Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: IL-9 and Th9–associated markers are elevated in TS patients. A Representative IHC pictures showing PU.1, IRF4, and IL-9 expression in TS patients’ tracheal tissues. IL-9 staining was single-color IHC, CD4/IL-9 co-localization was not assessed. B Quantitative analysis of IHC-positive regions showing PU.1, IRF4, and IL-9 expression in TS tissues ( n = 3). C qRT-PCR analysis of TS tissues and blood samples ( n = 10) for cytokines and fibrosis markers. D , E WB analysis of TS tissues for IFN-γ, fibrosis indicators, IL-9, and IL-4. F ELISA data showing collagen levels in the blood of TS patients. To determine statistical significance, unpaired t-tests were used. In comparison to controls, * P < 0.05, ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: Expressing, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: Th1/Th2 cytokine balance is skewed in patients with TS. A Method for gating peripheral blood samples and BALF CD3 + CD4 + T cells. Representative FCM plots showing the populations of IL-9 + , IL-4 + , and IFN-γ + CD4 + T cells in the control and TS groups are shown in ( B , D , and F ). C , E , and G Quantitative evaluation of IL-9, IL-4, and IFN-γ expression in CD4 + T cells ( n = 10). Unpaired t -tests were used to determine statistical significance. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. controls. TS: traumatic tracheal stenosis; BALF: bronchoalveolar lavage fluid; FCM: flow cytometry

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: Control, Expressing, Flow Cytometry

Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: IL-9 promotes fibroblast proliferation and activation via the TGF-β1 pathway in vitro. A Relative growth rate of HTFs treated with IL-9, IL-4 + TGF-β1, anti-IL-9, or SB at 24 h and 48 h, assessed by CCK-8 assay ( n = 5). B and C FCM histograms showing collagen I expression in CD3 − CD4 − HTFs following individual stimulation with IL-9, IL-4 + TGF-β1, anti-IL-9, or SB treatment. D and E ELISA and qRT-PCR analysis of collagen I secretion and COL1A1 mRNA expression in HTFs treated as above ( n = 5). F and G WB analysis of collagen III, collagen I, and α-SMA protein expression in HTFs following the same treatments ( n = 3). Statistical comparisons were performed using one-way ANOVA followed by Kruskal–Wallis or Newman–Keuls post hoc analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 vs. IL-9 group. HTF: human tracheal fibroblast; SB: SB-431,542; FCM: flow cytometry; ELISA: enzyme-linked immunosorbent assay; qRT-PCR: quantitative real-time polymerase chain reaction; WB: western blot; ANOVA: analysis of variance

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: Activation Assay, In Vitro, CCK-8 Assay, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control, Flow Cytometry, Real-time Polymerase Chain Reaction, Western Blot

Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: IL-9 blockade suppresses TGF-β1-induced fibroblast activation and collagen I production in vitro. A FCM histograms depict collagen I expression in CD3 − CD4 − HTFs subjected to individually applied IL-9, IL-4, TGF-β1, anti-IL-9, anti-IL-4, anti-TGF-β1, IL-9 + TGF-β1, or anti-IL-9 + TGF-β1 treatment. B Quantitative FCM analysis of collagen I levels in CD3 − CD4 − HTFs treated as above ( n = 5). C – E qRT-PCR analysis of COL3A1, COL1A1, and α-SMA mRNA expression in HTFs treated with the aforementioned agents ( n = 5). F and G WB analysis of collagen III, collagen I, and α-SMA protein expression in HTFs treated as above ( n = 3). H and I IF analysis of α-SMA and collagen I expression in HTFs following treatment with the conditions described above ( n = 3). Statistical comparisons were performed using one-way ANOVA followed by Kruskal–Wallis or Newman–Keuls post hoc analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 vs. IL-9 group. FCM: flow cytometry; qRT-PCR: quantitative real-time polymerase chain reaction; WB: western blot; IF: immunofluorescence; ANOVA: analysis of variance

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: Activation Assay, In Vitro, Expressing, Quantitative RT-PCR, Control, Flow Cytometry, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence

Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: IL-9 modulates Th1/Th2 cytokine profiles via TGF-β1 signaling in vitro . A , C , E , and G FCM scatter plots illustrate the distribution patterns of IL-9 + CD4 + , IL-4 + CD4 + , TGF-β1 + CD4 + , and IFN-γ + CD4 + T cell subsets across various treatment groups, including control, IL-9 alone, IL-4 + TGF-β1 co-stimulation, anti-IL-9 antibody alone, and SB alone. B , D , F , and H Quantification of FCM data for IL-9 + , IL-4 + , TGF-β1 + , and IFN-γ + CD4 + T cells in the treatment groups mentioned above ( n = 5). I and J ELISA and qRT-PCR analyses assessing protein and mRNA levels of IL-9, IL-4, IL-13, and IFN-γ following treatment with IL-9 or IL-4 + TGF-β1 co-stimulation, anti-IL-9 antibody, or SB ( n = 5). K and L WB analysis of IL-9, IL-4, IL-13, TGF-β1, and IFN-γ protein expression in the different treatment groups ( n = 3). Statistical comparisons were performed using one-way ANOVA followed by Kruskal–Wallis or Newman–Keuls post hoc analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 vs. IL-9 group. FCM: flow cytometry; ELISA: enzyme-linked immunosorbent assay; qRT-PCR: quantitative real-time polymerase chain reaction; SB: SB-431,542; WB: western blot; ANOVA: analysis of variance

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: In Vitro, Control, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction, Western Blot

Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: Th9 blockers inhibit collagen secretion by fibroblasts via the TGF-β1/SMAD2/3 pathway. A Representative WB results display the levels of phosphorylated SMAD2/3 (p-SMAD2/3) and total SMAD2/3 proteins in samples from the control, IL-9, IL-4 + TGF-β1, anti-IL-9, and SB treatment groups. B Quantification of p-SMAD2/3 and SMAD2/3 protein expression following treatment as described in ( A ) ( n = 3). C Representative WB results show the levels of p-SMAD2/3 and SMAD2/3 proteins in samples from the control, IL-9, IL-4, TGF-β1, anti-IL-9, anti-IL-4, anti-TGF-β1, IL-9 + TGF-β1, and anti-IL-9 + TGF-β1 treatment groups. D and E Quantification of p-SMAD2/3 and SMAD2/3 protein expression following treatments as described in ( C ) ( n = 3). Statistical comparisons were performed using one-way ANOVA followed by Kruskal–Wallis or Newman–Keuls post hoc analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 vs. IL-9 group. WB: western blot; SB: SB-431,542; ANOVA: analysis of variance

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: Control, Expressing, Western Blot

Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: IL-9 modulates the cytokine microenvironment in TS airways via the TGF-β1/SMAD2/3 pathway. A Tracheal tissues from the control, TS, TS + IL-9, TS + anti-IL-9, and TS + SB groups were immunohistochemically stained with IL-9, IL-4, IL-13, TGF-β1, and IFN-γ. IL-9 staining was single-color IHC, CD4/IL-9 co-localization was not assessed. Scale bar = 200 μm ( n = 3 per group). B The observed expression patterns were confirmed by quantitative analysis of the cytokine-positive region (%). Statistical comparisons were performed using one-way ANOVA followed by Kruskal–Wallis or Newman–Keuls post hoc analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 vs. TS group. TS: traumatic tracheal stenosis; SB: SB-431,542; ANOVA: analysis of variance

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: Control, Staining, Expressing

Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: IL-9 modulates IL-4, IFN-γ, and TGF-β1 in CD4 + T cells and reduces airway collagen I. A – F Representative FCM plots showing the percentages of CD3 − CD4 − collagen I + , IL-9 + , IL-4 + , IFN-γ + , and TGF-β1 + CD4 + T cells in various experimental groups ( n = 6). Statistical comparisons were performed using one-way ANOVA followed by Kruskal–Wallis or Newman–Keuls post hoc analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 vs. TS group. FCM: flow cytometry; SB: SB-431,542; ANOVA: analysis of variance

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: Control, Flow Cytometry

Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: IL-9 modulates the cytokine microenvironment in TS airways via the TGF-β1/SMAD2/3 pathway. A and B WB analysis was performed to examine the expression of fibrosis-related proteins, including collagen III, collagen I, phosphorylated SMAD2/3, total SMAD2/3, and α-SMA, as well as cytokines TGF-β1, IL-9, IL-4, IL-13, and IFN-γ, in tracheal tissues from control, TS, TS + IL-9, TS + anti-IL-9, and TS + SB groups ( n = 3). C qRT-PCR (tissue and blood) was used to assess the mRNA expression levels of IL-9, IRF4, PU.1, IL-4, IL-13, IFN-γ, COL3A1, and COL1A1 in the same experimental groups ( n = 6). D ELISA (BALF and blood) was used to assess the protein expression levels of IL-9, IL-4, IL-13, and IFN-γ in the same experimental groups ( n = 6). Statistical comparisons were performed using one-way ANOVA followed by Kruskal–Wallis or Newman–Keuls post hoc analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 vs. TS group. TS: traumatic tracheal stenosis; WB: western blot; SB: SB-431,542; qRT-PCR: quantitative real-time polymerase chain reaction; ELISA: enzyme-linked immunosorbent assay; bronchoalveolar lavage fluid; ANOVA: analysis of variance

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: Expressing, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Real-time Polymerase Chain Reaction

Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: Proteomic analysis of tracheal tissue from rats in five groups (rat TS model). Heatmap showing the patterns of protein abundance that differ across the five experimental groups. A Control, TS, TS + IL-9, TS + anti-IL-9, and TS + SB groups. B To identify important biological processes and functions, KEGG pathway and gene ontology enrichment analyses were conducted on differentially expressed proteins. C Grouped expression profiles showing patterns of grouped protein expression. TS: traumatic tracheal stenosis

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: Quantitative Proteomics, Control, Expressing

Journal: Respiratory Research

Article Title: MiR-493-5p inhibits Th9 cell differentiation in allergic asthma by targeting FOXO1

doi: 10.1186/s12931-022-02207-2

Figure Lengend Snippet: Down regulation of miR-493-5p and higher proportion of Th9 cells in murine asthma models. OVA was used to induce allergic airway inflammation in mice. The mice were euthanasia by dislocation of the cervical spine after the measurement of airway hyperreactivity, and then their lung tissues and BALF were collected (n = 8 for each group). A The IL-9 levels in lung tissue and BALF were measured by ELISA, and IL-9 level in lung tissue and BALF of asthma groups were significantly higher than the control groups. B The miR-493-5p expression in lung tissue and BALF were detected by RT-PCR, and the miR-493-5p expression were both obviously diminished in asthma groups compared with control groups. C MiR-493-5p, FOXO1, IRF4 and IL-9mRNA expression in lung tissues were detected by RT-PCR, and IL-9, FOXO1, IRF4 expression were all significantly upregulated in lung tissues from asthmatic mice. D , E The proportion of CD4 + T cells secreting IL-9 in PBMCs were analysed by flow cytometry, the results revealed that the higher proportion of CD4 + T cells secreting IL-9 in the PBMCs of the asthma mice . ** P < 0.01, compared to control group

Article Snippet: All samples were preserved at − 80 °C for subsequent assay of IL-9 by ELISA (Elabscience, E-EL-M0724c) according to the instructions.

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

Journal: Respiratory Research

Article Title: MiR-493-5p inhibits Th9 cell differentiation in allergic asthma by targeting FOXO1

doi: 10.1186/s12931-022-02207-2

Figure Lengend Snippet: MiR-493-5p negatively regulated the differentiation of Th9 cells in vitro. The CD4 + T cells were separated from widetype mice by density gradient centrifugation and magnetic beads. To investigate the effect of miR-493-5p on Th9 cells differentiation, both CD4 + T cells which were transfected with miR-493-5p mimic or treated with the inhibitor and their negative control (NC) were under the condition of inducing Th9 cell differentiation. A – C The mRNA level of IL-9, IRF4 and FOXO1 was detected by RT-qPCR. The data showed that the mRNA of FOXO1, IL-9 and IRF4 were downregulated in CD4 + T cells treated with miR-493-5p mimic and upregulated in CD4 + T cells treated with miR-493-5p inhibitor. D Groups. E – H The protein production of IL-9, IRF4 and FOXO1 was detected by western blot. The data showed that protein of FOXO1, IL-9 and IRF4 were downregulated in CD4 + T cells treated with miR-493-5p mimic and upregulated in CD4 + T cells treated with miR-493-5p inhibitor. I , J The proportion of CD4 + Th9 cells was analysed by flow cytometry and K IL-9 secretion in cell supernatant was measured by ELISA, and the data were all consistent with the proportion of Th9 cells. Data are from three experiments (mean and SD of three independent replicates). ** P < 0.01 compared to A group, # P < 0.05 compared to B group, ## P < 0.01 compared to B group

Article Snippet: All samples were preserved at − 80 °C for subsequent assay of IL-9 by ELISA (Elabscience, E-EL-M0724c) according to the instructions.

Techniques: In Vitro, Gradient Centrifugation, Magnetic Beads, Transfection, Negative Control, Cell Differentiation, Quantitative RT-PCR, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Central-European journal of immunology

Article Title: Targeting the serum marker interleukin 9 improves the underlying characterization and immune homeostasis in rheumatoid arthritis.

doi: 10.5114/ceji.2024.141695

Figure Lengend Snippet: Fig. 3. Interleukin 9 affected the severity of arthritis in CIA rats. A) ELISA for the detection of IL-9 levels. B) Rat arthritis score. C) Analysis of cartilage damage by saffron O-solid green staining. The descriptive statistics are pre- sented as mean ±SD. *p < 0.05 vs. the CIA group, n = 3

Article Snippet: We measured the levels of serum markers using ELISA with a specialized commercial human IL-9 ELISA Kit (CSB-E04642h, Cusabio, Wuhan, China), human Gal1 ELISA Kit (CSB-EL012882h, Cusabio), human anti-CII ELISA Kit (HB3371-Hu, Shanghai Hengyuan Biotechnology Co., Ltd, Shanghai, China), human sSR-A ELISA Kit (HB3370-Hu, Shanghai Hengyuan Biotechnology Co., Ltd), human anti-Sa ELISA Kit (ml382511V), human RF ELISA Kit (ml060678V), human ACPA ELISA Kit (ml233305V), rat IL-9 ELISA Kit (CSB-E07294r, Cusabio), rat Gal1 ELISA Kit (JL25762, Shanghai Jianglai Biotechnology Co., Ltd, Shanghai, China), rat anti-CII ELISA Kit (HB1364-Ra, Shanghai Hengyuan Biotechnology Co., Ltd), rat sSR-A ELISA Kit (HB1363-Ra, Shanghai Hengyuan Biotechnology Co., Ltd), rat anti-Sa ELISA Kit (ml202241V), rat RF ELISA Kit (ml059541V), and rat ACPA ELISA Kit (ml201458V).

Techniques: Enzyme-linked Immunosorbent Assay, Staining

Journal: Central-European journal of immunology

Article Title: Targeting the serum marker interleukin 9 improves the underlying characterization and immune homeostasis in rheumatoid arthritis.

doi: 10.5114/ceji.2024.141695

Figure Lengend Snippet: Fig. 3. Interleukin 9 affected the severity of arthritis in CIA rats. A) ELISA for the detection of IL-9 levels. B) Rat arthritis score. C) Analysis of cartilage damage by saffron O-solid green staining. The descriptive statistics are pre- sented as mean ±SD. *p < 0.05 vs. the CIA group, n = 3

Article Snippet: We measured the levels of serum markers using ELISA with a specialized commercial human IL-9 ELISA Kit (CSB-E04642h, Cusabio, Wuhan, China), human Gal1 ELISA Kit (CSB-EL012882h, Cusabio), human anti-CII ELISA Kit (HB3371-Hu, Shanghai Hengyuan Biotechnology Co., Ltd, Shanghai, China), human sSR-A ELISA Kit (HB3370-Hu, Shanghai Hengyuan Biotechnology Co., Ltd), human anti-Sa ELISA Kit (ml382511V), human RF ELISA Kit (ml060678V), human ACPA ELISA Kit (ml233305V), rat IL-9 ELISA Kit (CSB-E07294r, Cusabio), rat Gal1 ELISA Kit (JL25762, Shanghai Jianglai Biotechnology Co., Ltd, Shanghai, China), rat anti-CII ELISA Kit (HB1364-Ra, Shanghai Hengyuan Biotechnology Co., Ltd), rat sSR-A ELISA Kit (HB1363-Ra, Shanghai Hengyuan Biotechnology Co., Ltd), rat anti-Sa ELISA Kit (ml202241V), rat RF ELISA Kit (ml059541V), and rat ACPA ELISA Kit (ml201458V).

Techniques: Enzyme-linked Immunosorbent Assay, Staining

Journal:

Article Title: In vivo B 1 kinin-receptor upregulation. Evidence for involvement of protein kinases and nuclear factor ?B pathways

doi: 10.1038/sj.bjp.0702715

Figure Lengend Snippet: Effect of co-injection of the recombinant human IL-1 antagonist (IRA, A and B) or of IL-10 (C and D) on des-Arg9-BK-induced paw oedema in rats pre-treated with IL-1β (5 ng paw−1, 60 min beforehand; (A and C)) or with TNFα (5 ng paw−1, 30 min beforehand; (B and D)). Values represent the differences between volumes (in ml) of vehicle-injected (0.1 ml of PBS solution) and drug-injected paws. Each point represents the mean±s.e.mean of 4–6 rats. In some cases the error bars are hidden within the symbols. Significantly different from control values *P<0.05; **P<0.01 (A–C, Student's unpaired t-test) (D, ANOVA followed by Dunnett's test).

Article Snippet: Recombinant murine cytokines IL-1β (Lot BN091), TNFα (Lot CS184) and IL-10 (Lot BC110); anti-murine neutralizing antibodies anti-IL-1β (Lot B01D3), anti-TNFα (Lot CT101), anti-IL-6 (Lot BF 123), anti-IL-8 (Lot BB282) and human recombinant interleukin receptor antagonist (IRA) were obtained from R&D Systems Inc., Minneapolis, U.S.A.

Techniques: Injection, Recombinant, Control

Journal: Clinical and experimental immunology

Article Title: Targeting C3a/C5a receptors inhibits human mesangial cell proliferation and alleviates immunoglobulin A nephropathy in mice.

doi: 10.1111/cei.12961

Figure Lengend Snippet: Fig. 1. Cell proliferation and cytokine mRNA and protein expression in cultured human mesangial cells (HMCs). The HMCs were treated with blank medium or medium containing 100 lg/ml immunoglobulin (Ig)A, 100 lg/ml IgA 1 100 lM C3aR antagonist (C3aRA) or 100 lg/ml IgA 1 100 lM C5aR antagonist (C5aRA) for 48 h. The data are expressed as the mean 6 standard deviation (s.d.). (a) HMC proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (n 5 6 for each group). (b) Interleukin (IL)-6 (left row) and monocyte chemotactic protein 1 (MCP-1) (right row) gene expression levels relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by reverse transcription–quantitative polymerase chain reaction (RT–qPCR). (c) Western blot analysis of IL-6, MCP-1 and GAPDH protein expression. (d) Quantitative analysis for Western blot of IL-6 and MCP-1 protein expression. *P < 005, **P < 001, ***P < 0001 versus negative control.

Article Snippet: The membranes were blocked with 5% skimmed milk solution in 0 1% Tris-buffered saline Tween-20 (Dingguo Bioscience) for 1 h. The membranes were incubated subsequently with either mouse antihuman C3aR monoclonal antibodies (1 : 500 dilution; AbD Serotec, Oxford, UK), mouse anti-human C5aR monoclonal antibodies (1 : 500 dilution; AbD Serotec), rabbit anti-human interleukin (IL)-6 monoclonal antibodies (1 : 800 dilution; Abcam, Cambridge, UK), rabbit antihuman MCP-1 polyclonal antibodies (1:1000 dilution, Abcam) or rabbit anti-human GAPDH polyclonal antibodies (1 : 800 dilution; Good Here Bioscience, Hangzhou, China) overnight at 4 C with gentle shaking.

Techniques: Expressing, Cell Culture, Standard Deviation, MTT Assay, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Clinical and experimental immunology

Article Title: Targeting C3a/C5a receptors inhibits human mesangial cell proliferation and alleviates immunoglobulin A nephropathy in mice.

doi: 10.1111/cei.12961

Figure Lengend Snippet: Fig. 2. Wild-type (WT), C3aR deficient (C3aR–/–) and C5aR–/– mice were immunized with inactivated Sendai virus to induce immunoglobulin (Ig)A nephropathy (IgAN). After 14 weeks of immunization, IgA and C3 deposition in the mesangium and the associated histological changes were analysed. (a,b) Glomerular IgA (upper row) and C3 (low row) deposition was measured by immunofluorescence staining of kidney sections from WT, C3aR–/– and C5aR–/– IgAN mice and negative controls. (c) Mesangial matrix expansion and cell proliferation were demonstrated by periodic acid-Schiff (PAS) staining of renal tissue in the following four groups of mice: negative controls and WT, C3aR–/– and C5aR–/– IgAN mice. Magnification 3400. Scale bars represent 100 lM.

Article Snippet: The membranes were blocked with 5% skimmed milk solution in 0 1% Tris-buffered saline Tween-20 (Dingguo Bioscience) for 1 h. The membranes were incubated subsequently with either mouse antihuman C3aR monoclonal antibodies (1 : 500 dilution; AbD Serotec, Oxford, UK), mouse anti-human C5aR monoclonal antibodies (1 : 500 dilution; AbD Serotec), rabbit anti-human interleukin (IL)-6 monoclonal antibodies (1 : 800 dilution; Abcam, Cambridge, UK), rabbit antihuman MCP-1 polyclonal antibodies (1:1000 dilution, Abcam) or rabbit anti-human GAPDH polyclonal antibodies (1 : 800 dilution; Good Here Bioscience, Hangzhou, China) overnight at 4 C with gentle shaking.

Techniques: Virus, Immunofluorescence, Staining

Journal: Clinical and experimental immunology

Article Title: Targeting C3a/C5a receptors inhibits human mesangial cell proliferation and alleviates immunoglobulin A nephropathy in mice.

doi: 10.1111/cei.12961

Figure Lengend Snippet: Fig. 3. Representative images of immunohistochemical staining for interleukin (IL)-6 and monocyte chemotactic protein 1 (MCP-1) in the kidney sections of wild-type (WT), C3aR deficient (C3aR–/–) and C5aR–/– immunoglobulin (Ig)A nephropathy (IgAN) mice and negative controls. C3aR and C5aR deficiency reduced renal IL-6 and MCP-1 expression compared to WT mice. Scale bars represent 100 lM.

Article Snippet: The membranes were blocked with 5% skimmed milk solution in 0 1% Tris-buffered saline Tween-20 (Dingguo Bioscience) for 1 h. The membranes were incubated subsequently with either mouse antihuman C3aR monoclonal antibodies (1 : 500 dilution; AbD Serotec, Oxford, UK), mouse anti-human C5aR monoclonal antibodies (1 : 500 dilution; AbD Serotec), rabbit anti-human interleukin (IL)-6 monoclonal antibodies (1 : 800 dilution; Abcam, Cambridge, UK), rabbit antihuman MCP-1 polyclonal antibodies (1:1000 dilution, Abcam) or rabbit anti-human GAPDH polyclonal antibodies (1 : 800 dilution; Good Here Bioscience, Hangzhou, China) overnight at 4 C with gentle shaking.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Clinical and experimental immunology

Article Title: Targeting C3a/C5a receptors inhibits human mesangial cell proliferation and alleviates immunoglobulin A nephropathy in mice.

doi: 10.1111/cei.12961

Figure Lengend Snippet: Fig. 4. Cytokine and chemokine gene expression in the renal tissues of the following four groups of mice: negative controls and wild-type (WT), C3aR deficient (C3aR–/–) and C5aR–/– immunoglobulin (Ig)A nephropathy (IgAN) mice (n5 7 for each group). Reverse transcription–quantitative polymerase chain reaction (RT–qPCR) was used to quantify the mRNA expression levels of (a) tumour necrosis factor (TNF)-a, (b) transforming growth factor (TGF)-b, (c) interleukin (IL)-1b, (d) interleukin (IL)-6 and (e) monocyte chemotactic protein 1 (MCP-1) relative to glyceraldehyde-3- phosphate dehydrogenase (GAPDH). Data are expressed as the mean 6 standard deviation. *P< 005, **P< 001, ***P< 0001 versus negative control.

Article Snippet: The membranes were blocked with 5% skimmed milk solution in 0 1% Tris-buffered saline Tween-20 (Dingguo Bioscience) for 1 h. The membranes were incubated subsequently with either mouse antihuman C3aR monoclonal antibodies (1 : 500 dilution; AbD Serotec, Oxford, UK), mouse anti-human C5aR monoclonal antibodies (1 : 500 dilution; AbD Serotec), rabbit anti-human interleukin (IL)-6 monoclonal antibodies (1 : 800 dilution; Abcam, Cambridge, UK), rabbit antihuman MCP-1 polyclonal antibodies (1:1000 dilution, Abcam) or rabbit anti-human GAPDH polyclonal antibodies (1 : 800 dilution; Good Here Bioscience, Hangzhou, China) overnight at 4 C with gentle shaking.

Techniques: Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Standard Deviation, Negative Control