catalogue no: PA5–105895, 1:300, Thermofischer), Rat anti-mouse IL-6 mAb (Clone: MP5–20F3, Catalogue no: BE0046, 1:500, Bioxcell), anti-CD28 (Clone: 37.51, Catalogue no: 122004, ,1.1000, Biolegend) , anti-IL-4 (Catalogue no: 500-P54, 1:1000, Peprotech).

Techniques: Fluorescence, Expressing

Journal: Molecular medicine reports

Article Title: GDF15 promotes osteosarcoma cell migration and invasion by regulating the TGF‑β signaling pathway.

doi: 10.3892/mmr.2019.10664

Figure Lengend Snippet: Figure 1. GDF15 is overexpressed in metastatic osteosarcoma tissues. (A) GDF15 mRNA expression levels were significantly upregulated in metastatic osteosar- coma compared with non‑metastatic osteosarcoma samples in the GSE9508 dataset. *P<0.05, as indicated. (B) GDF15 mRNA (upper panel) and protein (lower panel) expression levels in benign (B1‑2), non‑metastatic (P1‑4) and pulmonary metastatic (P5‑8) osteosarcoma tissue samples were determined via RT‑qPCR and western blot analyses. *P<0.05, as indicated. (C) RT‑qPCR (upper panel), western blot (lower panel) and (D) ELISA analyses of GDF15 expression in human osteosarcoma cell lines, human FOB osteoblasts and mouse NIH3T3 fibroblasts. *P<0.05 vs. FOB. Data are presented as the mean ± standard deviation of three independent experiments. GDF15, growth and differentiation factor 15; RT‑qPCR, reverse transcription‑quantitative polymerase chain reaction.

Article Snippet: Briefly, the supernatant was transferred to a well coated with GdF15 monoclonal antibody (cat. no. MaB957; r&d Systems, inc.) and immunosorbed using biotinylated polyclonal anti-human GdF15 antibody (1:1,000; cat. no. BaF940, r&d Systems, inc.) at room temperature for 1 h. The color development was catalyzed by horseradish peroxidase, and the absorption was detected at 450 nm.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation, Polymerase Chain Reaction

Journal: Molecular medicine reports

Article Title: GDF15 promotes osteosarcoma cell migration and invasion by regulating the TGF‑β signaling pathway.

doi: 10.3892/mmr.2019.10664

Figure Lengend Snippet: Figure 3. GDF15 knockdown attenuates the migration and invasion of osteosarcoma cells. (A) Reverse transcription‑quantitative PCR (upper panel), western blot (lower panel) and (B) ELISA analyses of GDF15 expression in transduced MG‑63 and U‑2 OS cells. *P<0.05 vs. Vector. (C) Quantification of wound‑healing assays for the indicated cell lines after 24 h. *P<0.05 as indicated.(D) Representative micrographs (magnification, x200) and quantification of the invasive abilities of cells, as determined by Transwell invasion assays. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05, as indicated. GDF15, growth and differentiation factor 15; GDF15‑Ri, GDF15 shRNA treatment; Vector, vector control; rhGDF15, recombinant human GDF15.

Article Snippet: Briefly, the supernatant was transferred to a well coated with GdF15 monoclonal antibody (cat. no. MaB957; r&d Systems, inc.) and immunosorbed using biotinylated polyclonal anti-human GdF15 antibody (1:1,000; cat. no. BaF940, r&d Systems, inc.) at room temperature for 1 h. The color development was catalyzed by horseradish peroxidase, and the absorption was detected at 450 nm.

Techniques: Knockdown, Migration, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Plasmid Preparation, Standard Deviation, shRNA, Control, Recombinant

Journal: Molecular medicine reports

Article Title: GDF15 promotes osteosarcoma cell migration and invasion by regulating the TGF‑β signaling pathway.

doi: 10.3892/mmr.2019.10664

Figure Lengend Snippet: Figure 2. High serum levels of GDF15 are associated with poor clinical outcomes in patients with osteosarcoma. (A and B) Kaplan‑Meier analysis of the (A) overall survival and (B) pulmonary metastasis‑free survival of patients with osteosarcoma possessing high (n=35) or low GDF15 serum levels (n=71). P‑values were determined using log‑rank tests. Low serum GDF15 level: Patients with serum GDF15 concentrations <1 ng/ml. High serum GDF15 level: Patients with serum GDF15 concentrations ≥1 ng/ml. GDF15, growth and differentiation factor 15; HR, hazard ratio.

Article Snippet: Briefly, the supernatant was transferred to a well coated with GdF15 monoclonal antibody (cat. no. MaB957; r&d Systems, inc.) and immunosorbed using biotinylated polyclonal anti-human GdF15 antibody (1:1,000; cat. no. BaF940, r&d Systems, inc.) at room temperature for 1 h. The color development was catalyzed by horseradish peroxidase, and the absorption was detected at 450 nm.

Techniques:

Journal: Molecular medicine reports

Article Title: GDF15 promotes osteosarcoma cell migration and invasion by regulating the TGF‑β signaling pathway.

doi: 10.3892/mmr.2019.10664

Figure Lengend Snippet: Figure 4. GDF15 knockdown suppresses the TGF‑β signaling pathway. (A) Nuclear p‑SMAD2/3 expression as determined by western blot analysis. P84 was used as a loading control. (B) Transcriptional activity of a TGF‑β/SMAD‑responsive luciferase reporter, as determined by a luciferase assay. (C) Fold‑change in the mRNA expression of TGF‑β signaling‑associated genes, as determined via reverse transcription‑quantitative PCR analysis. (D) Schematic model. GDF15 activates the TGF‑β signaling pathway, leading to the metastasis of osteosarcoma. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05, as indicated. GDF15, growth and differentiation factor 15; GDF15‑Ri or Ri, GDF15 shRNA treatment; Vector/V, vector control; rhGDF15/rh, recombinant human GDF15; TGF‑β, transforming growth factor‑β; p‑, phosphorylated; SNAI, snail family transcriptional repressor; IL11, interleukin 11; MMP13, matrix metallopeptidase 13; TWIST1, twist family bHLH transcription factor 1; ZEB1, zinc finger E‑box binding homeobox 1; COL1A1, collagen type I α1 chain; VEGFA, vascular endothelial growth factor A.

Article Snippet: Briefly, the supernatant was transferred to a well coated with GdF15 monoclonal antibody (cat. no. MaB957; r&d Systems, inc.) and immunosorbed using biotinylated polyclonal anti-human GdF15 antibody (1:1,000; cat. no. BaF940, r&d Systems, inc.) at room temperature for 1 h. The color development was catalyzed by horseradish peroxidase, and the absorption was detected at 450 nm.

Techniques: Knockdown, Expressing, Western Blot, Control, Activity Assay, Luciferase, Standard Deviation, shRNA, Plasmid Preparation, Recombinant, Binding Assay