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Assaypro polyclonal rabbit anti gdf15

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 , <t>GDF15</t> 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.

Polyclonal Rabbit Anti Gdf15, supplied by Assaypro, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma"

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

Journal: Nature immunology

doi: 10.1038/s41590-020-0777-3

Figure Legend 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.

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

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.

Figure Legend 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.

Techniques Used: Staining, Isolation, Blocking Assay, Expressing

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.

Figure Legend 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.

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

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.

Figure Legend 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.

Techniques Used: Fluorescence, Expressing

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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.

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.

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.

Techniques: Fluorescence, Expressing

a Expression levels of eIF2α kinase genes ( EIF2AK1 , EIF2AK2 , EIF2AK3 , and EIF2AK4 ) were compared in each patient with acute kidney injury (GSE30718). b– e Expression of PKR (b, c) or Gdf15 ( d , e ) was determined in patients with renal injuries, including acute kidney injury (GSE30718, b and d) or chronic kidney disease (GSE66494, c and e). The results are shown as a plot with Tukey whiskers. The asterisks (∗) indicate significant differences between the two groups (∗ p < 0.05, *** p < 0.001). f – g Based on PKR or C/EBP homologous protein ( CHOP ) levels, we selected the 20 highest and 20 lowest level samples, which were further evaluated based on the Gdf15 levels in patients with renal injuries, including acute kidney injury (GSE30718, f ) or chronic kidney disease (GSE66494, g). The results are shown as a plot with Tukey whiskers and outliers (orange circles). The asterisks (∗) indicate significant differences between groups (∗ p < 0.05, ∗∗ p < 0.01 using a two-tailed unpaired Student’s t -test). h– i HK-2 cells ( h ) and HCT8 cells ( i ) transfected with control (the negative control shRNA) or shPERK or shPKR plasmid were treated with vehicle or 10 μmol/L cisplatin (CP) for 48 h. Cellular lysates were subjected to western blot analysis. eIF2α, eukaryotic initiation factor 2 alpha; Gdf15, growth differentiation factor 15; PKR, protein kinase R; PERK, protein kinase-like endoplasmic reticulum kinase.

Journal: Communications Biology

Article Title: Stress-responsive Gdf15 counteracts renointestinal toxicity via autophagic and microbiota reprogramming

doi: 10.1038/s42003-023-04965-1

Figure Lengend Snippet: a Expression levels of eIF2α kinase genes ( EIF2AK1 , EIF2AK2 , EIF2AK3 , and EIF2AK4 ) were compared in each patient with acute kidney injury (GSE30718). b– e Expression of PKR (b, c) or Gdf15 ( d , e ) was determined in patients with renal injuries, including acute kidney injury (GSE30718, b and d) or chronic kidney disease (GSE66494, c and e). The results are shown as a plot with Tukey whiskers. The asterisks (∗) indicate significant differences between the two groups (∗ p < 0.05, *** p < 0.001). f – g Based on PKR or C/EBP homologous protein ( CHOP ) levels, we selected the 20 highest and 20 lowest level samples, which were further evaluated based on the Gdf15 levels in patients with renal injuries, including acute kidney injury (GSE30718, f ) or chronic kidney disease (GSE66494, g). The results are shown as a plot with Tukey whiskers and outliers (orange circles). The asterisks (∗) indicate significant differences between groups (∗ p < 0.05, ∗∗ p < 0.01 using a two-tailed unpaired Student’s t -test). h– i HK-2 cells ( h ) and HCT8 cells ( i ) transfected with control (the negative control shRNA) or shPERK or shPKR plasmid were treated with vehicle or 10 μmol/L cisplatin (CP) for 48 h. Cellular lysates were subjected to western blot analysis. eIF2α, eukaryotic initiation factor 2 alpha; Gdf15, growth differentiation factor 15; PKR, protein kinase R; PERK, protein kinase-like endoplasmic reticulum kinase.

Article Snippet: The following antibodies were used for western blotting: mouse monoclonal anti-β-actin (1:1000; SC4778, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-PARP1/2 (1:1000; SC7150, Santa Cruz Biotechnology), and rabbit polyclonal anti-Gdf15 (Abclonal, Woburn, MA, USA).

Techniques: Expressing, Two Tailed Test, Transfection, Negative Control, shRNA, Plasmid Preparation, Western Blot

Eight-week-old wild-type and Gdf15 knockout (KO) mice were treated with vehicle or CP (20 mg/kg, intraperitoneal) for 72 h ( n = 3 − 5). a Kaplan-Meier’s survival analysis of wild-type and Gdf15 KO mice treated with CP ( n = 3 − 5, p < 0.01). b Gross anatomy of kidney sections from untreated mice and CP-treated mice (periodic acid-Schiff [PAS] staining) (Magnification, 5×; Scale bars(s), 1 mm). Serum levels of creatinine ( c ) and blood urea nitrogen (BUN) ( d ) were measured at 72 h after CP treatment using a colorimetric assay kit, as described in the method section. The results are shown as a bar graph with average and standard deviation, and different letters over each bar represent significant differences between groups ( p < 0.05). e Histological examination of PAS-stained kidney sections (Magnification, 400×; Scale bars(s), 50 μm). Quantitative analysis of tubular dilation ( f ), tubular vacuolation ( g ), and cyst formation ( h ). The results are shown as a plot with Tukey whiskers and outliers (orange circles). Different letters over each bar represent significant differences between groups ( p < 0.05). Gdf15, growth differentiation factor 15.

Journal: Communications Biology

Article Title: Stress-responsive Gdf15 counteracts renointestinal toxicity via autophagic and microbiota reprogramming

doi: 10.1038/s42003-023-04965-1

Figure Lengend Snippet: Eight-week-old wild-type and Gdf15 knockout (KO) mice were treated with vehicle or CP (20 mg/kg, intraperitoneal) for 72 h ( n = 3 − 5). a Kaplan-Meier’s survival analysis of wild-type and Gdf15 KO mice treated with CP ( n = 3 − 5, p < 0.01). b Gross anatomy of kidney sections from untreated mice and CP-treated mice (periodic acid-Schiff [PAS] staining) (Magnification, 5×; Scale bars(s), 1 mm). Serum levels of creatinine ( c ) and blood urea nitrogen (BUN) ( d ) were measured at 72 h after CP treatment using a colorimetric assay kit, as described in the method section. The results are shown as a bar graph with average and standard deviation, and different letters over each bar represent significant differences between groups ( p < 0.05). e Histological examination of PAS-stained kidney sections (Magnification, 400×; Scale bars(s), 50 μm). Quantitative analysis of tubular dilation ( f ), tubular vacuolation ( g ), and cyst formation ( h ). The results are shown as a plot with Tukey whiskers and outliers (orange circles). Different letters over each bar represent significant differences between groups ( p < 0.05). Gdf15, growth differentiation factor 15.

Techniques: Knock-Out, Staining, Colorimetric Assay, Standard Deviation

Eight-week-old wild-type and Gdf15 knockout (KO) mice ( n = 3–5) were treated with vehicle or cisplatin (CP; 20 mg/kg) for 72 h. a The average length of the small intestine from each group. b– c Average lengths of villus (left) and crypt (right) in the jejunum ( b ) and ileum ( c ). The results are shown as a plot with Tukey whiskers and outliers (orange circles). Different letters over bars represent significant differences between groups ( p < 0.05). d–g Histological examination of hematoxylin and eosin (H&E)-stained sections of the jejunum ( d ) and ileum ( e ) (Magnification, 100×; Scale bar(s), 100 μm). Quantitation of pathological severity of the jejunum ( f ) and ileum ( g ). The results are shown as a plot with Tukey whiskers and outliers (orange circles). The asterisks (∗) indicate significant differences between groups (∗∗ p < 0.01, ∗∗∗ p < 0.001 using a two-tailed unpaired Student’s t -test). Gdf15, growth differentia t ion factor 15.

Journal: Communications Biology

Article Title: Stress-responsive Gdf15 counteracts renointestinal toxicity via autophagic and microbiota reprogramming

doi: 10.1038/s42003-023-04965-1

Figure Lengend Snippet: Eight-week-old wild-type and Gdf15 knockout (KO) mice ( n = 3–5) were treated with vehicle or cisplatin (CP; 20 mg/kg) for 72 h. a The average length of the small intestine from each group. b– c Average lengths of villus (left) and crypt (right) in the jejunum ( b ) and ileum ( c ). The results are shown as a plot with Tukey whiskers and outliers (orange circles). Different letters over bars represent significant differences between groups ( p < 0.05). d–g Histological examination of hematoxylin and eosin (H&E)-stained sections of the jejunum ( d ) and ileum ( e ) (Magnification, 100×; Scale bar(s), 100 μm). Quantitation of pathological severity of the jejunum ( f ) and ileum ( g ). The results are shown as a plot with Tukey whiskers and outliers (orange circles). The asterisks (∗) indicate significant differences between groups (∗∗ p < 0.01, ∗∗∗ p < 0.001 using a two-tailed unpaired Student’s t -test). Gdf15, growth differentia t ion factor 15.

Techniques: Knock-Out, Staining, Quantitation Assay, Two Tailed Test

Eight-week-old wild-type and Gdf15 knockout (KO) mice ( n = 3-5) were treated with vehicle or 3% DSS for 8 days. Histological examination of hematoxylin and eosin (H&E)-stained colon sections ( a ). Quantitation of pathological severity of colons ( b ) (Magnification, 200×; Scale bar(s), 100 μm). The results are shown as a plot with Tukey whiskers and outliers (orange circles). The asterisks (∗) indicate significant differences between groups (∗∗ p < 0.01, ∗∗∗ p < 0.001). c Histological examination of periodic acid-Schiff (PAS)-stained kidney sections (Magnification, 400×; Scale bars(s), 50 μm). d Blood urea nitrogen (BUN) was measured 48 h after DSS treatment using a colorimetric assay kit. The results are shown as a plot with Tukey whiskers. The asterisks (∗) indicate significant differences between groups (** p < 0.01). e Quantitative analysis of tubular dilation, glomerular shrinkage, and glomerular sclerosis. The results are shown as a plot with Tukey whiskers and outliers (orange circles). The asterisks (∗) indicate significant differences between the two groups (* p < 0.05, ** p < 0.01, *** p < 0.001). Gdf15, growth differentiation factor 15.

Journal: Communications Biology

Article Title: Stress-responsive Gdf15 counteracts renointestinal toxicity via autophagic and microbiota reprogramming

doi: 10.1038/s42003-023-04965-1

Figure Lengend Snippet: Eight-week-old wild-type and Gdf15 knockout (KO) mice ( n = 3-5) were treated with vehicle or 3% DSS for 8 days. Histological examination of hematoxylin and eosin (H&E)-stained colon sections ( a ). Quantitation of pathological severity of colons ( b ) (Magnification, 200×; Scale bar(s), 100 μm). The results are shown as a plot with Tukey whiskers and outliers (orange circles). The asterisks (∗) indicate significant differences between groups (∗∗ p < 0.01, ∗∗∗ p < 0.001). c Histological examination of periodic acid-Schiff (PAS)-stained kidney sections (Magnification, 400×; Scale bars(s), 50 μm). d Blood urea nitrogen (BUN) was measured 48 h after DSS treatment using a colorimetric assay kit. The results are shown as a plot with Tukey whiskers. The asterisks (∗) indicate significant differences between groups (** p < 0.01). e Quantitative analysis of tubular dilation, glomerular shrinkage, and glomerular sclerosis. The results are shown as a plot with Tukey whiskers and outliers (orange circles). The asterisks (∗) indicate significant differences between the two groups (* p < 0.05, ** p < 0.01, *** p < 0.001). Gdf15, growth differentiation factor 15.

Techniques: Knock-Out, Staining, Quantitation Assay, Colorimetric Assay

a Eight-week-old wild-type and Gdf15 knockout (KO) mice ( n = 3-5) were treated with vehicle or cisplatin (CP; 20 mg/kg) for 72 h. Staining of ileum mucosa with Alcian blue (Magnification, 200×; Scale bar(s), 100 μm) and its quantitation (Magnification, 200×; Scale bar(s), 100 μm). b– d Eight-week-old wild-type and Gdf15 KO mice ( n = 3–5) were treated with vehicle or 3% DSS for 8 days. Staining of ileum mucosa with Alcian blue (Magnification, 200×; Scale bar(s), 100 μm) and its quantitation (Magnification, 200×; Scale bar(s), 100 μm) ( b ). Representative images of the intestinal mucosal bacteria using Gram staining ( c ) or 16 rRNA in situ staining ( d ) (Magnification, 200×; Scale bar(s), 100 μm). The mucosal layer thickness was measured (the right graph). The quantitation analysis was shown as plots with Tukey whiskers and outliers (orange circles). Different letters with bars represent significant differences between groups ( p < 0.05). Gdf15, growth differentiation factor 15.

Journal: Communications Biology

Article Title: Stress-responsive Gdf15 counteracts renointestinal toxicity via autophagic and microbiota reprogramming

doi: 10.1038/s42003-023-04965-1

Figure Lengend Snippet: a Eight-week-old wild-type and Gdf15 knockout (KO) mice ( n = 3-5) were treated with vehicle or cisplatin (CP; 20 mg/kg) for 72 h. Staining of ileum mucosa with Alcian blue (Magnification, 200×; Scale bar(s), 100 μm) and its quantitation (Magnification, 200×; Scale bar(s), 100 μm). b– d Eight-week-old wild-type and Gdf15 KO mice ( n = 3–5) were treated with vehicle or 3% DSS for 8 days. Staining of ileum mucosa with Alcian blue (Magnification, 200×; Scale bar(s), 100 μm) and its quantitation (Magnification, 200×; Scale bar(s), 100 μm) ( b ). Representative images of the intestinal mucosal bacteria using Gram staining ( c ) or 16 rRNA in situ staining ( d ) (Magnification, 200×; Scale bar(s), 100 μm). The mucosal layer thickness was measured (the right graph). The quantitation analysis was shown as plots with Tukey whiskers and outliers (orange circles). Different letters with bars represent significant differences between groups ( p < 0.05). Gdf15, growth differentiation factor 15.

Techniques: Knock-Out, Staining, Quantitation Assay, In Situ

Journal: Communications Biology

Article Title: Stress-responsive Gdf15 counteracts renointestinal toxicity via autophagic and microbiota reprogramming

doi: 10.1038/s42003-023-04965-1

Figure Lengend Snippet: The fecal bacteria were subjected to 16 S rRNA analysis to determine the phylogenetic composition. a,b The bacteria of each top 30 abundant taxa are listed along with the corresponding abundance ( a ) and the relative abundance ( b ). Re-marked microbes are potent mucin-foraging genera or species. c Genes related to mucin degradation enzymes reconstructed from 16 S rRNA profile with PICRUSt2. Data represent the mean ± standard deviation (SD) ( N = 3) with all datapoints (circles) (results are shown as the mean values ± SD. Asterisks (∗) indicate significant differences between groups (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). Gdf15, growth differentiation factor 15.

Techniques: Standard Deviation

Control or Gdf15-deficient cells (HCT-8 ( a ) or HK-2 ( b ) cells) were transfected with pDEST-CMV mCherry-GFP-LC3B WT. At 24 h after transfection, cells were treated with vehicle or 10 μmol/L cisplatin (CP) for 48 h. Cells were observed under the confocal microscope to monitor the pH-responsive autophagic flux (Magnification, 400×; Scale bar(s), 10 μm). The quantitation analysis was shown as plots with Tukey whiskers (lower graphs) and all datapoints (circles). Different letters with bars represent significant differences between groups ( p < 0.05). Gdf15, growth differentiation factor 15.

Journal: Communications Biology

Article Title: Stress-responsive Gdf15 counteracts renointestinal toxicity via autophagic and microbiota reprogramming

doi: 10.1038/s42003-023-04965-1

Figure Lengend Snippet: Control or Gdf15-deficient cells (HCT-8 ( a ) or HK-2 ( b ) cells) were transfected with pDEST-CMV mCherry-GFP-LC3B WT. At 24 h after transfection, cells were treated with vehicle or 10 μmol/L cisplatin (CP) for 48 h. Cells were observed under the confocal microscope to monitor the pH-responsive autophagic flux (Magnification, 400×; Scale bar(s), 10 μm). The quantitation analysis was shown as plots with Tukey whiskers (lower graphs) and all datapoints (circles). Different letters with bars represent significant differences between groups ( p < 0.05). Gdf15, growth differentiation factor 15.

Techniques: Transfection, Microscopy, Quantitation Assay

a HCT8 cells transfected with control or shGdf15 vector were treated with vehicle or 10 μmol/L cisplatin (CP) for 48 h. Cellular lysates were subjected to western blot analysis. b HCT-8 cells transfected with control (the negative control shRNA) or shGdf15 plasmid were treated with vehicle or 10 μmol/L CP in the absence or presence of 20 μmol/L 3-methyladenine (3-MA) for 48 h, and mRNA levels of mucin 4 or mucin 2 were measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Data values are presented as the mean ± standard deviation (SD) and all datapoints (circles). Asterisks (∗) indicate significant differences from the CP-treated group (∗∗∗ p < 0.001). Boxed blots indicate the efficient suppression of Gdf15 using shRNA. c Cluster evaluation of autophagy regulatory network (ARN; https://autophagyregulation.org ) in response to Gdf15 levels in human cells. The border thickness indicates the levels of betweenness centrality of each node. d The HCT-8 cells transfected with control (the negative control shRNA) or shGdf15 plasmid were treated with vehicle or 20 μmol/L CP for 12 h, and mRNA levels were measured using RT-qPCR analysis. Data values are presented as the mean ± SD and all datapoints (circles). Different letters over bars represent significant differences between groups ( p < 0.05). Gdf15, growth differentiation factor 15.

Journal: Communications Biology

Article Title: Stress-responsive Gdf15 counteracts renointestinal toxicity via autophagic and microbiota reprogramming

doi: 10.1038/s42003-023-04965-1

Figure Lengend Snippet: a HCT8 cells transfected with control or shGdf15 vector were treated with vehicle or 10 μmol/L cisplatin (CP) for 48 h. Cellular lysates were subjected to western blot analysis. b HCT-8 cells transfected with control (the negative control shRNA) or shGdf15 plasmid were treated with vehicle or 10 μmol/L CP in the absence or presence of 20 μmol/L 3-methyladenine (3-MA) for 48 h, and mRNA levels of mucin 4 or mucin 2 were measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Data values are presented as the mean ± standard deviation (SD) and all datapoints (circles). Asterisks (∗) indicate significant differences from the CP-treated group (∗∗∗ p < 0.001). Boxed blots indicate the efficient suppression of Gdf15 using shRNA. c Cluster evaluation of autophagy regulatory network (ARN; https://autophagyregulation.org ) in response to Gdf15 levels in human cells. The border thickness indicates the levels of betweenness centrality of each node. d The HCT-8 cells transfected with control (the negative control shRNA) or shGdf15 plasmid were treated with vehicle or 20 μmol/L CP for 12 h, and mRNA levels were measured using RT-qPCR analysis. Data values are presented as the mean ± SD and all datapoints (circles). Different letters over bars represent significant differences between groups ( p < 0.05). Gdf15, growth differentiation factor 15.

Techniques: Transfection, Plasmid Preparation, Western Blot, Negative Control, shRNA, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation

a Expression of BECN1 in patients with acute kidney injury (GSE30718). b Based on the Gdf15 levels in patients with acute kidney injury (GSE30718, left) or chronic kidney disease (GSE66494, right), we selected the 20 highest and 20 lowest level samples, followed by a comparison of BECN1 expression levels. The results are shown as a plot with Tukey whiskers. The asterisks (∗) indicate significant differences from the low-expression group (∗ p < 0.05 using a two-tailed unpaired Student’s t -test). c Eight-week-old wild-type and Gdf15 knockout (KO) mice ( n = 3–5) were treated with vehicle or cisplatin (CP; 20 mg/kg) for 72 h. Kidney tissue lysates were subjected to western blotting. The results are shown as a plot with Tukey whiskers. The graph shows the relative densities of light chain 3 B (LC3B) from the western blot. Different letters over bars represent significant differences between groups ( p < 0.05, n = 3). d HK-2 cells transfected with control or shGdf15 vector were treated with vehicle or 10 μmol/L CP for 48 h. Cellular lysates were subjected to western blot analysis. e HK-2 cells were treated with vehicle or 10 μmol/L CP in the absence or presence of 20 μmol/L 3-methyladenine (3-MA) for 48 h. Cellular lysates were subjected to western blot analysis. Gdf15, growth differentiation factor 15.

Journal: Communications Biology

Article Title: Stress-responsive Gdf15 counteracts renointestinal toxicity via autophagic and microbiota reprogramming

doi: 10.1038/s42003-023-04965-1

Figure Lengend Snippet: a Expression of BECN1 in patients with acute kidney injury (GSE30718). b Based on the Gdf15 levels in patients with acute kidney injury (GSE30718, left) or chronic kidney disease (GSE66494, right), we selected the 20 highest and 20 lowest level samples, followed by a comparison of BECN1 expression levels. The results are shown as a plot with Tukey whiskers. The asterisks (∗) indicate significant differences from the low-expression group (∗ p < 0.05 using a two-tailed unpaired Student’s t -test). c Eight-week-old wild-type and Gdf15 knockout (KO) mice ( n = 3–5) were treated with vehicle or cisplatin (CP; 20 mg/kg) for 72 h. Kidney tissue lysates were subjected to western blotting. The results are shown as a plot with Tukey whiskers. The graph shows the relative densities of light chain 3 B (LC3B) from the western blot. Different letters over bars represent significant differences between groups ( p < 0.05, n = 3). d HK-2 cells transfected with control or shGdf15 vector were treated with vehicle or 10 μmol/L CP for 48 h. Cellular lysates were subjected to western blot analysis. e HK-2 cells were treated with vehicle or 10 μmol/L CP in the absence or presence of 20 μmol/L 3-methyladenine (3-MA) for 48 h. Cellular lysates were subjected to western blot analysis. Gdf15, growth differentiation factor 15.

Techniques: Expressing, Two Tailed Test, Knock-Out, Western Blot, Transfection, Plasmid Preparation

a Based on protein kinase R ( PKR ) levels in patients with acute kidney injury (GSE30718), we selected the 20 highest and 20 lowest samples, followed by a comparison of gamma-aminobutyric acid receptor-associated protein-like 1 ( GABARAPL1 ). b Based on the GABARAPL1 levels in patients with acute kidney injury (GSE30718), we selected the 20 highest and 20 lowest samples, followed by a comparison of BECN1 expression. c Based on the Gdf15 levels, we selected the 20 highest and 20 lowest samples, followed by a comparison of GABARAPL1 (left) and PPAR-γ (right) expression in patients with acute kidney injury (GSE30718). The results are shown as a plot with Tukey whiskers and outliers (orange circles). The asterisks (∗) indicate significant differences from the low-expression group (* p < 0.05, ** p < 0.01 using a two-tailed unpaired Student’s t -test). d The HK-2 cells transfected with control (the negative control shRNA) or shGdf15 plasmid were treated with vehicle or 20 μmol/L cisplatin (CP) for 12 h, and mRNA levels were measured using reverse transcription-quantitative polymerase chain reaction analysis. Data values are presented as the mean ± standard deviation (SD), and different letters over bars represent significant differences between groups ( p < 0.05). e Mechanistic scheme of Gdf15-linked protection. Cells activate integrated stress responses following tubular or mucosal insults, leading to Gdf15 production. Stress-responsive Gdf15 plays crucial roles in reorganizing the autophagy regulatory network for endogenous adaptation, including intestinal mucin production and renal cell survival against cytotoxic injuries. Moreover, the protection-linked Gdf15 counteracted the mucus-degrading microbiota in the distressed gut. Gdf15, Growth differentiation factor 15; PPAR-γ, peroxisome proliferator-activated receptor gamma.

Journal: Communications Biology

Article Title: Stress-responsive Gdf15 counteracts renointestinal toxicity via autophagic and microbiota reprogramming

doi: 10.1038/s42003-023-04965-1

Figure Lengend Snippet: a Based on protein kinase R ( PKR ) levels in patients with acute kidney injury (GSE30718), we selected the 20 highest and 20 lowest samples, followed by a comparison of gamma-aminobutyric acid receptor-associated protein-like 1 ( GABARAPL1 ). b Based on the GABARAPL1 levels in patients with acute kidney injury (GSE30718), we selected the 20 highest and 20 lowest samples, followed by a comparison of BECN1 expression. c Based on the Gdf15 levels, we selected the 20 highest and 20 lowest samples, followed by a comparison of GABARAPL1 (left) and PPAR-γ (right) expression in patients with acute kidney injury (GSE30718). The results are shown as a plot with Tukey whiskers and outliers (orange circles). The asterisks (∗) indicate significant differences from the low-expression group (* p < 0.05, ** p < 0.01 using a two-tailed unpaired Student’s t -test). d The HK-2 cells transfected with control (the negative control shRNA) or shGdf15 plasmid were treated with vehicle or 20 μmol/L cisplatin (CP) for 12 h, and mRNA levels were measured using reverse transcription-quantitative polymerase chain reaction analysis. Data values are presented as the mean ± standard deviation (SD), and different letters over bars represent significant differences between groups ( p < 0.05). e Mechanistic scheme of Gdf15-linked protection. Cells activate integrated stress responses following tubular or mucosal insults, leading to Gdf15 production. Stress-responsive Gdf15 plays crucial roles in reorganizing the autophagy regulatory network for endogenous adaptation, including intestinal mucin production and renal cell survival against cytotoxic injuries. Moreover, the protection-linked Gdf15 counteracted the mucus-degrading microbiota in the distressed gut. Gdf15, Growth differentiation factor 15; PPAR-γ, peroxisome proliferator-activated receptor gamma.

Techniques: Expressing, Two Tailed Test, Transfection, Negative Control, shRNA, Plasmid Preparation, Real-time Polymerase Chain Reaction, Standard Deviation

Induction of GDF15 and an activation of its downstream signaling molecule during the differentiation of hESCs into BAs. ( a ) Results of microarray analyses regarding TGFB/BMP/GDF family are shown. The vertical and horizontal axes indicate the signal intensity and the time duration after an induction of differentiation, respectively. The cubic polynomial approximation curve is shown on the graph. ( b ) Western blotting studies for GDF15 protein and beta-tubulin (TUBB) proteins using the differentiating hESCs or human umbilical vein endothelial cells (HUVEC) as a positive control ( upper ), Western blotting for phosphorylated SMAD2 (pSMAD2), total SMAD2, and TUBB proteins at indicated time points ( lower ) are shown. ( c ) qRT-PCR for PAI mRNA. The vertical axis indicates “fold increments” when compared to the data of the samples at 0 h. Data are shown as mean ± SD ( n = 3). ( d ) Western blotting for PAI precursor protein and mature PAI protein. ( e ) qRT-PCR for GDF15 mRNA. The vertical axis indicates the fold increments compared to the data of the samples at Day 3. Data are shown as mean ± SD ( n = 3). ( f ) Immunostaining studies for GDF15 protein (green) using the terminally differentiated samples at Day 10. Nuclei were counterstained by DAPI (blue).

Journal: Cells

Article Title: New Role for Growth/Differentiation Factor 15 in the Survival of Transplanted Brown Adipose Tissues in Cooperation with Interleukin-6

doi: 10.3390/cells9061365

Figure Lengend Snippet: Induction of GDF15 and an activation of its downstream signaling molecule during the differentiation of hESCs into BAs. ( a ) Results of microarray analyses regarding TGFB/BMP/GDF family are shown. The vertical and horizontal axes indicate the signal intensity and the time duration after an induction of differentiation, respectively. The cubic polynomial approximation curve is shown on the graph. ( b ) Western blotting studies for GDF15 protein and beta-tubulin (TUBB) proteins using the differentiating hESCs or human umbilical vein endothelial cells (HUVEC) as a positive control ( upper ), Western blotting for phosphorylated SMAD2 (pSMAD2), total SMAD2, and TUBB proteins at indicated time points ( lower ) are shown. ( c ) qRT-PCR for PAI mRNA. The vertical axis indicates “fold increments” when compared to the data of the samples at 0 h. Data are shown as mean ± SD ( n = 3). ( d ) Western blotting for PAI precursor protein and mature PAI protein. ( e ) qRT-PCR for GDF15 mRNA. The vertical axis indicates the fold increments compared to the data of the samples at Day 3. Data are shown as mean ± SD ( n = 3). ( f ) Immunostaining studies for GDF15 protein (green) using the terminally differentiated samples at Day 10. Nuclei were counterstained by DAPI (blue).

Article Snippet: The 1st antibody reaction was performed by using either a rabbit polyclonal anti-human GDF15 antibody (sc66904; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or a normal IgG (sc-2027; Santa Cruz Biotechnology, Dallas, TX, USA), and the 2nd antibody reaction was performed by using an Alexa Fluor ® 594-conjugated goat anti-rabbit IgG antibody (A11036; Thermo Fisher Scientific Inc., Waltham, MA, USA).

Techniques: Activation Assay, Microarray, Western Blot, Positive Control, Quantitative RT-PCR, Immunostaining

GDF15 - /- mice have normal brown adipose tissue (BAT) and are metabolically healthy. Results of the PCR-based genotyping of the offspring ( a ). GDF15 mRNA expressions ( b , left ) and macroscopic features ( b , right ) of the iBAT of wild-type (WT) or GDF15 -/- mice are shown. There were no significant differences in body weight ( c ) or glucose tolerance, which was evaluated by an oral glucose tolerance test (OGTT) ( d ), between WT and GDF15 - /- mice (WT: n = 8; GDF15 -/- : n = 15) after an 8-week high-fat diet. Data are shown as mean ± SD.

Journal: Cells

Article Title: New Role for Growth/Differentiation Factor 15 in the Survival of Transplanted Brown Adipose Tissues in Cooperation with Interleukin-6

doi: 10.3390/cells9061365

Figure Lengend Snippet: GDF15 - /- mice have normal brown adipose tissue (BAT) and are metabolically healthy. Results of the PCR-based genotyping of the offspring ( a ). GDF15 mRNA expressions ( b , left ) and macroscopic features ( b , right ) of the iBAT of wild-type (WT) or GDF15 -/- mice are shown. There were no significant differences in body weight ( c ) or glucose tolerance, which was evaluated by an oral glucose tolerance test (OGTT) ( d ), between WT and GDF15 - /- mice (WT: n = 8; GDF15 -/- : n = 15) after an 8-week high-fat diet. Data are shown as mean ± SD.

Techniques: Metabolic Labelling

GDF15 - /- hESCs normally differentiated into BAs. GDF15 -/ - hESCs were subjected to differentiation into BAs. The expressions of the two major BA-selective genes, PRDM16 and PPARG , were properly induced in GDF15 -/- hESCs despite the lack of GDF15 expression ( n = 3 experiments) ( a ). There were no differences in the activities of the BA-SUP to enhance insulin secretion from murine pancreatic beta cells between WT and GDF15 -/- hESCs ( n = 3 experiments) ( b ). Data are shown as mean ± SD.

Journal: Cells

Article Title: New Role for Growth/Differentiation Factor 15 in the Survival of Transplanted Brown Adipose Tissues in Cooperation with Interleukin-6

doi: 10.3390/cells9061365

Figure Lengend Snippet: GDF15 - /- hESCs normally differentiated into BAs. GDF15 -/ - hESCs were subjected to differentiation into BAs. The expressions of the two major BA-selective genes, PRDM16 and PPARG , were properly induced in GDF15 -/- hESCs despite the lack of GDF15 expression ( n = 3 experiments) ( a ). There were no differences in the activities of the BA-SUP to enhance insulin secretion from murine pancreatic beta cells between WT and GDF15 -/- hESCs ( n = 3 experiments) ( b ). Data are shown as mean ± SD.

Techniques: Expressing

IL6 expressions were reduced by the absence of GDF15 . The expression of IL6 mRNA was measured in the iBATs of WT and GDF15 -/- mice ( n = 3 mice) ( a ) or in the hESCs that were subjected to the differentiation into BAs ( n = 3 experiments) ( b ). Data are shown as mean ± SD.

Journal: Cells

Article Title: New Role for Growth/Differentiation Factor 15 in the Survival of Transplanted Brown Adipose Tissues in Cooperation with Interleukin-6

doi: 10.3390/cells9061365

Figure Lengend Snippet: IL6 expressions were reduced by the absence of GDF15 . The expression of IL6 mRNA was measured in the iBATs of WT and GDF15 -/- mice ( n = 3 mice) ( a ) or in the hESCs that were subjected to the differentiation into BAs ( n = 3 experiments) ( b ). Data are shown as mean ± SD.

Techniques: Expressing

GDF15 expressions were blunted by the absence of IL6. Using three clones of IL6 - /- hESCs, PRDM16 and GDF15 expressions were examined in the course of differentiation into BAs. Despite the induction of PRDM16 ( upper panels ), GDF15 mRNA was markedly repressed ( middle panels ) ( n = 3 experiments) by the absence of IL6 ( lower panels ). Data are shown as mean ± SD.

Journal: Cells

Article Title: New Role for Growth/Differentiation Factor 15 in the Survival of Transplanted Brown Adipose Tissues in Cooperation with Interleukin-6

doi: 10.3390/cells9061365

Figure Lengend Snippet: GDF15 expressions were blunted by the absence of IL6. Using three clones of IL6 - /- hESCs, PRDM16 and GDF15 expressions were examined in the course of differentiation into BAs. Despite the induction of PRDM16 ( upper panels ), GDF15 mRNA was markedly repressed ( middle panels ) ( n = 3 experiments) by the absence of IL6 ( lower panels ). Data are shown as mean ± SD.

Techniques: Clone Assay

GDF15 is required for long-term survival of BAT grafts. The iBATs of either GDF15 -/- or WT mice were intraperitoneally transplanted into GDF15 - /- mice according to the method presented by Stanford et al. . After 12 weeks, the grafts were removed. ( a ) Macroscopic observations. The BAT grafts of GDF15 -/- mice were shrunk and no angiogenesis was detected ( upper ), whereas those of WT mice retained their sizes and neo-angiogenesis was detected in the soft tissue around the grafts ( lower ). ( b ) Histological observations. Tissue slices of BAT grafts derived from GDF15 - /- mice ( left ) and those of WT mice ( right ) were subjected to HE staining. Structures of the elastic membranes of the arteries were well preserved even in GDF15 -/- -derived BAT grafts ( left , arrows).

Journal: Cells

Article Title: New Role for Growth/Differentiation Factor 15 in the Survival of Transplanted Brown Adipose Tissues in Cooperation with Interleukin-6

doi: 10.3390/cells9061365

Figure Lengend Snippet: GDF15 is required for long-term survival of BAT grafts. The iBATs of either GDF15 -/- or WT mice were intraperitoneally transplanted into GDF15 - /- mice according to the method presented by Stanford et al. . After 12 weeks, the grafts were removed. ( a ) Macroscopic observations. The BAT grafts of GDF15 -/- mice were shrunk and no angiogenesis was detected ( upper ), whereas those of WT mice retained their sizes and neo-angiogenesis was detected in the soft tissue around the grafts ( lower ). ( b ) Histological observations. Tissue slices of BAT grafts derived from GDF15 - /- mice ( left ) and those of WT mice ( right ) were subjected to HE staining. Structures of the elastic membranes of the arteries were well preserved even in GDF15 -/- -derived BAT grafts ( left , arrows).

Techniques: Derivative Assay, Staining

Model. IL6 induces GDF15 in the former phase of the differentiation of hESCs into BAs, whereas GDF15 and IL6 create a mutually inducing loop in the later phase. The GDF15, which may be secreted as a component of large-sized EVs, and IL6 potentiate and stabilize neovascularization, guaranteeing long-term survival of BAT grafts.

Journal: Cells

Article Title: New Role for Growth/Differentiation Factor 15 in the Survival of Transplanted Brown Adipose Tissues in Cooperation with Interleukin-6

doi: 10.3390/cells9061365

Figure Lengend Snippet: Model. IL6 induces GDF15 in the former phase of the differentiation of hESCs into BAs, whereas GDF15 and IL6 create a mutually inducing loop in the later phase. The GDF15, which may be secreted as a component of large-sized EVs, and IL6 potentiate and stabilize neovascularization, guaranteeing long-term survival of BAT grafts.

Techniques:

Correlations between GDF15 IHC score and clinical characteristics in patients with breast cancer

Journal: Oncotarget

Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

doi: 10.18632/oncotarget.21765

Figure Lengend Snippet: Correlations between GDF15 IHC score and clinical characteristics in patients with breast cancer

Article Snippet: TMAs were then stained with rabbit polyclonal anti-human GDF15 (also known as MIC-1; antibody 3249; dilution 1:100; Cell Signaling Technology, Inc., 3 Trask Lane, Danvers, MA 01923) overnight at 4°C.

Techniques:

(A) Western blotting ( above ) and real-time PCR ( graph ) for GDF15 in BT474, JIMT1 and MDA-MB-231 (MDA231) breast cancer cell lines. PCR values reflect fold change in GDF15 transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (B) Real-time PCR for GDF15 in BT474 pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Values reflect fold change in GDF15 transcript level normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (C-D) BT474 parental, pCMV empty vector control clone (pCMV), and GDF15 stable clones 2 and 3 (C2, C3) were fixed, stained with propidium iodide, and analyzed for DNA content by flow cytometry. The percentage of cells in each cell cycle phase is shown per cell line (C) (white, G0/G1; gray, S; black, G2/M). Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. Representative cell cycle histograms are shown per line (D).

Journal: Oncotarget

Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

doi: 10.18632/oncotarget.21765

Figure Lengend Snippet: (A) Western blotting ( above ) and real-time PCR ( graph ) for GDF15 in BT474, JIMT1 and MDA-MB-231 (MDA231) breast cancer cell lines. PCR values reflect fold change in GDF15 transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (B) Real-time PCR for GDF15 in BT474 pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Values reflect fold change in GDF15 transcript level normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (C-D) BT474 parental, pCMV empty vector control clone (pCMV), and GDF15 stable clones 2 and 3 (C2, C3) were fixed, stained with propidium iodide, and analyzed for DNA content by flow cytometry. The percentage of cells in each cell cycle phase is shown per cell line (C) (white, G0/G1; gray, S; black, G2/M). Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. Representative cell cycle histograms are shown per line (D).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation, Plasmid Preparation, Control, Clone Assay, Staining, Flow Cytometry

(A) Total protein whole-cell lysates were collected from BT474 pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Western blots were performed for E-Cadherin, N-Cadherin, vimentin, and FoxM1; actin was measured as a loading control. Blots were repeated at least three times, and representative blots are shown. (B) Real-time PCR for Snail, Zeb-1 and Slug in BT474 parental, pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Values reflect fold change in transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (C) BT474 pCMV empty vector control clone (pCMV) and GDF15 stable clones 3 and 5 (C3 and C5) were imaged at 10× magnification to evaluate changes in morphology. (D) Representative images of spheroid cultures are shown for BT474 parental and GDF15 stable clone 2 (C2). (E) BT474 stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5) were plated in basement membrane matrix mimic (Matrigel)-coated Boyden chambers in serum-free media; 10% FBS was added to the well below each chamber as a chemo-attractant. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005, * p<0.05.

Journal: Oncotarget

Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

doi: 10.18632/oncotarget.21765

Figure Lengend Snippet: (A) Total protein whole-cell lysates were collected from BT474 pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Western blots were performed for E-Cadherin, N-Cadherin, vimentin, and FoxM1; actin was measured as a loading control. Blots were repeated at least three times, and representative blots are shown. (B) Real-time PCR for Snail, Zeb-1 and Slug in BT474 parental, pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Values reflect fold change in transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (C) BT474 pCMV empty vector control clone (pCMV) and GDF15 stable clones 3 and 5 (C3 and C5) were imaged at 10× magnification to evaluate changes in morphology. (D) Representative images of spheroid cultures are shown for BT474 parental and GDF15 stable clone 2 (C2). (E) BT474 stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5) were plated in basement membrane matrix mimic (Matrigel)-coated Boyden chambers in serum-free media; 10% FBS was added to the well below each chamber as a chemo-attractant. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005, * p<0.05.

Techniques: Plasmid Preparation, Control, Clone Assay, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation, Stable Transfection, Membrane, Staining

(A) Total protein lysates were collected from BT474 stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Western blots were performed for p-Tyr1131 IGF-1R and total IGF-1R; actin was probed as loading control. Experiments were repeated 3 times; representative blots are shown. Quantification (shown beneath each band) was normalized to actin and performed using Odyssey Li-Cor imaging software. (B) BT474 GDF15 stable clone 2 (C2) cells were treated with normal mouse IgG control or 0.25 μg/mL alpha IR3 (aIR3) IGF-1R monoclonal antibody for 48 hours. Western blots of total protein lysates were performed for total IGF-1R, vimentin, FoxM1, and E-Cadherin. Bar graphs show quantification relative to actin loading control, and was performed using Odyssey Li-Cor imaging software. Error bars represent standard deviation between triplicates; experiments were performed at least 3 times. (C) BT474 stable empty vector control clone (pCMV) and BT474 GDF15 stable clone 5 (C5) cells were pre-treated with normal mouse IgG or 0.25 μg/mL alpha IR3 (aIR3) IGF-1R monoclonal antibody for 24 hours. Cells were then seeded in Matrigel-coated Boyden chambers in serum-free media plus control IgG or aIR3; 10% FBS was added to the well as a chemo-attractant. After 24 hours of invasion, cells in chambers were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, * p<0.05.

Journal: Oncotarget

Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

doi: 10.18632/oncotarget.21765

Figure Lengend Snippet: (A) Total protein lysates were collected from BT474 stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Western blots were performed for p-Tyr1131 IGF-1R and total IGF-1R; actin was probed as loading control. Experiments were repeated 3 times; representative blots are shown. Quantification (shown beneath each band) was normalized to actin and performed using Odyssey Li-Cor imaging software. (B) BT474 GDF15 stable clone 2 (C2) cells were treated with normal mouse IgG control or 0.25 μg/mL alpha IR3 (aIR3) IGF-1R monoclonal antibody for 48 hours. Western blots of total protein lysates were performed for total IGF-1R, vimentin, FoxM1, and E-Cadherin. Bar graphs show quantification relative to actin loading control, and was performed using Odyssey Li-Cor imaging software. Error bars represent standard deviation between triplicates; experiments were performed at least 3 times. (C) BT474 stable empty vector control clone (pCMV) and BT474 GDF15 stable clone 5 (C5) cells were pre-treated with normal mouse IgG or 0.25 μg/mL alpha IR3 (aIR3) IGF-1R monoclonal antibody for 24 hours. Cells were then seeded in Matrigel-coated Boyden chambers in serum-free media plus control IgG or aIR3; 10% FBS was added to the well as a chemo-attractant. After 24 hours of invasion, cells in chambers were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, * p<0.05.

Techniques: Plasmid Preparation, Control, Clone Assay, Western Blot, Imaging, Software, Stable Transfection, Standard Deviation, Staining

(A) Real-time PCR of total RNA from BT474 pCMV empty vector control clone (pCMV) and GDF15 stable clones (C2, C3, and C5) for MMP2 ( left graph ) and MMP9 ( right graph ). Values reflect average fold in transcript normalized to internal control RPLPO relative to pCMV group. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (B) BT474 pCMV empty vector control clone (pCMV) and GDF15 stable clones (C2, C3 and C5) were plated in serum-free media in Matrigel-coated Boyden chambers and treated with vehicle control or 1 μg pan-MMP inhibitor GM6001 for 24 hours, after which cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005, * p<0.05.

Journal: Oncotarget

Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

doi: 10.18632/oncotarget.21765

Figure Lengend Snippet: (A) Real-time PCR of total RNA from BT474 pCMV empty vector control clone (pCMV) and GDF15 stable clones (C2, C3, and C5) for MMP2 ( left graph ) and MMP9 ( right graph ). Values reflect average fold in transcript normalized to internal control RPLPO relative to pCMV group. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (B) BT474 pCMV empty vector control clone (pCMV) and GDF15 stable clones (C2, C3 and C5) were plated in serum-free media in Matrigel-coated Boyden chambers and treated with vehicle control or 1 μg pan-MMP inhibitor GM6001 for 24 hours, after which cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005, * p<0.05.

Techniques: Real-time Polymerase Chain Reaction, Plasmid Preparation, Control, Clone Assay, Standard Deviation, Staining

(A) BT474 stable empty vector control clone (pCMV) and GDF15 stable clone 5 (C5) were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 48 hours, and then plated in serum-free media in Matrigel-coated Boyden chambers. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005. (B) BT474 GDF15 stable clone 5 (C5) cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (FoxM1) for 48 hours. Real-time PCR was performed for FoxM1, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times, ** p<0.005.

Journal: Oncotarget

Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

doi: 10.18632/oncotarget.21765

Figure Lengend Snippet: (A) BT474 stable empty vector control clone (pCMV) and GDF15 stable clone 5 (C5) were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 48 hours, and then plated in serum-free media in Matrigel-coated Boyden chambers. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005. (B) BT474 GDF15 stable clone 5 (C5) cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (FoxM1) for 48 hours. Real-time PCR was performed for FoxM1, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times, ** p<0.005.

Techniques: Plasmid Preparation, Control, Stable Transfection, Transfection, Staining, Real-time Polymerase Chain Reaction, Standard Deviation

(A) JIMT1 cells were serum starved for 24 hours, and then stimulated with 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for p-Tyr1131 IGF-1R and total IGF-1R; actin was probed as loading control. Experiments were repeated 3 times; representative blots are shown. Quantification (shown beneath each band) was normalized to actin and performed using Odyssey Li-Cor imaging software. (B) JIMT-1 cells were pretreated for 24 hours with control IgG or 0.25 μg/ml alpha IR3 (aIR3) in serum-free media, and then stimulated with 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for total IGF-1R with actin as loading control. Experiments were repeated 3 times; representative blots are shown. Quantification (shown beneath each band) was normalized to actin and performed using Odyssey Li-Cor imaging software. (C) JIMT-1 cells were pretreated for 24 hours with control IgG or 0.25 μg/ml alpha IR3 (aIR3) in serum-free media, and then stimulated with 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) for another 24 hours. Real-time PCR was performed for SNAIL, SLUG, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (D) JIMT-1 cells were pretreated for 24 hours with control IgG or 0.25 μg/ml alpha IR3 (aIR3) in serum-free media, and then seeded in Matrigel-coated Boyden chambers in serum-free media with 10% FBS in the lower chamber as chemoattractant. Drug treatment was continued (in indicated samples), and 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) was added to the chambers of treatment groups where indicated. After 24 hours of invasion, photos were taken at 20× magnification; representative photos are shown. The number of invaded cells is shown per group; error bars represent standard deviation between triplicate samples, * p<0.05.

Journal: Oncotarget

Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

doi: 10.18632/oncotarget.21765

Figure Lengend Snippet: (A) JIMT1 cells were serum starved for 24 hours, and then stimulated with 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for p-Tyr1131 IGF-1R and total IGF-1R; actin was probed as loading control. Experiments were repeated 3 times; representative blots are shown. Quantification (shown beneath each band) was normalized to actin and performed using Odyssey Li-Cor imaging software. (B) JIMT-1 cells were pretreated for 24 hours with control IgG or 0.25 μg/ml alpha IR3 (aIR3) in serum-free media, and then stimulated with 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for total IGF-1R with actin as loading control. Experiments were repeated 3 times; representative blots are shown. Quantification (shown beneath each band) was normalized to actin and performed using Odyssey Li-Cor imaging software. (C) JIMT-1 cells were pretreated for 24 hours with control IgG or 0.25 μg/ml alpha IR3 (aIR3) in serum-free media, and then stimulated with 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) for another 24 hours. Real-time PCR was performed for SNAIL, SLUG, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (D) JIMT-1 cells were pretreated for 24 hours with control IgG or 0.25 μg/ml alpha IR3 (aIR3) in serum-free media, and then seeded in Matrigel-coated Boyden chambers in serum-free media with 10% FBS in the lower chamber as chemoattractant. Drug treatment was continued (in indicated samples), and 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) was added to the chambers of treatment groups where indicated. After 24 hours of invasion, photos were taken at 20× magnification; representative photos are shown. The number of invaded cells is shown per group; error bars represent standard deviation between triplicate samples, * p<0.05.

Techniques: Recombinant, Western Blot, Control, Imaging, Software, Real-time Polymerase Chain Reaction, Standard Deviation

(A) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then stimulated with 2 or 20 ng/mL recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for total FoxM1 with actin as loading control. (B) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then stimulated with 2 or 20 ng/mL recombinant human GDF15 (rhGDF15) for another 24 hours. Real-time PCR was performed for Snail, Slug, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (C) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then plated in serum-free media in Matrigel-coated Boyden chambers. Recombinant human GDF15 (rhGDF15; 2 or 20 ng/mL) was added to lower chambers where indicated. After 24 hours, cells were fixed and stained; representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, ** p<0.005, * p<0.05.

Journal: Oncotarget

Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

doi: 10.18632/oncotarget.21765

Figure Lengend Snippet: (A) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then stimulated with 2 or 20 ng/mL recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for total FoxM1 with actin as loading control. (B) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then stimulated with 2 or 20 ng/mL recombinant human GDF15 (rhGDF15) for another 24 hours. Real-time PCR was performed for Snail, Slug, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (C) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then plated in serum-free media in Matrigel-coated Boyden chambers. Recombinant human GDF15 (rhGDF15; 2 or 20 ng/mL) was added to lower chambers where indicated. After 24 hours, cells were fixed and stained; representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, ** p<0.005, * p<0.05.

Techniques: Transfection, Control, Recombinant, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation, Staining

(A) BT474 stable empty vector control clone (pCMV), BT474 GDF15 stable clone 5 (C5), and MDA-MB-231 cells were transfected with 100 nM siRNA control (siCtrl) or GDF15 siRNA (siGDF15). After 24 hours, transfected cells were plated in serum-free media in Matrigel-coated Boyden chambers with 10% FBS in the wells as a chemoattractant. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, * p<0.05. (B) BT474 stable empty vector control clone (pCMV), BT474 GDF15 stable clone 5 (C5), and MDA-MB-231 cells were transfected with 100 nM siRNA control (siCtrl) or GDF15 siRNA (siGDF15) for 48 hours. Real-time PCR was performed to confirm GDF15 knockdown. Values reflect the fold change in transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated twice; ** p<0.005, * p<0.05.

Journal: Oncotarget

Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

doi: 10.18632/oncotarget.21765

Figure Lengend Snippet: (A) BT474 stable empty vector control clone (pCMV), BT474 GDF15 stable clone 5 (C5), and MDA-MB-231 cells were transfected with 100 nM siRNA control (siCtrl) or GDF15 siRNA (siGDF15). After 24 hours, transfected cells were plated in serum-free media in Matrigel-coated Boyden chambers with 10% FBS in the wells as a chemoattractant. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, * p<0.05. (B) BT474 stable empty vector control clone (pCMV), BT474 GDF15 stable clone 5 (C5), and MDA-MB-231 cells were transfected with 100 nM siRNA control (siCtrl) or GDF15 siRNA (siGDF15) for 48 hours. Real-time PCR was performed to confirm GDF15 knockdown. Values reflect the fold change in transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated twice; ** p<0.005, * p<0.05.

Techniques: Plasmid Preparation, Control, Stable Transfection, Transfection, Staining, Real-time Polymerase Chain Reaction, Knockdown, Standard Deviation

GDF15 activates IGF-1R signaling and induces expression of FoxM1, which upregulates expression of EMT transcription factors (TFs), including Snail and Slug, and induces expression of matrix metalloproteinases (MMPs), stimulating EMT and breast cancer cell invasion.

Journal: Oncotarget

Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

doi: 10.18632/oncotarget.21765

Figure Lengend Snippet: GDF15 activates IGF-1R signaling and induces expression of FoxM1, which upregulates expression of EMT transcription factors (TFs), including Snail and Slug, and induces expression of matrix metalloproteinases (MMPs), stimulating EMT and breast cancer cell invasion.

Techniques: Expressing

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