Journal: PeerJ

Article Title: CircHIPK3 regulates cardiac fibroblast proliferation, migration and phenotypic switching through the miR-152-3p/TGF-β2 axis under hypoxia

doi: 10.7717/peerj.9796

Figure Lengend Snippet: To determine the role of miR-152-3p in CFs, CFs were transfected with the miR-152-3p mimic or inhibitor. (A–G and J–P) Representative images of EdU-stained CFs from different groups. Quantification of EdU+ cells presented as the % EdU-positive cells and Hoechst-stained nuclei. The orange color is EdU-positive cells, and the blue color is nuclei stained by Hoechst 33342. Scale bar = 20 μm. n = 10. * P < 0.05, vs. MNC group or INC group. (H, I and Q, R) The protein expression levels of Col I and Col III were measured by western blot analysis. n = 3. * P < 0.05, vs. MNC group or INC group. (S) Relative expression of miR-152-3p in CFs after transfection with miR-152-3p mimic or inhibitor was examined by RT-qPCR analysis. n = 3. * P < 0.05, vs. hypoxia group. (T) TGF-β2 was predicted as a target gene of miR-152-3p using the TargetScan database. HEK-293T cells were cotransfected with RLuc-TGF-β2-WT or RLuc-TGF-β2-Mut and miR-152-3p mimics or NC mimic. Luciferase activity was detected using the dual luciferase reporter assay at 48 h post-transfection. n = 3. * P < 0.05, vs. TGF-β2-WT + NC group. # P < 0.05, vs. TGF-β2-WT + miR-152-3p group. (U–X) The protein expression level of TGF-β2 was measured by western blot analysis. n = 3. * P < 0.05, vs. MNC group or INC group.

Article Snippet: The primary anti-bodies: Anti-Collagen I (Cell Signaling Technology, 91144, dilution: 1:1,000), Anti-Collagen III (Abcam, ab7778, dilution: 1:7,000), Anti-α-SMA (Cell Signaling Technology, 19245, dilution: 1:1,000), Anti-p-Smad2 (Cell Signaling Technology, 18338, dilution: 1:1,000), Anti-p-Smad3 (Cell Signaling Technology, 9520, dilution: 1:1,000), Anti-TGF-β2 (Bioss, bs-20412R, dilution: 1:1,000) and Anti-β-actin Antibody (Abcam, ab8227, dilution: 1:1,000), were used.

Techniques: Transfection, Staining, Expressing, Western Blot, Quantitative RT-PCR, Luciferase, Activity Assay, Reporter Assay

Journal: PeerJ

Article Title: CircHIPK3 regulates cardiac fibroblast proliferation, migration and phenotypic switching through the miR-152-3p/TGF-β2 axis under hypoxia

doi: 10.7717/peerj.9796

Figure Lengend Snippet: (A–E) The cell cycle was investigated by a cell cycle assay in cardiac fibroblasts. The results represent the percentage of cells in G1 and S + G2/M phases for the indicated conditions. n = 3. * P < 0.05, # P < 0.05. (F–J) Wound scratch assay for the different groups. The average sizes of the gaps were measured at 48 h. Scale bar = 200 μm. n = 5. * P < 0.05, # P < 0.05. (K and L) The protein expression levels of Col I, Col III, TGF-β2, p-Smad2 and p-Smad3 were measured by western blot analysis. n = 3. * P < 0.05, # P < 0.05. * P < 0.05, vs. hypoxia group, # P < 0.05, vs. si-circHIPK3 group.

Article Snippet: The primary anti-bodies: Anti-Collagen I (Cell Signaling Technology, 91144, dilution: 1:1,000), Anti-Collagen III (Abcam, ab7778, dilution: 1:7,000), Anti-α-SMA (Cell Signaling Technology, 19245, dilution: 1:1,000), Anti-p-Smad2 (Cell Signaling Technology, 18338, dilution: 1:1,000), Anti-p-Smad3 (Cell Signaling Technology, 9520, dilution: 1:1,000), Anti-TGF-β2 (Bioss, bs-20412R, dilution: 1:1,000) and Anti-β-actin Antibody (Abcam, ab8227, dilution: 1:1,000), were used.

Techniques: Cell Cycle Assay, Wound Healing Assay, Expressing, Western Blot

Journal: Journal of Veterinary Science

Article Title: In vitro effects of meloxicam on the number, Foxp3 expression, production of selected cytokines, and apoptosis of bovine CD25+CD4+ and CD25-CD4+ cells

doi: 10.4142/jvs.2013.14.2.125

Figure Lengend Snippet: The effect of MEL on IL-10 (A), TGF-β (B), and IFN-γ (C) production. The results are expressed as a percentage of CD25 high CD4+, CD25 low CD4+, and CD25-CD4+ cells expressing IL-10, TGF-β, or IFN-γ. Typical cytograms (D) illustrating IFN-γ expression in particular lymphocyte subpopulations are shown. Results are presented as the mean ± SE (n = 18) of three independent experiments (each one using six animals). * p < 0.01, MEL-treated cells versus the control cells.

Article Snippet: After extracellular staining (as described above) and fixation (200 μL 2% paraformaldehyde in Dulbecco's PBS per sample for 15 min on ice; both reagents were purchased from Sigma-Aldrich, USA), the cells were permeabilized with 2 mL SAP buffer [0.1% (w/v) saponin and 0.05% (w/v) NaN 3 in Hanks' balanced salt solution (HBSS; all from Sigma-Aldrich, USA)] and stained with APC (allophycocyanin) -conjugated mouse anti-TGF-β mAb (1 : 20, 1D11, IgG1, this antibody reacts with human, mouse, and bovine TGF-β1 and TGF-β2; R&D Systems, USA) for 45 min at RT in the dark.

Techniques: Expressing, Control

Journal: International Journal of Ophthalmology

Article Title: Pirfenidone suppresses the abnormal activation of human Müller cells after platelet-derived growth factor-BB stimulation

doi: 10.18240/ijo.2019.07.04

Figure Lengend Snippet: A: The cells of the normal group and the model group were respectively treated by 0, 0.2 and 0.3 mg/mL pirfenidone. The relative mRNA levels of TGF-β1, TGF-β2 and PEDF were determined by real-time PCR. Results are shown as indicated. The expression levels of TGF-β1, TGF-β2 and PEDF under the indicated treatment of pirfenidone were detected by Western blotting (B) and immunofluorescence (C). Green fluorescence, for the indicated proteins; blue fluorescence for the nucleus. aP<0.05 vs normal+0 mg/mL pirfenidone group, bP<0.01 vs normal+0 mg/mL pirfenidone group, cP<0.0001 vs normal+0 mg/mL pirfenidone group; dP<0.05 vs model+0 mg/mL pirfenidone group, eP<0.01 vs model+0 mg/mL pirfenidone group, fP<0.0001 vs model+0 mg/mL pirfenidone group; gP<0.05 vs model+0.2 mg/mL pirfenidone group, hP<0.01 vs model+0.2 mg/mL pirfenidone group, iP<0.0001 vs model+0.2 mg/mL pirfenidone group.

Article Snippet: The expression of TGF-β1, TGF-β2, PEDF, and β-actin was respectively tested by anti-TGF-β1 antibody (Boster, USA, {"type":"entrez-nucleotide","attrs":{"text":"A04630","term_id":"411029","term_text":"A04630"}} A04630 ), anti-TGF-β2 antibody (Boster, USA, {"type":"entrez-protein","attrs":{"text":"A00892","term_id":"77211","term_text":"pir||A00892"}} A00892 ), anti-PEDF antibody (Abcam, USA, ab233120) and anti-β-actin antibody (Invitrogen, USA, PA1183).

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

Journal:

Article Title: Characterisation of corneal fibrotic wound repair at the LASIK flap margin

doi:

Figure Lengend Snippet: Immunohistochemistry of the LASIK flap edge (arrows) demonstrating the expression of TGF-β1 (A; red) and TGF-β2 (B; red) at 4 days, and TGF-β2 (C; red), TGF-βRII (D; red), and CTGF (E; red) at 2 weeks post-surgery. All sections are counterstained for nuclei (blue), and oriented with the corneal periphery to the left. (C–E) Represent serial cross sections. Bar indicates 100 μm.

Article Snippet: To detect selected growth factors and receptors, sections were incubated overnight with one of the following four primary antibodies: goat anti-human transforming growth factor β1 (TGF-β1; 1:100; Santa Cruz Biotechnology, CA, USA); mouse anti-human transforming growth factor β2 (TGF-β2; clone 8607.211; 1:75; R&D Systems, Minneapolis, MN, USA); goat anti-human transforming growth factor β receptor II (TGF-βRII; 1:100; Santa Cruz Biotechnology, CA, USA); and goat anti-human connective tissue growth factor (CTGF; 1:12500; a generous gift from Dr Gary Grotendorst).

Techniques: Immunohistochemistry, Expressing

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: TGF-β levels are elevated in melioidosis patients. (A) On admission, TGF-β levels in plasma of patients with culture-proven melioidosis (n = 33) were elevated compared to healthy controls (n = 30). (B) Plasma levels of TGF-β decreased in melioidosis patients 2 weeks after successful antibiotic treatment (n = 6). *, P < 0.05.

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques:

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: Increased expression of TGF-β in lung tissue of wild-type mice infected with B. pseudomallei. Elevated pulmonary TGF-β levels were observed in mice (n = 8) 72 h after intranasal infection with 7.5 × 102 CFU of B. pseudomallei compared to 24 h after infection. ***, P < 0.001.

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques: Expressing, Infection

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: Decreased expression of phosphorylated Smad2 in mice receiving anti-TGF-β treatment. TGF-β signaling was evaluated by quantifying the total levels of Smad2 and Smad3 and their phosphorylation in lung tissue by Western blot 48 h after intranasal infection with B. pseudomallei. The left 4 lanes show the expression of Smad2 (A) and Smad3 (B) in lung tissue of control mice and the right 4 lanes of mice that received anti-TGF-β treatment (2 × 200 μg TGFβ antibody). The bar graph shows the ratio of expressed phosphorylated Smad/total Smad corrected for actin loading levels. (C) A significant decrease in p-Smad2 is demonstrated in mice that were treated with anti-TGF-β antibody. *, P < 0.05.

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques: Expressing, Western Blot, Infection

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: Cytokine profile during experimental melioidosis in mice treated with control antibody or anti-TGF-β a

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques:

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: Less organ damage in mice receiving anti-TGF-β treatment. Levels of (A) BUN, (B) creatinine, (C) AST, and (D) ALT were measured in B. pseudomallei-infected mice (n = 8/group) that were treated with anti-TGF-β antibody and their controls at 72 h as parameters for organ damage. Expression of AST and BUN were decreased, demonstrating less organ damage. *, P < 0.05. Data shown from single independent experiments.

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques: Infection, Expressing

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: Anti-TGF-β treatment results in diminished bacterial growth during experimental melioidosis. Mice (n = 8/group) were treated with anti-TGF-β antibody or control IgG antibody and inoculated with 7.5 × 102 CFU of B. pseudomallei intranasally. Bacterial numbers were quantified 72 h postinfection. Less growth was seen in the lungs (A) and spleens (B) of mice treated with anti-TGF-β antibody. *, P < 0.05. Data shown from single independent experiments.

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques:

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: No influence on survival of anti-TGF-β treatment during murine melioidosis. Mice (n = 12/group) received either 2× 200 μg control antibody (black squares) or 2× 200 μg anti-TGF-β antibody (white squares) and were infected with 7.5 × 102 CFU, a lethal dose, of B. pseudomallei intranasally, after which they were followed until death. Mortality was assessed every 4 h. No difference in survival was seen.

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques: Infection

Journal: Journal of Translational Autoimmunity

Article Title: Antiphospholipid antibodies induce proinflammatory and procoagulant pathways in endothelial cells

doi: 10.1016/j.jtauto.2023.100202

Figure Lengend Snippet: Methodology, results and validation of RNA-seq in HUVECs treated with IgG and β2GPI isolated from APS patients A. Schematic representation of HUVECs' treatment and RNA sequencing. B. Transcriptomics and Bioinformatics analysis uncovered 395 genes that were activated during the first 6 h of treatment. Inflammatory and immune response genes as well as known APS-markers including genes that encode for transcription factors (e.g NF-κB, SMAD, YAP1), chemokines (e.g IL-8), growth factors (TGFβ-2), adhesion molecules (e.g E-Selectin, VCAM-1, ICAM-1), signal transduction molecules (e.g MAP2K3, MAP3K5) are upregulated. C. Verification of the results from transcriptomics analyses using gene-specific qPCR assays for selected genes (left panel). High correlation of the RNA-seq/qPCR results is detected (right panel).

Article Snippet: Coverslips were incubated overnight at 4 °C with primary antibodies against IL-6 (5 μg/ml, CSB-PA06757A0Rb, Cusabio), IL-8 (5 μg/ml, CSB-MA083271A0m, Cusabio), NF-κB1 (5 μg/ml, CSB-PA190132, Cusabio), TGF-β2 (5 μg/ml, CSB- PA07319A0Rb, Cusabio), Tissue Factor (5 μg/ml, 4509, American Diagnostica), ICAM-1 (5 μg/ml, AF796, R&D Systems), VCAM-1 (4 μg/ml, sc-18854, Santa Cruz Biotechnology), E-selectin (4 μg/ml, sc-271267, Santa Cruz Biotechnology), P-selectin (4 μg/ml,sc-137054, Santa Cruz Biotechnology) and TGFR1 (5 μg/ml,CSB- PA023451LA01HU, Cusabio).

Techniques: Biomarker Discovery, RNA Sequencing, Isolation, Transduction

Journal: Journal of Translational Autoimmunity

Article Title: Antiphospholipid antibodies induce proinflammatory and procoagulant pathways in endothelial cells

doi: 10.1016/j.jtauto.2023.100202

Figure Lengend Snippet: RNA-seq intersection with published data and gene ontology analysis. A. Snapshots from Genome Browser show that sequencing reads (signal) covering exon sequences of NFKB1, SELE, VCAM1, BMP2, IL7R are increased in treated samples compared to control samples whereas signals derived from ACTB exons, remain unaffected B. Direct intersection of previously published microarray results with our RNA-seq results revealed 31 common upregulated genes (2 B)·C-D KEGG pathway and Gene Ontology analysis (Biological Process) for the upregulated genes reveals that signal transduction pathways such as TNF-signaling, TGF-β signaling, MAPK38- signaling and Hippo pathway operate simultaneously upon HUVECs activation.

Article Snippet: Coverslips were incubated overnight at 4 °C with primary antibodies against IL-6 (5 μg/ml, CSB-PA06757A0Rb, Cusabio), IL-8 (5 μg/ml, CSB-MA083271A0m, Cusabio), NF-κB1 (5 μg/ml, CSB-PA190132, Cusabio), TGF-β2 (5 μg/ml, CSB- PA07319A0Rb, Cusabio), Tissue Factor (5 μg/ml, 4509, American Diagnostica), ICAM-1 (5 μg/ml, AF796, R&D Systems), VCAM-1 (4 μg/ml, sc-18854, Santa Cruz Biotechnology), E-selectin (4 μg/ml, sc-271267, Santa Cruz Biotechnology), P-selectin (4 μg/ml,sc-137054, Santa Cruz Biotechnology) and TGFR1 (5 μg/ml,CSB- PA023451LA01HU, Cusabio).

Techniques: RNA Sequencing, Sequencing, Control, Derivative Assay, Microarray, Transduction, Activation Assay

Journal: Journal of Translational Autoimmunity

Article Title: Antiphospholipid antibodies induce proinflammatory and procoagulant pathways in endothelial cells

doi: 10.1016/j.jtauto.2023.100202

Figure Lengend Snippet: Immunofluorescent antibody staining in placenta biopsies from APS patients and healthy women. A-K Placenta biopsies derived from APS patients as well as Healthy Donors show increased signal intensity for IL-6, IL-8, NF-κB1, ICAM1, VCAM-1, E-selectin, P-selectin, TGF-β2, and TGFR1 (5A-5D, 5F-5J). Slight difference in fluorescence intensity between HD and APS patient was observed for Tissue Factor (5E). Increased signal intensity was observed as well for the TNF-α molecule in the APS placenta biopsies (5 K).

Article Snippet: Coverslips were incubated overnight at 4 °C with primary antibodies against IL-6 (5 μg/ml, CSB-PA06757A0Rb, Cusabio), IL-8 (5 μg/ml, CSB-MA083271A0m, Cusabio), NF-κB1 (5 μg/ml, CSB-PA190132, Cusabio), TGF-β2 (5 μg/ml, CSB- PA07319A0Rb, Cusabio), Tissue Factor (5 μg/ml, 4509, American Diagnostica), ICAM-1 (5 μg/ml, AF796, R&D Systems), VCAM-1 (4 μg/ml, sc-18854, Santa Cruz Biotechnology), E-selectin (4 μg/ml, sc-271267, Santa Cruz Biotechnology), P-selectin (4 μg/ml,sc-137054, Santa Cruz Biotechnology) and TGFR1 (5 μg/ml,CSB- PA023451LA01HU, Cusabio).

Techniques: Staining, Derivative Assay, Fluorescence