Fig 2: Influence of HUWE1 on the ubiquitination and degradation of Ets-1 protein. (A) Jurkat T cells were transfected with Hb-HA and Lenti-HUWE1 or Lenti-HUWE1C4341A, respectively. Ubiquitin experiment was conducted to assess the ubiquitination of Ets-1. (B) Jurkat T cells were transfected with Lenti-HUWE1 for 48 h and then were treated with 50 mmol/L CHX for 0, 1, 2 and 4 h. Western blot was performed to measure the protein levels of Ets-1 and HUWE1. (C) Jurkat T cells were transfected with Lenti-HUWE1 for 48 h and then were treated with 10 µm MG132 4 h before the sample harvesting. Western blot was applied to detect the protein level of Ets-1 and HUWE1. (D) Jurkat T cells were transfected with Lenti-HUWE1 for 48 h. An immunofluorescence assay was conducted to analyze the expression of Ets-1 and the nuclear export of Ets-1 (scale bar: 10 µm). (E) Jurkat T cells were transfected with Lenti-HUWE1 and/or Lenti-MEK1. Western blot was applied to detect the protein levels of Ets-1 and MEK1. (F) Jurkat T cells were treated with TAK-733 (1 µM) and/or HUWE1 specific inhibitor BI8622 (1 µM), GDC0994 (1 µM) and/or BI8622 (1 µM). Western blot was performed to detect the protein level of Ets-1. **p < 0.01 vs. Lenti. The experiment was repeated three times. CHX: cycloheximide.

Fig 3: 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 ß (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.

Fig 4: 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.

Fig 5: 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.