Journal: Frontiers in Cell and Developmental Biology

Article Title: HUWE1 Causes an Immune Imbalance in Immune Thrombocytopenic Purpura by Reducing the Number and Function of Treg Cells Through the Ubiquitination Degradation of Ets-1

doi: 10.3389/fcell.2021.708562

Figure Lengend Snippet: Expression of HUWE1 in CD4 + T cells in peripheral blood from immune thrombocytopenic purpura patients. (A) Quantitative real-time PCR (qRT-PCR) and Western blot assays were performed to detect the mRNA and protein levels of HUWE1 in CD4 + T cells in the peripheral blood from healthy controls and immune thrombocytopenic purpura (ITP) patients; and the quantitative analysis of HUWE1 protein level (mean ± SEM, n = 5). (B) Correlation analysis of the mRNA level of HUWE1 and platelet counts in CD4 + T cells in the peripheral blood from ITP patients ( r = −0.890, p < 0.01), n = 30. (C) Flow cytometry was applied to analyze the percentage of Treg cells in CD4 + T cells in peripheral blood from ITP patients and correlation analysis of the mRNA level of HUWE1 and the percentage of Treg cells in CD4 + T cells in peripheral blood from ITP patients ( r = −0.858, p < 0.01), n = 30. (D) Western blot was performed to assess the Ets-1 protein level in the CD4 + T cells in the peripheral blood from ITP patients. The experiment was repeated three times. GAPDH is applied as the loading control. ** p < 0.01 vs. control. ITP, immune thrombocytopenic purpura.

Article Snippet: Given the standard procedure of the reagent manufacturer, a human CD4 + T Cell Enrichment Kit (Stemcell, Beijing, China) was applied to isolate CD4 + T cells from PBMCs or a MagCellect Human Naive CD4 + T Cell Isolation Kit (Bio-Techne, MN, United States).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: HUWE1 Causes an Immune Imbalance in Immune Thrombocytopenic Purpura by Reducing the Number and Function of Treg Cells Through the Ubiquitination Degradation of Ets-1

doi: 10.3389/fcell.2021.708562

Figure Lengend Snippet: Influence of HUWE1 on Treg cell percentage, Foxp3 expression, IL-10 production and Treg cell immunosuppressive function. CD4 + T cells in peripheral blood from ITP patients were transfected with HUWE1 shRNA for 96 h. (A) Analysis of the HUWE1 mRNA and protein levels using qRT-PCR and Western blot; and the quantitative analysis of HUWE1 protein level (mean ± SEM, n = 5). (B) Flow cytometry was conducted to analyze the percentage of Treg cells in CD4 + T cells (mean ± SEM, n = 8). (C) qRT-PCR and Western blot were performed to quantify the mRNA and protein levels of Foxp3 in CD4 + T cells; and the quantitative analysis of Foxp3 protein level (mean ± SEM, n = 5). (D) Enzyme-linked immunosorbent assay (ELISA) was applied to detect the concentration of IL-10 in CD4 + T cell culture supernatant (mean ± SEM, n = 8). (E) Treg cells from ITP patients were transfected with HUWE1-interfering lentivirus (HUWE1 shRNA) and then cultured with effector T cells in a ratio of 1:4. Immunosuppression assay was performed to analyze the inhibitory effect of Treg cells on the proliferation of effector T cells (mean ± SEM, n = 8). ** p < 0.01 vs. shRNA. The experiment was repeated three times.

Article Snippet: Given the standard procedure of the reagent manufacturer, a human CD4 + T Cell Enrichment Kit (Stemcell, Beijing, China) was applied to isolate CD4 + T cells from PBMCs or a MagCellect Human Naive CD4 + T Cell Isolation Kit (Bio-Techne, MN, United States).

Techniques: Expressing, Transfection, shRNA, Quantitative RT-PCR, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture, Immunosuppression Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: HUWE1 Causes an Immune Imbalance in Immune Thrombocytopenic Purpura by Reducing the Number and Function of Treg Cells Through the Ubiquitination Degradation of Ets-1

doi: 10.3389/fcell.2021.708562

Figure Lengend Snippet: Effect of HUWE1 overexpression on Treg cell percentage, Foxp3 expression, IL-10 production and Treg cell immunosuppressive function. CD4 + T cells in peripheral blood from healthy controls were transfected with Lenti-HUWE1 for 96 h. (A) Flow cytometry was performed to detect the percentage of Treg cells in CD4 + T cells (mean ± SEM, n = 8). (B) qRT-PCR was conducted to measure the mRNA level of Foxp3 in CD4 + T cells and Western blot was carried out to measure the protein levels of Foxp3 and HUWE1 in CD4 + T cells; and the quantitative analysis of Foxp3 protein level (mean ± SEM, n = 5). (C) ELISA was applied to detect the concentration of IL-10 in the CD4 + T cell culture supernatant (mean ± SEM, n = 8). (D) An immunosuppression experiment was conducted to assess the inhibitory effect of Treg cells on the proliferation of effector T cells (mean ± SEM, n = 8). ** p < 0.01 vs. Lenti. The experiment was repeated three times.

Article Snippet: Given the standard procedure of the reagent manufacturer, a human CD4 + T Cell Enrichment Kit (Stemcell, Beijing, China) was applied to isolate CD4 + T cells from PBMCs or a MagCellect Human Naive CD4 + T Cell Isolation Kit (Bio-Techne, MN, United States).

Techniques: Over Expression, Expressing, Transfection, Flow Cytometry, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture

Journal: Frontiers in Cell and Developmental Biology

Article Title: HUWE1 Causes an Immune Imbalance in Immune Thrombocytopenic Purpura by Reducing the Number and Function of Treg Cells Through the Ubiquitination Degradation of Ets-1

doi: 10.3389/fcell.2021.708562

Figure Lengend Snippet: The interaction between HUWE1 and E26 transformation-specific-1 (Ets-1). (A) Correlation analysis of the protein level of HUWE1 and the protein level of Ets-1 in the CD4 + T cells in the peripheral blood from ITP patients ( r = −0.904, p < 0.001), n = 30. (B) Immunoprecipitation (IP) was performed to analyze the binding of HUWE1 to Ets-1 in CD4 + T cells in peripheral blood from healthy controls or ITP patients. (C) Lenti-MEK1 (a constitutively activated MEK1) was transfected into Jurkat T cells and the transfection efficiency was verified by Western blot. IP was conducted to assess the binding of HUWE1 to Ets-1 and a Western bolt assay was applied to detect the protein levels of p-Ets-1 (T38 sites), Ets-1 and HUWE1. (D) 293A cells were transfected with WT (Ets-1 wild-type plasmid) and HUWE1-Flag, T38A (point mutant plasmid phosphorylation inactivation at T38 of Ets-1: mutates T to A) and HUWE1-Flag, or T38D (point mutant plasmid phosphorylation activation at T38 of Ets-1: mutates T to D) and HUWE1-Flag for 24 h, HA for IP followed by Flag for immunoblotting (IB) or Flag for IP followed by HA for IB. The analysis of the binding of HUWE1 to Ets-1 WT, to Ets-1 T38A or Ets-1 T38D. HA: Both the WT plasmid and Mut plasmid of Ets-1 had HA tags. (E) Jurkat T cells were treated with 1 μM MEK inhibitor TAK-733, 1 μM ERK inhibitor GDC0994, and 10 μM the Src family kinase inhibitor dasatinib for 0, 1, 2, 4, 6 and 8 h, respectively. The analysis of the binding of HUWE1 to Ets-1. (F) Immunofluorescence confirmed the overexpression of HUWE1 and its co-localization with ETS-1 in the nucleus (scale bar: 10 μm). IP, immunoprecipitation; IB, immunoblotting; WCL, whole cell lysate. The experiment was repeated three times.

Article Snippet: Given the standard procedure of the reagent manufacturer, a human CD4 + T Cell Enrichment Kit (Stemcell, Beijing, China) was applied to isolate CD4 + T cells from PBMCs or a MagCellect Human Naive CD4 + T Cell Isolation Kit (Bio-Techne, MN, United States).

Techniques: Transformation Assay, Immunoprecipitation, Binding Assay, Transfection, Western Blot, Plasmid Preparation, Mutagenesis, Activation Assay, Immunofluorescence, Over Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: HUWE1 Causes an Immune Imbalance in Immune Thrombocytopenic Purpura by Reducing the Number and Function of Treg Cells Through the Ubiquitination Degradation of Ets-1

doi: 10.3389/fcell.2021.708562

Figure Lengend Snippet: Regulation of HUWE1 on the differentiation and function of Treg cells through Ets-1. Naive CD4 + T cells were isolated from the peripheral blood of healthy controls and transfected with HUWE1 lentivirus and/or Ets-1 lentivirus for 48 h, and were cultured in Treg polarization conditions (cells in the presence of plate-bound anti-CD3, solid anti-CD28, TGF β (5 ng/ml), and IL-2 (50 IU/ml) for 72 h. (A) Western blot was applied to measure the protein levels of HUWE1 and Ets-1. (B) qRT-PCR was performed to quantify the mRNA level of Foxp3 (mean ± SEM, n = 3). (C) Flow cytometry was conducted to assess the percentage of Treg cells in Naive CD4 + T cells (mean ± SEM, n = 8). (D) Treg cells from the peripheral blood of healthy controls were transfected with HUWE1 lentivirus and/or Ets-1 lentivirus for 48 h and then cultured with effector T cells in a ratio of 1:4. Immunosuppression assay was performed to analyze the inhibitory effect of Treg cells on the proliferation of effector T cells (mean ± SEM, n = 8). ** p < 0.01 vs. Lenti or Lenti-HUWE1. The experiment was repeated three times.

Article Snippet: Given the standard procedure of the reagent manufacturer, a human CD4 + T Cell Enrichment Kit (Stemcell, Beijing, China) was applied to isolate CD4 + T cells from PBMCs or a MagCellect Human Naive CD4 + T Cell Isolation Kit (Bio-Techne, MN, United States).

Techniques: Isolation, Transfection, Cell Culture, Western Blot, Quantitative RT-PCR, Flow Cytometry, Immunosuppression Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: HUWE1 Causes an Immune Imbalance in Immune Thrombocytopenic Purpura by Reducing the Number and Function of Treg Cells Through the Ubiquitination Degradation of Ets-1

doi: 10.3389/fcell.2021.708562

Figure Lengend Snippet: The function of HUWE1 inhibitor on ITP mice. 0.1 mg/kg HUWE1-specific inhibitor BI8622 was intraperitoneally injected into mice during ITP modeling (three times a week). Sixteen male C57BL/6J mice (6–8 weeks old) were divided into the following two groups: ITP group and ITP + BI8622 group, and eight mice were randomly assigned to each group. (A) Hematoxylin-eosin (HE) staining was performed to analyze the pathological changes of the spleen in ITP mice (scale bar: 5.0 μm), n = 8. (B) Analysis of platelet counts in ITP mice (mean ± SEM, n = 8). (C) Flow cytometry was conducted to analyze the percentage of Treg cells in spleens of ITP mice (mean ± SEM, n = 8). (D) After the CD4 + T cells were isolated from the spleen cells of ITP mice, a Western blot was conducted to detect the protein levels of Ets-1 and p-Ets-1 in cells, n = 3. (E) The interaction between HUWE1 and Ets-1 in spleen CD4 + T cells was confirmed using IP assay. ** p < 0.01 vs. ITP.

Article Snippet: Given the standard procedure of the reagent manufacturer, a human CD4 + T Cell Enrichment Kit (Stemcell, Beijing, China) was applied to isolate CD4 + T cells from PBMCs or a MagCellect Human Naive CD4 + T Cell Isolation Kit (Bio-Techne, MN, United States).

Techniques: Injection, Staining, Flow Cytometry, Isolation, Western Blot