Journal: Molecular Medicine Reports

Article Title: Next-generation sequencing of miRNAs and lncRNAs from rat femur and tibia under mechanical stress

doi: 10.3892/mmr.2021.12200

Figure Lengend Snippet: Primers (mRNAs) used in reverse transcription-quantitative PCR.

Article Snippet: Anti-TGFβR1 , BIOSS , bs-0638R , Rabbit , 1:1,000.

Techniques: Sequencing

Journal: Molecular Medicine Reports

Article Title: Next-generation sequencing of miRNAs and lncRNAs from rat femur and tibia under mechanical stress

doi: 10.3892/mmr.2021.12200

Figure Lengend Snippet: Antibodies used in western blotting.

Article Snippet: Anti-TGFβR1 , BIOSS , bs-0638R , Rabbit , 1:1,000.

Techniques: Western Blot

Journal: Molecular Medicine Reports

Article Title: Next-generation sequencing of miRNAs and lncRNAs from rat femur and tibia under mechanical stress

doi: 10.3892/mmr.2021.12200

Figure Lengend Snippet: Measurement of biological indices in a rat exercise model. The mRNA expression of (A) Runx2 , (B) PPAR γ, (C) Smad2 , (D) TGF β 1 and (E) TGF β R1 in the femur and tibia of rats in both exercise and control groups was detected by reverse transcription-quantitative PCR. (F) The protein expression of Runx2, PPARγ, Smad2, TGFβ1 and TGFβR1 in the femur and tibia of rats in both an exercise and control group was detected by western blotting. Data are presented as the mean ± SD (n≥3), *P<0.05, **P<0.01 (t-test). Runx2 , runt-related transcription factor 2; PPAR γ, peroxisome proliferator-activated receptor γ; TGF β R1 , TGFβ receptor 1.

Article Snippet: Anti-TGFβR1 , BIOSS , bs-0638R , Rabbit , 1:1,000.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal:

Article Title: Antisense Oligonucleotides Against Thrombospondin-1 Inhibit Activation of TGF-? in Fibrotic Renal Disease in the Rat in Vivo

doi:

Figure Lengend Snippet: Transfer of phosphorothioate ODN against TSP1 decreases activation, but not expression of TGF-β in nephritic glomeruli. In all figures treated kidneys (solid bars) were compared to nontreated control kidneys (open bars). Glomerular TGF-β1 or TGF-β2 protein (by brown immunostaining) was not changed in any group of nephritic rats (A and B). In agreement, equal levels of total TGF-β levels were determined using the PAI-1 luciferase assay. Active TGF-β levels were significantly reduced in the antisense-treated kidneys (C). Additionally, active TGF-β in nephritic glomeruli was determined by an antibody specifically recognizing the active form of TGF-β1 (D, gray cytoplasmic staining) and by an antibody specific for the phosphorylated form of the TGF-β signal-transduction molecule Smad 2/3 (H, black nuclear staining; arrowheads indicate examples for P-Smad2/3-positive nuclei). Inhibition of TSP1 expression after antisense ODN therapy but not scrambled ODN therapy was associated with a markedly decreased glomerular TGF-β activity in the left kidney as reflected by immunostaining for active TGF-β1 (E, evaluated by scoring system; F, evaluated by computerized morphometry) and by a marked reduction of glomerular cells showing positive nuclei for the TGF-β signaling molecule phospho-Smad2/3 (G). * marks significant differences (P < 0.01) of antisense groups versus the control (scrambled) group.

Article Snippet: 25 Immunostaining for matrix proteins was conducted with polyclonal antibodies to collagen I (rabbit anti-rat collagen I; Quartett, Berlin, Germany), 25 collagen IV (goat anti-human/bovine collagen IV; Southern Biotechnology Associates, Inc., Birmingham, AL), 25 active TGF-β1 (chicken anti-human active TGF-β1; R&D systems, Wiesbaden-Nordenstadt, Germany), 28 TGF-β1 (rabbit anti-human TGF-β1; Santa Cruz Biotechnology Inc., Santa Cruz, CA), 24 TGF-β2 (rabbit anti-human TGF-β2; Santa Cruz Biotechnology), 24 and a murine IgG 1 mAb against TSP1 (Dunn, Labortechnik GmbH, Asbach, Germany), 25 P-Smad2/3 (rabbit anti-human Smad2 peptide phosphorylated at Ser-433/435; Santa Cruz).

Techniques: Activation Assay, Expressing, Immunostaining, Luciferase, Staining, Transduction, Inhibition, Activity Assay

Journal: Frontiers in Oncology

Article Title: Identification of a core EMT signature that separates basal-like breast cancers into partial- and post-EMT subtypes

doi: 10.3389/fonc.2023.1249895

Figure Lengend Snippet: Generation and RNA-sequencing of epithelial and mesenchymal (post-EMT) subpopulations of mammary epithelial cell-derived EMT models. (A) EpCAM-positive and EpCAM-negative subpopulations of HMLE cells were separated using anti-EpCAM-conjugated capture beads. The cell fractions were flow cytometry-sorted into an epithelial (CD24 High /CD44 Low /EpCAM High ) and a mesenchymal (CD24 Low /CD44 High /EpCAM Low ) population. (B) MCF10A cells were left untreated or treated with 10 ng/ml TGF-β for 8 days. (C) D492 and D492M were cultured in 2D monolayers. (D) Flow chart of the RNA-Sequencing experiments. (E) Heatmap showing the fold change (post-EMT versus epithelial) of selected EMT marker genes, EMT transcription factors, and housekeeping genes. (F) Positively enriched Hallmarks with the post-EMT cells identified by Gene Set Enrichment Analysis (GSEA). The data from the three cell models are analyzed separately or combined (right panel). Ep, Epithelial; Mes, Mesenchymal; TFs, Transcription factors.

Article Snippet: To induce EMT, MCF10A cells were treated with 10 ng/ml recombinant human TGF-β for 8 days (Bio-Techne, CFQ-331134).

Techniques: RNA Sequencing Assay, Derivative Assay, Flow Cytometry, Cell Culture, Marker

Journal: Frontiers in Oncology

Article Title: Identification of a core EMT signature that separates basal-like breast cancers into partial- and post-EMT subtypes

doi: 10.3389/fonc.2023.1249895

Figure Lengend Snippet: ZEB1 acts as a transcriptional activator and is required for acquisition of post-EMT gene expression pattern. (A, B) SNAI1 , TWIST1 , ZEB1 , and ZEB2 expression in basal-like patients from the TCGA (A) post-EMT cluster 1 (C1) and partial-EMT cluster 5 (C5) and METABRIC post-EMT cluster 1 (C1) and partial-EMT cluster 3 (C3). Expression values are log2(RPKM+1). (C) and (D) Correlation analysis for individual genes withing the 265-gene mammary EMT signature with ZEB1 and SNAI1 in TCGA (C) and METABRIC (D) . (E) Hierarchical clustering of the mammary EMT signature in wild type and ZEB1 knockout MCF10A cells left untreated of treated with TGF-β. Genes found to be up- or downregulated in the EMT cell lines models (Up/Down), gene clusters from the breast cancer cell lines (GC-CL) clustering, and gene clusters from the TCGA (GC-TCGA) clustering are shown as side banners on the left side of the heatmap. Expression values are row-based z-normalized counts per million (CPM). (F) Hierarchical clustering of the mammary EMT signature in HMLER ZEB1 knock out cells (ZEB1 ko), ZEB1 ko cells that overexpress SNAI1 (SNAI1 oe), and SNAI1-expressing cells that are rescued by ectopic expression of ZEB1 (ZEB1 rescue). * P<0.05; *** P<0.001; **** P<0.0001. ns, not significant.

Article Snippet: To induce EMT, MCF10A cells were treated with 10 ng/ml recombinant human TGF-β for 8 days (Bio-Techne, CFQ-331134).

Techniques: Expressing, Knock-Out

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