Journal: The American Journal of Pathology

Article Title: Selective Cathepsin S Inhibition Attenuates Atherosclerosis in Apolipoprotein E–Deficient Mice with Chronic Renal Disease

doi: 10.1016/j.ajpath.2014.11.026

Figure Lengend Snippet: Cathepsin S inhibitor RO5444101 decreases plaque size, macrophage accumulation, and growth differentiation factor-15 (GDF15) expression in the atherosclerotic arteries of Apoe−/− mice with chronic renal disease (CRD). A and D: Quantification of atherosclerotic plaque size at the lesser curvature of the aortic arch using hematoxylin and eosin (H&E) staining. B and E: Mac3 staining in the aortic arch (B) shows macrophage accumulation on representative images; quantitative analysis of Mac3 staining (E). C and F: Immunohistochemical staining for a proinflammatory cytokine, GDF15, in the aortic arch (C) and its quantification (F). Data represent means ± SD. ∗P < 0.05, ∗∗∗P < 0.001.

Article Snippet: Immunohistochemical analysis for macrophages (anti-mouse mac3; BD Biosciences, San Jose, CA), cathepsin S (anti-mouse cathepsin S; Santa Cruz Biotechnology, Santa Cruz, CA), osteocalcin (goat anti-mouse polyclonal antibody; Serotech, Dusseldorf, Germany), and growth differentiation factor-15 (GDF15) (rabbit anti–Gdf-15 polyclonal antibody; Bioss Antibodies, Woburn, MA) was performed using the avidin-biotin peroxidase method.

Techniques: Expressing, Staining, Immunohistochemical staining

Journal: The American Journal of Pathology

Article Title: Selective Cathepsin S Inhibition Attenuates Atherosclerosis in Apolipoprotein E–Deficient Mice with Chronic Renal Disease

doi: 10.1016/j.ajpath.2014.11.026

Figure Lengend Snippet: Cathepsin S (CatS)–specific inhibition by RO5444101 attenuates interferon γ (IFN-γ)–induced expression of growth differentiation factor-15 (GDF15) and monocyte chemotactic protein-1 (MCP-1)/CCL2 in human and mouse macrophages. Cultured human primary macrophages (MΦ) differentiated from peripheral blood monocytes and mouse macrophage-like RAW264.7 cells were pretreated with cathepsin S inhibitor RO5444101 and then were incubated with IFN-γ. RO5444101 or siRNA against cathepsin S reduced IFN-γ–induced GDF15 (A) and MCP-1/CCL2 (B) expression in both human and mouse macrophages. N = 4 for all experiments. Data represent means ± SD. ∗P < 0.05 between comparisons. Ctrl, control; hIFN-γ, human IFN-γ; mIFN-γ, mouse IFN-γ.

Article Snippet: Immunohistochemical analysis for macrophages (anti-mouse mac3; BD Biosciences, San Jose, CA), cathepsin S (anti-mouse cathepsin S; Santa Cruz Biotechnology, Santa Cruz, CA), osteocalcin (goat anti-mouse polyclonal antibody; Serotech, Dusseldorf, Germany), and growth differentiation factor-15 (GDF15) (rabbit anti–Gdf-15 polyclonal antibody; Bioss Antibodies, Woburn, MA) was performed using the avidin-biotin peroxidase method.

Techniques: Inhibition, Expressing, Cell Culture, Incubation

Journal: The American Journal of Pathology

Article Title: Selective Cathepsin S Inhibition Attenuates Atherosclerosis in Apolipoprotein E–Deficient Mice with Chronic Renal Disease

doi: 10.1016/j.ajpath.2014.11.026

Figure Lengend Snippet: Cathepsin S inhibition in atherosclerotic plaques in chronic renal disease. Inhibition of cathepsin S may reduce lesion size via decreasing growth differentiation factor-15 (GDF15), a modulator of macrophage chemotaxis. Cathepsin S inhibition also may decrease osteogenic stimuli, such as osteopontin and osteocalcin, by suppressing elastin degradation. CCR2, C-C chemokine receptor type 2.

Article Snippet: Immunohistochemical analysis for macrophages (anti-mouse mac3; BD Biosciences, San Jose, CA), cathepsin S (anti-mouse cathepsin S; Santa Cruz Biotechnology, Santa Cruz, CA), osteocalcin (goat anti-mouse polyclonal antibody; Serotech, Dusseldorf, Germany), and growth differentiation factor-15 (GDF15) (rabbit anti–Gdf-15 polyclonal antibody; Bioss Antibodies, Woburn, MA) was performed using the avidin-biotin peroxidase method.

Techniques: Inhibition, Chemotaxis Assay

Journal: Cancer Medicine

Article Title: Angiogenesis enhanced by treatment damage to hepatocellular carcinoma through the release of GDF 15

doi: 10.1002/cam4.1330

Figure Lengend Snippet: GDF 15 was increased in the chemotherapy‐damaged HCC cells. (A) The protein expression of GDF 15 was upregulated in the cisplatin‐ or doxorubicin‐treated HCC cells (Huh7 and HepG2) detected by Western blots. The β‐actin served as the loading control. (B) Quantitative RT ‐ PCR analyses showed the VEGF expression in cisplatin‐ or doxorubicin‐treated HCC cells (Huh7 and HepG2). * P < 0.05, ** P < 0.01. HCC, hepatocellular carcinoma; GDF15, growth differentiation factor 15; VEGF, vascular endothelial growth factor.

Article Snippet: Recombinant human GDF15 was purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR

Journal: Cancer Medicine

Article Title: Angiogenesis enhanced by treatment damage to hepatocellular carcinoma through the release of GDF 15

doi: 10.1002/cam4.1330

Figure Lengend Snippet: In vitro angiogenesis promoted by GDF 15 from the chemotherapy‐treated HCC cells. (A) Migration of endothelial cells EA .hy926 was analyzed by Transwell assay (100× magnification). Serum‐starved endothelial cells EA .hy926 (6 × 10 4 cell/well) were seeded into the upper chamber of Transwell, and the conditioned medium from nontreated Huh7 cells or cisplatin‐treated Huh7 was added to the bottom chamber. The anti‐human GDF 15 neutralizing antibody was added into the conditioned medium from cisplatin‐treated Huh7. (B) Tube formation of endothelial cells. Endothelial cells EA .hy926 (4 × 10 4 cells/well) were seeded 24‐well plates coated with Matrigel matrix in the presence of conditioned medium of nontreated Huh7, or cisplatin‐treated Huh7. The anti‐human GDF 15 neutralizing antibody was added into the conditioned medium from cisplatin‐treated Huh7. The images were captured (100× magnification) from three random fields at 4 h. * P < 0.05, ** P < 0.01. HCC, hepatocellular carcinoma; GDF15, growth differentiation factor 15.

Article Snippet: Recombinant human GDF15 was purchased from R&D Systems (Minneapolis, MN).

Techniques: In Vitro, Migration, Transwell Assay

Journal: Cancer Medicine

Article Title: Angiogenesis enhanced by treatment damage to hepatocellular carcinoma through the release of GDF 15

doi: 10.1002/cam4.1330

Figure Lengend Snippet: GDF 15 promoted the proliferation, migration, and tube formation of endothelial cells. (A, B) The proliferation of endothelial cells EA .hy926 was detected by WST ‐1 and EdU assays (200× magnification). (C) Quantitative analyses of MMP 9 expression in the endothelial cells EA .hy926 treated with GDF 15. (D) Paxillin expression in GDF 15‐treated endothelial cells EA .hy926 was analyzed by immunofluorescence. Paxillin staining (red) and nuclei counterstained with DIPA (blue) (200× magnification). (E) Migration of endothelial cells was blocked by Src inhibitor or thalidomide. Endothelial cells EA .hy926 (6 × 10 4 cells/well) were seeded into the upper chamber; control medium, GDF 15 (100 ng/mL), GDF 15 (100 ng/mL) with Src inhibitor (Saracatinib) or GDF 15 (100 ng/ml) with thalidomide (100 μg/mL) was added to the bottom chamber. After 24 h, migration of endothelial cells EA .hy926 was analyzed by Transwell assay (100× magnification). (F) Tube formation of endothelial cells. Endothelial cells EA .hy926 (4 × 10 4 cells/well) were seeded 24‐well plates coated with Matrigel matrix in the presence of control medium, GDF 15 (100 ng/mL), GDF 15 (100 ng/mL) with Src inhibitor (Saracatinib) or GDF 15 (100 ng/mL) with thalidomide (100 μg/mL). The images were captured (100× magnification) from three random fields at 4 h. * P < 0.05, ** P < 0.01. GDF15, growth differentiation factor 15.

Article Snippet: Recombinant human GDF15 was purchased from R&D Systems (Minneapolis, MN).

Techniques: Migration, Expressing, Immunofluorescence, Staining, Control, Transwell Assay

Journal: Cancer Medicine

Article Title: Angiogenesis enhanced by treatment damage to hepatocellular carcinoma through the release of GDF 15

doi: 10.1002/cam4.1330

Figure Lengend Snippet: Pro‐angiogenic effects of GDF 15 through the activation of Src, AKT , MAPK , and NF ‐κB signaling (A) The phosphorylation of Src, AKT , P38 MAPK , JNK , Erk1/2, and NF ‐κB activation was detected by Western blots in the GDF 15‐treated endothelial cells EA .hy926. (B) Pretreatment with Src inhibitor Saracatinib (2.7 nmol/L), the phosphorylation of Src, AKT , P38 MAPK , JNK , Erk1/2, and NF ‐κB was detected by Western blots in the GDF 15‐treated endothelial cells EA .hy926. (C, D) Pretreatment with inhibitors of AKT ( LY 294002) (50 μmol/L), P38 MAPK ( SB 203580) (10 μmol/L), JNK ( SP 600125) (20 μmol/L), or Erk1/2 ( PD 98059) (50 μmol/L) in the GDF 15‐treated endothelial cells EA .hy926, activation of NF ‐κB was examined by western blots. GDF15, growth differentiation factor 15.

Article Snippet: Recombinant human GDF15 was purchased from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Phospho-proteomics, Western Blot

Journal: Cancer Medicine

Article Title: Angiogenesis enhanced by treatment damage to hepatocellular carcinoma through the release of GDF 15

doi: 10.1002/cam4.1330

Figure Lengend Snippet: Thalidomide suppressed the in vivo chemotherapy‐damaged HCC cells‐promoted angiogenesis. (A) The tumor size in the group of cisplatin‐treated Huh7 with endothelial cells EA .hy926 was larger than that of cisplatin‐treated Huh7 alone, but significantly smaller than that of nontreated Huh7 with endothelial cells EA .hy926. (B) In vivo angiogenesis stained by CD 31 was significantly increased in the group of cisplatin‐treated Huh7 with endothelial cells EA .hy926 when compared with two control groups. In parallel with increased angiogenesis, GDF 15 was increased significantly (200× magnification). (C) The tumor size in the thalidomide‐treated group was significantly decreased. (D) Compared with the control group, a significant reduction in CD 31 was observed after thalidomide treatment, but GDF 15 expression was not significantly different. * P < 0.05, ** P < 0.01. HCC, hepatocellular carcinoma; GDF15, growth differentiation factor 15.

Article Snippet: Recombinant human GDF15 was purchased from R&D Systems (Minneapolis, MN).

Techniques: In Vivo, Staining, Control, Expressing

Journal: Cancer Medicine

Article Title: Angiogenesis enhanced by treatment damage to hepatocellular carcinoma through the release of GDF 15

doi: 10.1002/cam4.1330

Figure Lengend Snippet: Thalidomide inhibited the GDF 15‐induced activation of Src and its downstream pathways. Src, AKT , P38 MAPK , JNK , Erk1/2, and NF ‐κB were detected by Western blots in the GDF 15‐treated endothelial cells EA .hy926 in the presence of thalidomide. GDF15, growth differentiation factor 15.

Article Snippet: Recombinant human GDF15 was purchased from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Western Blot

Journal: Journal for immunotherapy of cancer

Article Title: Loss of ADAR1 in macrophages in combination with interferon gamma suppresses tumor growth by remodeling the tumor microenvironment.

doi: 10.1136/jitc-2023-007402

Figure Lengend Snippet: Figure 3 ADAR1 loss in macrophage with IFN-γ treatment affects the secretion of key cytokines through PKR/EIF2α signaling. (A, C) Human XL cytokine arrays for detecting differential factors between THP-1 cells with scrambled shRNA and shADAR1#1 under the treatment of IFN-γ (A) and between THP-1 cells with empty vector and WT ADAR, co-cultured with A549 (C). (B, D) Bar plots showing the expression levels of differential factors on ADAR1 knockdown (B) and ADAR1 overexpression (D). (E) Venn gram showing key cytokines identified by ADAR1 knockdown and overexpression experiments and their potential effects on the tumor microenvironment. (F) RT-qPCR-based mRNA expression levels of ADAR, CCL20, GDF15, IFN-G, IL-18, IL-18BP, and HAVCR2 in different THP-1 cells (transfected with scrambled shRNA, shADAR1#1 or shADAR1#2) with IFN-γ treatment. β-actin was used as an internal control. (G) RT-qPCR-based mRNA expression levels of Ccl20, Gdf15, Il-18, Il-18bp, and Havcr2 in BMDMs from C57BL/6 mice (Adarfl/fl and Adarfl/flLyz2Cre) treated with IFN-γ. Gapdh was used as an internal control. (H) Immunofluorescent staining for anti-dsRNA (J2) in THP-1 cells treated with IFN-γ. RNase III treatment was used as the negative control for the dsRNA signal. Scale bars, 10 µm. (I) Western blot showing the protein expression of p-PKRThr446/

Article Snippet: 2- AP (GlpBio, GC61906), Phorbol 12- myristate 13- acetate (PMA) (MedChemExpress, HY- 18739), Recombinant Human IFN-γ (Novoprotein, C014), Recombinant Mouse IL- 2 (Novoprotein, P04351), Recombinant M- CSF (Novoprotein, CB34), Recombinant Mouse IFN-γ (Novoprotein, C746), Recombinant Mouse TIM- 3 (Novoprotein, CM54), Recombinant Mouse CCL20 (PeproTech, 250–2), Recombinant Mouse GDF15 (R&D Systems, 8944- GD025), Recombinant Mouse IL- 18 Binding Protein Isoform d (Novoprotein, CM45), ShortCut RNase III (NEB, M0245S), poly (I:C) (GlpBio, GC14710).

Techniques: shRNA, Plasmid Preparation, Cell Culture, Expressing, Knockdown, Over Expression, Quantitative RT-PCR, Transfection, Control, Staining, Negative Control, Western Blot

Journal: Journal for immunotherapy of cancer

Article Title: Loss of ADAR1 in macrophages in combination with interferon gamma suppresses tumor growth by remodeling the tumor microenvironment.

doi: 10.1136/jitc-2023-007402

Figure Lengend Snippet: Figure 5 The effect of ADAR1 loss and key cytokines on tube formation. (A) Schematic diagram of tube formation experiment of SVEC4-10 cells. (B) Tube formation of SVEC4-10 cells treated with conditioned media from BMDMs (Adarfl/fl vs Adarfl/flLyz2Cre) and CCL20, GDF15, or IFN-γ. Scale bar, 500 µm. (C) Bar plots showing relative vessel area, the total number of junctions, and the vessel length for SVEC4-10 tube formation. (D) Tube formation of SVEC4-10 cells treated with conditioned media from BMDMs (Adarfl/flLyz2Cre) and IFN-γ after pretreatment with or without 2-AP (5 mM). Scale bar, 500 µm. (E) Bar plots showing relative vessel area, the total number of junctions, and vessel length for SVEC4-10 tube formation. (F) Tube formation of HUVEC cells treated with conditioned media from THP-1 cells (transfected with shADAR1#1) and IFN-γ after pretreatment with or without 2-AP (5 mM). Scale bar, 200 µm. (G) Bar plots showing relative vessel area, the total number of junctions, and the vessel length for HUVEC tube formation. (C, E, and G) P values are based on unpaired Student’s t-test. Data are presented as mean±SD. ADAR, adenosine deaminases acting on RNA; BMDMs, bone marrow-derived macrophages; IFN, interferon; 2-AP, 2-aminopurine.

Article Snippet: 2- AP (GlpBio, GC61906), Phorbol 12- myristate 13- acetate (PMA) (MedChemExpress, HY- 18739), Recombinant Human IFN-γ (Novoprotein, C014), Recombinant Mouse IL- 2 (Novoprotein, P04351), Recombinant M- CSF (Novoprotein, CB34), Recombinant Mouse IFN-γ (Novoprotein, C746), Recombinant Mouse TIM- 3 (Novoprotein, CM54), Recombinant Mouse CCL20 (PeproTech, 250–2), Recombinant Mouse GDF15 (R&D Systems, 8944- GD025), Recombinant Mouse IL- 18 Binding Protein Isoform d (Novoprotein, CM45), ShortCut RNase III (NEB, M0245S), poly (I:C) (GlpBio, GC14710).

Techniques: Transfection, Derivative Assay

Journal: Journal for immunotherapy of cancer

Article Title: Loss of ADAR1 in macrophages in combination with interferon gamma suppresses tumor growth by remodeling the tumor microenvironment.

doi: 10.1136/jitc-2023-007402

Figure Lengend Snippet: Figure 8 Schematic summary of this study. ADAR1-deficient macrophages combined with IFN-γ treatment reprogram the tumor microenvironment by two mechanisms: (1) inhibit angiogenesis by decreased secretion of GDF15 and CCL20 and increased secretion of IFN-γ, and (2) activate CD8+ T cells by decreased secretion of TIM-3 and IL-18BP and increased secretion of IL-18. These effects collectively convert a “cold tumor” into a “hot tumor.” Combined treatment with ADAR1- deficient macrophages and IFN-γ may represent an effective therapeutic approach. dsRNA, double-stranded RNA; IFN, interferon; IL, interleukin; uORF, upstream Open Reading Frame.

Article Snippet: 2- AP (GlpBio, GC61906), Phorbol 12- myristate 13- acetate (PMA) (MedChemExpress, HY- 18739), Recombinant Human IFN-γ (Novoprotein, C014), Recombinant Mouse IL- 2 (Novoprotein, P04351), Recombinant M- CSF (Novoprotein, CB34), Recombinant Mouse IFN-γ (Novoprotein, C746), Recombinant Mouse TIM- 3 (Novoprotein, CM54), Recombinant Mouse CCL20 (PeproTech, 250–2), Recombinant Mouse GDF15 (R&D Systems, 8944- GD025), Recombinant Mouse IL- 18 Binding Protein Isoform d (Novoprotein, CM45), ShortCut RNase III (NEB, M0245S), poly (I:C) (GlpBio, GC14710).

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