Journal: EBioMedicine

Article Title: Autocrine CTHRC1 activates hepatic stellate cells and promotes liver fibrosis by activating TGF-β signaling

doi: 10.1016/j.ebiom.2019.01.009

Figure Lengend Snippet: CTHRC1 activates both TGF-β and Wnt signaling, while the promotive effect of CTHRC1 on HSC activation is mainly dependent on TGF-β signaling. A. The expression of α-SMA in primary HSCs after 7 days isolated from WT and CTHRC1 −/− mice. B. The expression of α-SMA in primary rat HSCs after 4 days, which were treated with vehicle, 20 nM rCTHRC1 protein alone, and 20 nM rCTHRC1 protein plus CTHRC1 mAb or IgG. C. Western blotting analysis of phosphorylation of Smad2, Smad3, JNK, total Smad4 and expression of Wnt5a in five liver tissues of WT or CTHRC1 −/− mice intraperitoneally injected with CCl 4 . GAPDH was the loading control. The densitometry of p-Smad2/Smad2 was shown below. D. Phosphorylation of Smad2, Smad3, JNK, total Smad4 and expression of Wnt5a in primary rat HSCs, which were treated with vehicle, 20 nM rCTHRC1 protein, and 20 nM rCTHRC1 protein plus CTHRC1 mAb or IgG for 1, 3, 5 days, individually. GAPDH was the loading control. The densitometry of p-Smad2/Smad2 was shown below. E and F. Representative immunofluorescence images of α-SMA (green in E, red in F) in primary rat HSCs after 4 days, which were treated with vehicle, 20 nM rCTHRC1 protein, and 20 nM rCTHRC1 protein plus neutralizing antibodies or inhibitor as follows: TGFBR2 neutralizing antibody or TGF-β receptor inhibitor (E), Wnt5a or Wnt3a neutralizing antibody (F). Nuclei are stained with DAPI (blue). Scale bars, 50 μm. G. The expression of α-SMA in primary rat HSCs after 4 days, which were treated with vehicle, 20 nM rCTHRC1 protein, and 20 nM rCTHRC1 protein plus TGFBR2 neutralizing antibody or TGF-β receptor inhibitor. GAPDH was the loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Recombinant CTHRC1 and/or TGFBR2 neutralizing antibody (R&D, AF-241-NA), TGF-βR inhibitor, LY2109761 (SELLECK, S2704) were added in the medium simultaneously.

Techniques: Activation Assay, Expressing, Isolation, Western Blot, Injection, Immunofluorescence, Staining

Journal: International immunopharmacology

Article Title: Foxp2 inhibits Th9 cell differentiation and attenuates allergic airway inflammation in a mouse model of ovalbumin-induced asthma.

doi: 10.1016/j.intimp.2022.109060

Figure Lengend Snippet: Fig. 1. Anti-IL-9 antibody inhibits Th9 cells differentiation in asthmatic mice. Forty BALB/c mice were randomly divided into four groups: control group (Control), OVA-induced asthma model group (OVA), mouse IgG injection treatment group (IgG) and anti-IL-9 mAb injection treatment group (anti-IL-9). Four hours after the last OVA stimulation, the mice were anesthetized and killed to collect bronchoalveolar lavage fluid (BALF) and lung tissue. A-B. The percentage of Th9 cells in BALF was evaluated by flow cytometry in each group. C-D. The percentage of Th9 cells in lung tissue was evaluated by flow cytometry in each group. E-F. The levels of IL-9 in BALF and lung tissue were measured by ELISA in each group. G-H. The mRNA expression of BATF and IRF4 in lung tissue were detected by RT-PCR. I-K. The protein expression of BATF and IRF4 in lung tissue were detected by western blot. **P < 0.01,***p < 0.001 vs Control group, #P < 0.05, ##P < 0.01,###p < 0.001 vs IgG group.

Article Snippet: Subsequently, CD4+ T cells were cultured in Th9 induction medium (10 ng/mL IL-4, 3 ng/mL TGF-β, and 10 mg/mL anti-IFN-γ, Proteintech).

Techniques: Control, Injection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: International immunopharmacology

Article Title: Foxp2 inhibits Th9 cell differentiation and attenuates allergic airway inflammation in a mouse model of ovalbumin-induced asthma.

doi: 10.1016/j.intimp.2022.109060

Figure Lengend Snippet: Fig. 3. Down regulation of Foxp2 expression in Th9 cells Spleen cells were isolated from normal mice and CD4+T cells were sorted by magnetic beads. IL-4, anti–IFN-ɣ and TGF-β were used to induce the differentiation of Th9 cells. After 4 days of induction, cells were collected. A.The levels of IL-9 in the supernatant of pre-induction (CD4+T) and post-induction (Th9) cells were were measured by ELISA. B-C. The mRNA expres sion of IL-9 and Foxp2 in pre-induction (CD4+T) and post-induction (Th9) cells were detected by RT-PCR in each group. D-E. The protein expression of IL-9 and Foxp2 in pre-induction (CD4+T) and post-induction (Th9) cells were analyzed by western blot in each group. **P < 0.01,***p < 0.001 vs CD4 + T cell group.

Article Snippet: Subsequently, CD4+ T cells were cultured in Th9 induction medium (10 ng/mL IL-4, 3 ng/mL TGF-β, and 10 mg/mL anti-IFN-γ, Proteintech).

Techniques: Expressing, Isolation, Magnetic Beads, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: International immunopharmacology

Article Title: Foxp2 inhibits Th9 cell differentiation and attenuates allergic airway inflammation in a mouse model of ovalbumin-induced asthma.

doi: 10.1016/j.intimp.2022.109060

Figure Lengend Snippet: Fig. 4. Overexpression of Foxp2 inhibits Th9 cell differentiation in vitro Th9 cells were infected withFoxp2 overexpressed lentivirus (LV-Foxp2) and control lentivirus (LV-NC), respectively, and no infected group as control. A-B. The percentage of Th9 cells was evaluated by Flow cytometry. C. The levels of IL-9 in cell supernatant was measured by ELISA. D. The mRNA expression of IL-9 in cells was detected by RT-PCR. E-F. The mRNA expression of BATF and IRF4 in cells were detected by RT- PCR. **P < 0.01, ***p < 0.001 vs LV-NC group.

Article Snippet: Subsequently, CD4+ T cells were cultured in Th9 induction medium (10 ng/mL IL-4, 3 ng/mL TGF-β, and 10 mg/mL anti-IFN-γ, Proteintech).

Techniques: Over Expression, Cell Differentiation, In Vitro, Infection, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: International immunopharmacology

Article Title: Foxp2 inhibits Th9 cell differentiation and attenuates allergic airway inflammation in a mouse model of ovalbumin-induced asthma.

doi: 10.1016/j.intimp.2022.109060

Figure Lengend Snippet: Fig. 5. Overexpression of Foxp2 improves airway inflammation in asthmatic mice by inhibiting Th9 cell differentiation. Ten BALB/c mice were fed adaptively for one week. In the second week, 10 mice were divided into two groups (OVA + NC group and OVA + Foxp2 group) to establish asthma model. In OVA + NC group, OVA sensitized/challenged asthmatic mice were given control lentivirus by tracheal delivery at 3 days before aerosol challenge. In OVA + Foxp2 group, OVA sensitized/challenged asthmatic mice were given Foxp2 overexpression lentivirus by tracheal delivery at 3 days before aerosol challenge. Four hours after the last challenge, the mice were anesthetized and killed to get the lung tissue. A. HE staining was used to observe the pathological changes of lung tissue. B. PAS glycogen staining was used to determine the airway mucus secretion. C-F. The mRNA expression of IL-9, FOXP2, BATF and IRF4 in lung tissue were detected by RT-PCR. *P < 0.05,**P < 0.01,***p < 0.001 vs OVA + NC group.

Article Snippet: Subsequently, CD4+ T cells were cultured in Th9 induction medium (10 ng/mL IL-4, 3 ng/mL TGF-β, and 10 mg/mL anti-IFN-γ, Proteintech).

Techniques: Over Expression, Cell Differentiation, Control, Aerosol, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Interleukin-6 and Type-I Collagen Production by Systemic Sclerosis Fibroblasts Are Differentially Regulated by Interleukin-17A in the Presence of Transforming Growth Factor-Beta 1

doi: 10.3389/fimmu.2018.01865

Figure Lengend Snippet: IL-6 and monocyte chemotactic protein-1 (MCP-1) are synergistically and specifically induced in human fibroblasts by the combined action of IL-17A and TGF-β. Primary human dermal fibroblasts from healthy donors (HD) and systemic sclerosis (SSc) patients were cultured in the presence of IL-17A (25 ng/ml), TGF-β (2.5 ng/ml), or their combination for 48 h, in 96-well plates, in triplicates. IL-6 (A) , MCP-1 (B) , IL-8 (C) were assessed by ELISA in culture supernatants. Results are expressed as fold change compared to spontaneous production in control (ctrl) cultures. Basal levels were: 22.7 (±7.3) and 40.7 (±16.7) pg/ml for IL-6; 328.1 (±33.3) and 377.2 (±85.4) pg/ml for MCP-1 and 211.8 (±56.6) and 207.7 (±56.9) pg/ml for IL-8, in HD and SSc, respectively. Significance was assessed by paired t test.

Article Snippet: rhIL-17A, rhTGF-β, monoclonal mouse IgG1 TGF-β1, 2, 3 antibody, IL-6, MCP-1, MMP-1, IL-8, pro-collagen Iα1 and fibronectin ELISA DuoSet kits were from R&D Systems (Abingdon, UK); DMEM, PBS, glutamine, penicillin, streptomycin, trypsin, dispase, collagenase type I from Gibco (Paisley, UK); FCS from Biowest (Nuaillé, France); BSA, α-ketoglutaric acid, β-amino propionitrile, l -ascorbic acid, p38 MAPK inhibitor SB203580, and PI3K inhibitor LY294002 from Sigma (St. Louis, MO, USA); MEK1/2 inhibitor U-0126 from Calbiochem (San Diego, CA, USA); TGF-βRI inhibitor SD 208, JNK inhibitor SP 600125 and IKK-2 inhibitor TPCA-1 from Tocris Bioscience (Bristol, UK); LEAF irrelevant control mAbs from Biolegend (San Diego, CA, USA); Complete Protease Inhibitor Cocktail and PhosSTOP phosphatase inhibitor from Roche (Basel, Switzerland); nitrocellulose membranes and chemiluminescence (ECL) blotting analysis system from GE Healthcare (Zurich, Switzerland); phospho-Akt (Ser473), phospho-Smad2 (Ser465/467), phospho-p38 MAPK (Thr180/Tyr182), phospho-NF-κB p65 (Ser536), phospho-IκB-α (Ser32), β-actin and BSA for Western blots from Cell Signaling (Danvers, MA, USA); TMB ELISA substrate from Abcam (Cambridge, UK); EZ4U cell proliferation assay from Biomedica (Vienna, Austria).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Interleukin-6 and Type-I Collagen Production by Systemic Sclerosis Fibroblasts Are Differentially Regulated by Interleukin-17A in the Presence of Transforming Growth Factor-Beta 1

doi: 10.3389/fimmu.2018.01865

Figure Lengend Snippet: TGF-β inhibition abrogates the synergistic response with IL-17A. HD fibroblasts were treated with (A) 10 µg/ml TGF-β 1 neutralizing antibody or 10 µg/ml of an irrelevant ctrl Ab, (B) SD208 (TGFβR1 inhibitor) or vehicle for 1 h prior to the addition of IL-17A (25 ng/ml) or TGF-β (2.5 ng/ml) and cultured for 48 h. IL-6 levels in SN were assessed by ELISA. Results are shown as fold change to untreated control cultures (basal level of IL-6 was 3.1 ± 1.1 pg/ml). (B) Square: TGF-β (10 ng/ml); empty circle: (SD2018, 1 µM); full circle: vehicle. Significant differences were assessed by paired t -test. Bars in (A) and symbols in (B) represent the mean + SEM of three experiments.

Article Snippet: rhIL-17A, rhTGF-β, monoclonal mouse IgG1 TGF-β1, 2, 3 antibody, IL-6, MCP-1, MMP-1, IL-8, pro-collagen Iα1 and fibronectin ELISA DuoSet kits were from R&D Systems (Abingdon, UK); DMEM, PBS, glutamine, penicillin, streptomycin, trypsin, dispase, collagenase type I from Gibco (Paisley, UK); FCS from Biowest (Nuaillé, France); BSA, α-ketoglutaric acid, β-amino propionitrile, l -ascorbic acid, p38 MAPK inhibitor SB203580, and PI3K inhibitor LY294002 from Sigma (St. Louis, MO, USA); MEK1/2 inhibitor U-0126 from Calbiochem (San Diego, CA, USA); TGF-βRI inhibitor SD 208, JNK inhibitor SP 600125 and IKK-2 inhibitor TPCA-1 from Tocris Bioscience (Bristol, UK); LEAF irrelevant control mAbs from Biolegend (San Diego, CA, USA); Complete Protease Inhibitor Cocktail and PhosSTOP phosphatase inhibitor from Roche (Basel, Switzerland); nitrocellulose membranes and chemiluminescence (ECL) blotting analysis system from GE Healthcare (Zurich, Switzerland); phospho-Akt (Ser473), phospho-Smad2 (Ser465/467), phospho-p38 MAPK (Thr180/Tyr182), phospho-NF-κB p65 (Ser536), phospho-IκB-α (Ser32), β-actin and BSA for Western blots from Cell Signaling (Danvers, MA, USA); TMB ELISA substrate from Abcam (Cambridge, UK); EZ4U cell proliferation assay from Biomedica (Vienna, Austria).

Techniques: Inhibition, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Interleukin-6 and Type-I Collagen Production by Systemic Sclerosis Fibroblasts Are Differentially Regulated by Interleukin-17A in the Presence of Transforming Growth Factor-Beta 1

doi: 10.3389/fimmu.2018.01865

Figure Lengend Snippet: Shared and private signaling pathways are preferentially used by IL-17A and TGF-β to induce IL-6. Healthy donors fibroblasts were treated with optimal doses of inhibitors (20 µM U0126, 20 µM SB203580, 10 µM SP600125, 10 µM Ly294002, or 0.37 µM TPCA-1) or vehicle for 1 h prior to the addition of IL-17A (25 ng/ml) or TGF-β (2.5 ng/ml) and cultured for an additional 48 h, in triplicates. (A) IL-6 levels in SN were assessed by ELISA. Results are shown as the percentage of IL-6 production induced by IL-17A or TGF-β in the absence of inhibitors (levels of IL-6 were: 22.8 ± 3.3 pg/ml for IL-17A and 8.8 ± 3.9 pg/ml for TGF-β). Bars represent the mean + SEM of three experiments. Significant differences versus control were assessed by paired t -test: * P < 0.05, **** P < 0.001. (B) . Fibroblast viability was assessed by EZ4U and found >90% for all culture conditions.

Article Snippet: rhIL-17A, rhTGF-β, monoclonal mouse IgG1 TGF-β1, 2, 3 antibody, IL-6, MCP-1, MMP-1, IL-8, pro-collagen Iα1 and fibronectin ELISA DuoSet kits were from R&D Systems (Abingdon, UK); DMEM, PBS, glutamine, penicillin, streptomycin, trypsin, dispase, collagenase type I from Gibco (Paisley, UK); FCS from Biowest (Nuaillé, France); BSA, α-ketoglutaric acid, β-amino propionitrile, l -ascorbic acid, p38 MAPK inhibitor SB203580, and PI3K inhibitor LY294002 from Sigma (St. Louis, MO, USA); MEK1/2 inhibitor U-0126 from Calbiochem (San Diego, CA, USA); TGF-βRI inhibitor SD 208, JNK inhibitor SP 600125 and IKK-2 inhibitor TPCA-1 from Tocris Bioscience (Bristol, UK); LEAF irrelevant control mAbs from Biolegend (San Diego, CA, USA); Complete Protease Inhibitor Cocktail and PhosSTOP phosphatase inhibitor from Roche (Basel, Switzerland); nitrocellulose membranes and chemiluminescence (ECL) blotting analysis system from GE Healthcare (Zurich, Switzerland); phospho-Akt (Ser473), phospho-Smad2 (Ser465/467), phospho-p38 MAPK (Thr180/Tyr182), phospho-NF-κB p65 (Ser536), phospho-IκB-α (Ser32), β-actin and BSA for Western blots from Cell Signaling (Danvers, MA, USA); TMB ELISA substrate from Abcam (Cambridge, UK); EZ4U cell proliferation assay from Biomedica (Vienna, Austria).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Interleukin-6 and Type-I Collagen Production by Systemic Sclerosis Fibroblasts Are Differentially Regulated by Interleukin-17A in the Presence of Transforming Growth Factor-Beta 1

doi: 10.3389/fimmu.2018.01865

Figure Lengend Snippet: NFκB and PI3K signaling pathways are preferentially used by IL-17A and TGF-β, respectively and together cooperate in inducing IL-6. Healthy donors fibroblasts were treated with the indicated concentrations of (A) TPCA-1; (B) Ly294002; or (C) suboptimal doses of TPCA-1 (0.03 µM) and/or Ly294002 (Ly, 2 µM) for 1 h prior to addition of IL-17A (25 ng/ml) and/or TGF-β (2.5 ng/ml). After 48 h, culture SNs were collected and IL-6 levels were assessed by ELISA. (A,B) Results are shown as fold change to untreated cells, mean + SEM is indicated ( N = 4). Please note the log 2 scale. (C) Results are shown as the percentage of IL-6 production induced by IL-17A and/or TGF-β in the absence of inhibitors (levels of IL-6 were: 86.2 ± 11.8 pg/ml for IL-17A, 49.6 ± 10.2 pg/ml for TGF-β, and 257.5 ± 82.5 pg/ml for IL-17A + TGF-β). Bars represent the mean + SEM. Significant differences versus control were assessed by paired t -test: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001.

Article Snippet: rhIL-17A, rhTGF-β, monoclonal mouse IgG1 TGF-β1, 2, 3 antibody, IL-6, MCP-1, MMP-1, IL-8, pro-collagen Iα1 and fibronectin ELISA DuoSet kits were from R&D Systems (Abingdon, UK); DMEM, PBS, glutamine, penicillin, streptomycin, trypsin, dispase, collagenase type I from Gibco (Paisley, UK); FCS from Biowest (Nuaillé, France); BSA, α-ketoglutaric acid, β-amino propionitrile, l -ascorbic acid, p38 MAPK inhibitor SB203580, and PI3K inhibitor LY294002 from Sigma (St. Louis, MO, USA); MEK1/2 inhibitor U-0126 from Calbiochem (San Diego, CA, USA); TGF-βRI inhibitor SD 208, JNK inhibitor SP 600125 and IKK-2 inhibitor TPCA-1 from Tocris Bioscience (Bristol, UK); LEAF irrelevant control mAbs from Biolegend (San Diego, CA, USA); Complete Protease Inhibitor Cocktail and PhosSTOP phosphatase inhibitor from Roche (Basel, Switzerland); nitrocellulose membranes and chemiluminescence (ECL) blotting analysis system from GE Healthcare (Zurich, Switzerland); phospho-Akt (Ser473), phospho-Smad2 (Ser465/467), phospho-p38 MAPK (Thr180/Tyr182), phospho-NF-κB p65 (Ser536), phospho-IκB-α (Ser32), β-actin and BSA for Western blots from Cell Signaling (Danvers, MA, USA); TMB ELISA substrate from Abcam (Cambridge, UK); EZ4U cell proliferation assay from Biomedica (Vienna, Austria).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Interleukin-6 and Type-I Collagen Production by Systemic Sclerosis Fibroblasts Are Differentially Regulated by Interleukin-17A in the Presence of Transforming Growth Factor-Beta 1

doi: 10.3389/fimmu.2018.01865

Figure Lengend Snippet: Phosphorylation of MAPK p38 is enhanced by the combined action of IL-17A and TGF-β. (A) Western blot (WB) of healthy donors fibroblasts treated with 1 µM TPCA-1 and/or 10 µM Ly294002 for 1 h prior to addition of IL-17A (25 ng/ml) and/or TGF-β (2.5 ng/ml) and cultured for an additional 10 min. Results are representative of three experiments with inhibitors and two additional experiments with cytokines only. (B) Quantification of Western blot (WB) analysis was performed with ImageJ software ( http://rsbweb.nih.gov/ij ) and values were normalized to β-actin, N = 5. Results are shown as fold change to IL-17A-treated cells (for p-p38, p-IκBα, and p-NF-κB p65) or to untreated cells (for p-Akt), N = 5. Significance assessed by paired t test.

Article Snippet: rhIL-17A, rhTGF-β, monoclonal mouse IgG1 TGF-β1, 2, 3 antibody, IL-6, MCP-1, MMP-1, IL-8, pro-collagen Iα1 and fibronectin ELISA DuoSet kits were from R&D Systems (Abingdon, UK); DMEM, PBS, glutamine, penicillin, streptomycin, trypsin, dispase, collagenase type I from Gibco (Paisley, UK); FCS from Biowest (Nuaillé, France); BSA, α-ketoglutaric acid, β-amino propionitrile, l -ascorbic acid, p38 MAPK inhibitor SB203580, and PI3K inhibitor LY294002 from Sigma (St. Louis, MO, USA); MEK1/2 inhibitor U-0126 from Calbiochem (San Diego, CA, USA); TGF-βRI inhibitor SD 208, JNK inhibitor SP 600125 and IKK-2 inhibitor TPCA-1 from Tocris Bioscience (Bristol, UK); LEAF irrelevant control mAbs from Biolegend (San Diego, CA, USA); Complete Protease Inhibitor Cocktail and PhosSTOP phosphatase inhibitor from Roche (Basel, Switzerland); nitrocellulose membranes and chemiluminescence (ECL) blotting analysis system from GE Healthcare (Zurich, Switzerland); phospho-Akt (Ser473), phospho-Smad2 (Ser465/467), phospho-p38 MAPK (Thr180/Tyr182), phospho-NF-κB p65 (Ser536), phospho-IκB-α (Ser32), β-actin and BSA for Western blots from Cell Signaling (Danvers, MA, USA); TMB ELISA substrate from Abcam (Cambridge, UK); EZ4U cell proliferation assay from Biomedica (Vienna, Austria).

Techniques: Western Blot, Cell Culture, Software

Journal: Frontiers in Immunology

Article Title: Interleukin-6 and Type-I Collagen Production by Systemic Sclerosis Fibroblasts Are Differentially Regulated by Interleukin-17A in the Presence of Transforming Growth Factor-Beta 1

doi: 10.3389/fimmu.2018.01865

Figure Lengend Snippet: p38 MAPK signaling pathway is common to IL-17A- and TGF-β-induced IL-6 production. Healthy donors fibroblasts were treated with the indicated concentrations of SB203580 (A) or 20 µM SB203580 (B) for 1 h prior to the addition of IL-17A (25 ng/ml) and/or TGF-β (2.5 ng/ml) in triplicates. After 48 h, culture SNs were collected and IL-6 levels were assessed by ELISA. (A) Results are shown as fold change to untreated cells, mean + SEM is indicated, N = 3. Please note the log 2 scale. (B) Results are shown as the percentage of IL-6 production induced by IL-17A and/or TGF- β in the absence of inhibitor (levels of IL-6 were: 14.7 ± 7.4 pg/ml for IL-17A, 11.4 ± 6.1 pg/ml for TGF-β, and 48.9 ± 13.7 pg/ml for IL-17A + TGF-β). Bars represent the mean + SEM of three experiments. Significant differences versus control were assessed by paired t -test.

Article Snippet: rhIL-17A, rhTGF-β, monoclonal mouse IgG1 TGF-β1, 2, 3 antibody, IL-6, MCP-1, MMP-1, IL-8, pro-collagen Iα1 and fibronectin ELISA DuoSet kits were from R&D Systems (Abingdon, UK); DMEM, PBS, glutamine, penicillin, streptomycin, trypsin, dispase, collagenase type I from Gibco (Paisley, UK); FCS from Biowest (Nuaillé, France); BSA, α-ketoglutaric acid, β-amino propionitrile, l -ascorbic acid, p38 MAPK inhibitor SB203580, and PI3K inhibitor LY294002 from Sigma (St. Louis, MO, USA); MEK1/2 inhibitor U-0126 from Calbiochem (San Diego, CA, USA); TGF-βRI inhibitor SD 208, JNK inhibitor SP 600125 and IKK-2 inhibitor TPCA-1 from Tocris Bioscience (Bristol, UK); LEAF irrelevant control mAbs from Biolegend (San Diego, CA, USA); Complete Protease Inhibitor Cocktail and PhosSTOP phosphatase inhibitor from Roche (Basel, Switzerland); nitrocellulose membranes and chemiluminescence (ECL) blotting analysis system from GE Healthcare (Zurich, Switzerland); phospho-Akt (Ser473), phospho-Smad2 (Ser465/467), phospho-p38 MAPK (Thr180/Tyr182), phospho-NF-κB p65 (Ser536), phospho-IκB-α (Ser32), β-actin and BSA for Western blots from Cell Signaling (Danvers, MA, USA); TMB ELISA substrate from Abcam (Cambridge, UK); EZ4U cell proliferation assay from Biomedica (Vienna, Austria).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Interleukin-6 and Type-I Collagen Production by Systemic Sclerosis Fibroblasts Are Differentially Regulated by Interleukin-17A in the Presence of Transforming Growth Factor-Beta 1

doi: 10.3389/fimmu.2018.01865

Figure Lengend Snippet: IL-17A decreases the col-I to MMP-1 ratio enhanced by TGF-β. Primary human dermal fibroblasts from healthy donors (HD) (left panel) and systemic sclerosis (SSc) patients (right panel) were cultured in the presence of IL-17A (25 ng/ml), TGF-β (2.5 ng/ml), or their combination for 48 h, in 96-well plates, in triplicates. MMP-1 levels (A) were assessed by ELISA in culture supernatants. Results are expressed as fold change compared to spontaneous production in control (ctrl) cultures. Basal levels for MMP-1 were 15.71 (±1.3) and 20.1 (±2.3) ng/ml, in HD and SSc, respectively. (B) The ratio of col-I levels from Figure C to MMP-1 was calculated. Significance was assessed by paired t -test.

Article Snippet: rhIL-17A, rhTGF-β, monoclonal mouse IgG1 TGF-β1, 2, 3 antibody, IL-6, MCP-1, MMP-1, IL-8, pro-collagen Iα1 and fibronectin ELISA DuoSet kits were from R&D Systems (Abingdon, UK); DMEM, PBS, glutamine, penicillin, streptomycin, trypsin, dispase, collagenase type I from Gibco (Paisley, UK); FCS from Biowest (Nuaillé, France); BSA, α-ketoglutaric acid, β-amino propionitrile, l -ascorbic acid, p38 MAPK inhibitor SB203580, and PI3K inhibitor LY294002 from Sigma (St. Louis, MO, USA); MEK1/2 inhibitor U-0126 from Calbiochem (San Diego, CA, USA); TGF-βRI inhibitor SD 208, JNK inhibitor SP 600125 and IKK-2 inhibitor TPCA-1 from Tocris Bioscience (Bristol, UK); LEAF irrelevant control mAbs from Biolegend (San Diego, CA, USA); Complete Protease Inhibitor Cocktail and PhosSTOP phosphatase inhibitor from Roche (Basel, Switzerland); nitrocellulose membranes and chemiluminescence (ECL) blotting analysis system from GE Healthcare (Zurich, Switzerland); phospho-Akt (Ser473), phospho-Smad2 (Ser465/467), phospho-p38 MAPK (Thr180/Tyr182), phospho-NF-κB p65 (Ser536), phospho-IκB-α (Ser32), β-actin and BSA for Western blots from Cell Signaling (Danvers, MA, USA); TMB ELISA substrate from Abcam (Cambridge, UK); EZ4U cell proliferation assay from Biomedica (Vienna, Austria).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Interleukin-6 and Type-I Collagen Production by Systemic Sclerosis Fibroblasts Are Differentially Regulated by Interleukin-17A in the Presence of Transforming Growth Factor-Beta 1

doi: 10.3389/fimmu.2018.01865

Figure Lengend Snippet: Proposed model linking IL-17A, TGF-β, and IL-6 in the context of extracellular matrix deposition and Th17 cell differentiation. Blue arrows: stimulatory signal; red arrows: inhibitory signal. The relevant references are reported in the discussion. For tissue inhibitor of metalloproteinases 1 (TIMP-1), we refer to Fineschi et al. .

Article Snippet: rhIL-17A, rhTGF-β, monoclonal mouse IgG1 TGF-β1, 2, 3 antibody, IL-6, MCP-1, MMP-1, IL-8, pro-collagen Iα1 and fibronectin ELISA DuoSet kits were from R&D Systems (Abingdon, UK); DMEM, PBS, glutamine, penicillin, streptomycin, trypsin, dispase, collagenase type I from Gibco (Paisley, UK); FCS from Biowest (Nuaillé, France); BSA, α-ketoglutaric acid, β-amino propionitrile, l -ascorbic acid, p38 MAPK inhibitor SB203580, and PI3K inhibitor LY294002 from Sigma (St. Louis, MO, USA); MEK1/2 inhibitor U-0126 from Calbiochem (San Diego, CA, USA); TGF-βRI inhibitor SD 208, JNK inhibitor SP 600125 and IKK-2 inhibitor TPCA-1 from Tocris Bioscience (Bristol, UK); LEAF irrelevant control mAbs from Biolegend (San Diego, CA, USA); Complete Protease Inhibitor Cocktail and PhosSTOP phosphatase inhibitor from Roche (Basel, Switzerland); nitrocellulose membranes and chemiluminescence (ECL) blotting analysis system from GE Healthcare (Zurich, Switzerland); phospho-Akt (Ser473), phospho-Smad2 (Ser465/467), phospho-p38 MAPK (Thr180/Tyr182), phospho-NF-κB p65 (Ser536), phospho-IκB-α (Ser32), β-actin and BSA for Western blots from Cell Signaling (Danvers, MA, USA); TMB ELISA substrate from Abcam (Cambridge, UK); EZ4U cell proliferation assay from Biomedica (Vienna, Austria).

Techniques: Cell Differentiation

Journal: The Journal of Experimental Medicine

Article Title: Allergen-responsive CD4 + CD25 + Regulatory T Cells in Children who Have Outgrown Cow's Milk Allergy

doi: 10.1084/jem.20032121

Figure Lengend Snippet: Generation of the immunosuppressive cytokines TGF-β1-LAP and IL-10 by PBMCs purified from blood of allergic or tolerant children before and 1 wk after in vivo challenge with cow's milk. (A) Production of TGF-β1-LAP determined in supernatants from PBMCs stimulated for 96 h with β-LG (1 mg/ml) or PHA (50 μg/ml) by subtracting the TGF-β1-LAP concentration in unstimulated wells. (B) Production of IL-10 under the same culture conditions as in A. Student's t test was used for statistical analysis.

Article Snippet: A biotinylated mouse mAb to human TGF-β1-LAP (125 ng/ml, clone 27240; RD System Europe) was added to the wells and incubated overnight at 4°C.

Techniques: Purification, In Vivo, Concentration Assay