Journal: Oncotarget

Article Title: Induction of specific T helper-9 cells to inhibit glioma cell growth

doi: 10.18632/oncotarget.13981

Figure Lengend Snippet: Glioma-bearing mice were treated with Ag (the GL261 cell extracts; 100 μg/mouse; cAg, the NG108-15 cell extracts, 100 μg/mouse; used as a control Ag) or/and SEB (2 μg/mouse; ip). Samples of serum, glioma tissue and the spleens were collected at sacrifice. ( A ) the bars indicate the serum levels of IL-4, IL-9 and IFN-γ (by ELISA). ( B ) the bars indicate the proliferation rate of Teff cells in the glioma tissue and the spleen (by flow cytometry). ( C – N ), CD4 + CD25 − T cells were isolated from the spleen and glioma tissue by MACS, stained with CFSE, and cultured with DCs in the presence of glioma cell extracts (5 μg/ml) for 3 days. The histograms show the representative results of Teff cell proliferation (the summarized data are presented in B). ( O – U ), the histograms show the frequency of IL-4 + cells, IL-9 + cells, IFN-γ + cells and IL-17 + cells in panel F (O-R) and panel K ( S – V ) respectively. Each group consists of 9 mice. The sera from individual mice were analyzed separately; the data represent 3 independent experiments. Teff cells isolated from 3 glioma tissue were pooled to one sample (due to the small amount of cell number). The data of C-G represent 3 samples per group performed in one experiment. Teff cells isolated from individual mice were processed separately. The cell ratio in the proliferation assay is 5 × 10 4 Teff cells: 1 × 10 4 DCs. Phorbol-12-myristate-13-acetate (PMA; 20 ng/ml) was added to the culture to activate the Teff cells. Each group consists of 9 mice.

Article Snippet: The ELISA kits of IL-4 (Cat#M4000B), IL-9 (DY409), IFN-γ (MIF00) and recombinant IL-9 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Isolation, Staining, Cell Culture, Proliferation Assay

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: European journal of immunology

Article Title: Critical role of IL-33 receptor ST2 in experimental cerebral malaria development.

doi: 10.1002/eji.201445206

Figure Lengend Snippet: Figure 6. ST2 deficiency does not affect lung or spleen inflammation after PbA sporozoite infection. WT- or ST2-deficient mice were infected with 1000 PbA sporozoites. (A) Lung inflammation with alveolar septae thickening with mononuclear cell infiltration, hemorrhage in alveoli, and interstitial edema on day 9 post infection in WT or ST2−/−mice; H&E staining. Magnification 20×; scale bar = 100 μm. Images are representative of two independent experiments. (B) Semiquantitative score of lung pathology, expressed in arbitrary unit (AUs). The bar graphs show the mean ± SEM of n = 9 mice per group and are pooled from two independent experiments. (C–F) PbA-induced spleen inflammation in WT or ST2−/−mice. The absolute numbers of CD8+ (C) and CD4+ (D) T cells, respectively, CD8+CD69+ and CD4+CD69+ T cells, CD8+CXCR3+ and CD4+CXCR3+ T cells per spleen are shown. IFN-γ (E) and IL-10 (F) levels were measured by ELISA in spleen homogenates on days 0 and 9 post infection. (C–F) Data are expressed as mean ± SEM of n = 5–6 mice per condition and are representative of two independent experiments; *p < 0.05; statistical analysis with Kruskal–Wallis test followed by Dunn’s comparison test.

Article Snippet: After centrifugation IL-33, IL-10, and IFN-γ levels were determined in supernatants using ELISA assay kits (Mouse DuoSet, R&D Systems, Minneapolis, USA).

Techniques: Infection, Staining, Enzyme-linked Immunosorbent Assay, Comparison

Journal: PLoS ONE

Article Title: A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity

doi: 10.1371/journal.pone.0069405

Figure Lengend Snippet: ( A ) GIFT9 amino acid sequence. The signal peptides of each part are underscored, IL9 signal peptide serves as the linker of the two parts. ( B ) Western blot of GIFT9 protein in the condition media from 293T cells retrovirally transduced to express GIFT9. Recombinant mouse GMCSF and IL9 were used as controls. ( C ) Western Blot of phospho-STAT1, STAT3, and STAT5 in JawsII cells after GIFT9 stimulation. Total STAT protein was used as a loading control. ( D ) Western Blot of phospho-STAT1, STAT3, and STAT5 in MC/9 cells after GIFT9 stimulation without or with GMCSF-Rα antibody blocking. Total STAT protein was used as a loading control. Normalized STAT1 phosphorylation level is shown, *: P<0.05, **: P<0.01.

Article Snippet: Recombinant mouse GMCSF and IL9 were purchased from R&D systems.

Techniques: Sequencing, Western Blot, Recombinant, Control, Blocking Assay, Phospho-proteomics

Journal: PLoS ONE

Article Title: A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity

doi: 10.1371/journal.pone.0069405

Figure Lengend Snippet: ( A ) JAK1, JAK2, and JAK3 phosphorylation levels remain same after GIFT9 stimulation. Total JAK protein was used as a loading control. Normalized JAK phosphorylation levels in GMCSF+IL9 and GIFT9 treatments are shown. ( B ) Inhibition of lipid rafts did not affect the hyperphosphorylation of STAT1 induced by GIFT9. Total STAT protein was used as a loading control. Normalized STAT1 phosphorylation level is shown.

Article Snippet: Recombinant mouse GMCSF and IL9 were purchased from R&D systems.

Techniques: Phospho-proteomics, Control, Inhibition

Journal: PLoS ONE

Article Title: A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity

doi: 10.1371/journal.pone.0069405

Figure Lengend Snippet: ( A ) GMCSFR βc was co-immunoprecipitated by α-common γc antibody after GIFT9 stimulation. *: P<0.05. ( B ) Common γc was co-immunoprecipitated by α-GMCSF-Rβ antibody after GIFT9 stimulation. *: P<0.05. ( C ) Representative confocal microscopy images of immunofluorescence staining showed the colocalization of GMCSF receptor and IL9 receptor after GIFT9 stimulation. Scale bar: 5 µm. ( D ) Percentages of color pixel colocalization after different cytokine treatment. **: P<0.01.

Article Snippet: Recombinant mouse GMCSF and IL9 were purchased from R&D systems.

Techniques: Immunoprecipitation, Confocal Microscopy, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity

doi: 10.1371/journal.pone.0069405

Figure Lengend Snippet: ( A ) JAK2 inhibitor TG101348 treatment abolished hyperphosphorylation of STAT1 after GIFT9 stimulation. Total STAT protein was used as a loading control. ( B ) JAK3 inhibitor CP690550 treatment had a minor effect of STAT1 hyperphosphorylation induced by JAK2. Total STAT protein was used as a loading control. ( C ) Double inhibition of JAK1 and JAK2 by INCB018424 inhibited STAT5 phosphorylation after GMCSF/IL9 but not GIFT9 stimulation. Total STAT protein was used as a loading control. ( D ) Inhibition of all JAKs by INCB018424 and CP690550 depleted STATs phosphorylation. Total STAT protein was used as a loading control. Normalized STAT1 phosphorylation level is shown, *: P<0.05, **: P<0.01.

Article Snippet: Recombinant mouse GMCSF and IL9 were purchased from R&D systems.

Techniques: Control, Inhibition, Phospho-proteomics

Journal: PLoS ONE

Article Title: A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity

doi: 10.1371/journal.pone.0069405

Figure Lengend Snippet: ( A ) Flow cytometry analysis of BMMCs. ( B ) MTT assay of BMMCs after 5 days of culture with GMCSF/IL9 or GIFT9. *: P<0.05. ( C ) Model of GMCSF and IL9 receptor clustering and downstream signaling after GIFT9 stimulation. See text for further details.

Article Snippet: Recombinant mouse GMCSF and IL9 were purchased from R&D systems.

Techniques: Flow Cytometry, MTT Assay