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

Journal: Current Issues in Molecular Biology

Article Title: Oncological Properties of Intravenous Leiomyomatosis: Involvement of Mesenchymal Tumor Stem-Like Cells

doi: 10.3390/cimb43020084

Figure Lengend Snippet: Differential expressions of PSMB9/β1i, CALPONIN h1 and CD44 in human uterine mesenchymal tumors and uterine LANT-like tumor.

Article Snippet: We isolated a candidate population of CD44-positive SK-LMS-1 subclone as human Ut-LMS stem-like cells from human Ut-LMS primary cells and SK-LMS-1 cells, using the CD44-positive selection method with MagCellect Plus Human CD44+ Cell Isolation Kit (R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: Expressing

Journal: Current Issues in Molecular Biology

Article Title: Oncological Properties of Intravenous Leiomyomatosis: Involvement of Mesenchymal Tumor Stem-Like Cells

doi: 10.3390/cimb43020084

Figure Lengend Snippet: Differential expressions of factors cyclin B, cyclin E, caveolin 1, Ki-67, LMP2/b1i, and CD44 as potential biomarkers in intravenous leiomyomatosis and intravenous leiomyosarcoma. ( A ) Extensive intravascular growth of focal cellular smooth muscle is noted, forming worm-like plugs within veins. Focal hydropic change and components of endometrial glands are also observed. CD44-positive cells in the external tissue of intravenous are shown in the box a . CD44-positive cells in the internal tissue of intravenous are shown in the box b . ( B ) Intravenous leiomyosarcoma is shown in the upper right photograph. In uterine leiomyosarcoma, spindle-shaped tumor cells with round nuclei proliferate solidly. Compared with leiomyoma, uterine leiomyosarcoma shows increased nuclear density, nuclear hypertrophy, nuclear irregularities, and increased fission. ( C ) Immunohistochemistry of intravenous leiomyomatosis and intravenous leiomyosarcoma tissue sections was performed using appropriate monoclonal antibodies with standard procedures. The five tissue sites were randomly selected from intravenous leiomyomatosis’s internal and external tissues (i.e., normal uterine leiomyoma). As with uterine leiomyoma, the five tissue sites were randomly selected from the internal tissue of intravenous leiomyosarcoma. In a 40× field of view, the positive rates of the six factors were calculated in the five tissue sites. The positive rates at the sites of each tissue are shown in the scatter plot. Int.UL: Intravenous leiomyomatosis, Int.uLMS: Intravenous uterine leiomyosarcoma.

Article Snippet: We isolated a candidate population of CD44-positive SK-LMS-1 subclone as human Ut-LMS stem-like cells from human Ut-LMS primary cells and SK-LMS-1 cells, using the CD44-positive selection method with MagCellect Plus Human CD44+ Cell Isolation Kit (R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: Immunohistochemistry

Journal: Current Issues in Molecular Biology

Article Title: Oncological Properties of Intravenous Leiomyomatosis: Involvement of Mesenchymal Tumor Stem-Like Cells

doi: 10.3390/cimb43020084

Figure Lengend Snippet: Induction of micrometastases in alveolar tissues derived from BALB/c nu/nu mice xenografted with human CD44-positive SK-LMS-1 stem-like cells. ( A ) Photographs show xenografts derived from BALB/c nu/nu mice inoculated with CD44-negative SK-LMS-1 cells or CD44-positive SK-LMS-1 cells at 2 months after the injection. Xenografts derived from BALB/c nu/nu mice inoculated with CD44-positive SK-LMS-1 cells were highly angiogenic. Data are representative of one of ten independent experiments obtained with different xenograft samples derived from CD44-negative SK-LMS-1 cells or CD44-positive SK-LMS-1 cells. The photographs show the pathology in metastasizes of respiratory tissue derived from the primary tumor on the left side of the second mammary fat pad, and micrometastases in the alveolar tissues of BALB/c nu/nu mice xenografted with CD44-negative SK-LMS-1 cells or CD44-positive SK-LMS-1 cells at 2 months after injection. In xenograft experiments using BALB/c nu/nu mice, the tumorigenicity of CD44-negative SK-LMS-1 cell and CD44-positive SK-LMS-1 cell xenografts was similar. Data are representative of one of ten independent experiments obtained with different xenograft samples derived from CD44-negative SK-LMS-1 cells or CD44-positive SK-LMS-1 cells. Metastatic tissues are indicated by black arrows. Micrometastasis in the alveoli are indicated by red arrows. ( B ) CD44-negative SK-LMS-1 cells or CD44-positive SK-LMS-1 cells were harvested from the cell culture using trypsin, washed, and resuspended in PBS (2 × 10 7 cells/mL). Nude mice (BALB/cSlc- nu/nu , female, 7–8 weeks old; Japan SLC, Shizuoka, Japan) were injected with 1 × 10 6 CD44-negative SK-LMS-1 cells or CD44-positive SK-LMS-1 cells in 5 mg/mL BD Matrigel Matrix (BD Biosciences) in culture medium containing 15% fetal calf serum plus SmGM-2 SingleQuots (Lonza, Basel) at a volume of 100 μL on the left side of the second mammary fat pad. Tumor formation was assessed every day. Tumor volumes were calculated as (L × W × W)/2, where W represents the width, and L represents the length. Statistical analyses were performed on mean tumor volumes at the end of the study using Student’s t -test. ( C ) The upper graph shows the number of mice in which micrometastases were observed in alveoli. The lower graph shows the number of micrometastatic sites found in alveolar sections in each xenografted mouse. It is important to note that in CD44-positive SK-LMS-1 cell-xenografted mice, the frequency of micrometastases in alveolar tissue was significantly higher than that in CD44-negative SK-LMS-1 cell-xenografted mice. Compared with CD44-negative SK-LMS-1 cells, CD44-positive SK-LMS-1 cells exhibit the ability to induce tumor angiogenesis and have higher hematogenous metastatic potential.

Article Snippet: We isolated a candidate population of CD44-positive SK-LMS-1 subclone as human Ut-LMS stem-like cells from human Ut-LMS primary cells and SK-LMS-1 cells, using the CD44-positive selection method with MagCellect Plus Human CD44+ Cell Isolation Kit (R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: Derivative Assay, Injection, Cell Culture

Journal: Current Issues in Molecular Biology

Article Title: Oncological Properties of Intravenous Leiomyomatosis: Involvement of Mesenchymal Tumor Stem-Like Cells

doi: 10.3390/cimb43020084

Figure Lengend Snippet: Differential expression of cyclin B, cyclin E, caveolin 1, Ki-67, LMP2/b1i, and CD44 as potential biomarkers in leiomyoma, leiomyosarcoma, and intravenous leiomyomatosis. ( A ) The photograph shows spindle cell leiomyoma. The low power view (10× field) shows a well-circumscribed tumor nodule in the myometrium composed of broad fascicles of spindle cells. High power view (40× field) shows that uterine leiomyoma (spindle cell) has bland cytological features, with elongated nuclei and fine nuclear chromatin. Immunohistochemistry of leiomyoma tissue was performed using appropriate monoclonal antibodies with standard procedures. ( B ) The photograph shows epithelioid leiomyosarcoma. The low power view (10× field) shows the uterine mass irregular interface with the myometrium, composed of round to polygonal cells with granular eosinophilic cytoplasms. Notice the presence of significant nuclear atypia and clear mitoses. The high power view (40× field) shows that the tumor cells are round to ovoid, with eosinophilic granular cytoplasm and irregularly shaped nuclei. Immunohistochemistry of leiomyoma tissue was performed using appropriate monoclonal antibodies with standard procedures. ( C ) Intravenous leiomyomatosis is a rare neoplasm characterized by nodular masses of histologically benign smooth muscle cells growing within the venous system. Significant smooth muscle hyperplasia was observed in the myometrium. Some tumors invade venous blood vessels. The low power view (10× field) shows no obvious high-grade nuclear atypia or mitotic cell proliferation and necrosis. The high power view (40× field) shows interlaced bundles of homogeneous spindle cells with oval nuclei, eosinophilic cytoplasm, rare mitotic figures, and decorated with several thick-walled small blood vessels, which are consistent with features of leiomyoma. Immunohistochemistry of leiomyoma tissue was performed using appropriate monoclonal antibodies with standard procedures. The areas surrounded by the dotted squares are enlarged.

Article Snippet: We isolated a candidate population of CD44-positive SK-LMS-1 subclone as human Ut-LMS stem-like cells from human Ut-LMS primary cells and SK-LMS-1 cells, using the CD44-positive selection method with MagCellect Plus Human CD44+ Cell Isolation Kit (R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: Expressing, Immunohistochemistry

Journal: Current Issues in Molecular Biology

Article Title: Oncological Properties of Intravenous Leiomyomatosis: Involvement of Mesenchymal Tumor Stem-Like Cells

doi: 10.3390/cimb43020084

Figure Lengend Snippet: CD44-positive uterine mesenchymal tumor stem-like cells in intravenous leiomyomatosis and intravenous uterine leiomyosarcoma. Immunohistochemistry of normal myometrium, uterine leiomyoma, uterine leiomyosarcoma, intravenous leiomyomatosis, and intravenous leiomyosarcoma tissues was performed using appropriate monoclonal antibodies with standard procedures. The five tissue sites were randomly selected: normal myometrium, uterine leiomyoma, uterine leiomyosarcoma, internal tissue of intravenous leiomyomatosis, and intravenous leiomyosarcoma. In a 40× field of view, the positive rates of the six factors were calculated in the five tissue sites under microscopy (Panthera Shimadzu Co. Ltd., Kyoto, Japan); the positive rates at the sites of each tissue are shown in the scatter plot. Int.UL: Intravenous leiomyomatosis, Int.uLMS: Intravenous uterine leiomyosarcoma.

Article Snippet: We isolated a candidate population of CD44-positive SK-LMS-1 subclone as human Ut-LMS stem-like cells from human Ut-LMS primary cells and SK-LMS-1 cells, using the CD44-positive selection method with MagCellect Plus Human CD44+ Cell Isolation Kit (R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: Immunohistochemistry, Microscopy

Journal: Current Issues in Molecular Biology

Article Title: Oncological Properties of Intravenous Leiomyomatosis: Involvement of Mesenchymal Tumor Stem-Like Cells

doi: 10.3390/cimb43020084

Figure Lengend Snippet: The invasion and metastatic process of uterine mesenchymal tumor stem-like cells. The nature of malignant tumors varies from patient to patient. Malignant tumor tissue comprises a heterogeneous cell population, containing many fibroblasts and tumor stem cells in addition to tumor cells. The heterogeneity of malignant tumor cells is one of the main reasons for the difficulty in treating malignant tumors. In particular, malignant tumor stem cells undergo distant metastasis and have resistance to antitumor agents. Understanding the oncological properties of malignant tumor stem cells contributes to the development of new targeted antitumor agents. The complex process of distant metastasis includes detachment, invasion of the tumor microenvironment, and shedding of invasive tumor cells (ITCs) and CD44-positive invasive tumor stem cells (ITSCs) into the bloodstream (intravasation). The majority of ITCs relate to differentiated cancer cells with low tumorigenicity and instead undergo apoptosis. However, CD44-positive ITSCs bear cancer stem cell features, i.e., CD44-positive circulating tumor stem cells (CTSC), survive in the circulating blood, escape from immune surveillance, and go home to secondary sites, extravasate, and ultimately form distant metastatic lesions. The application of cytotoxic drugs for ITSCs and/or CTSCs, such as celecoxib, greatly reduces the incidence of tumor metastases and tumor recurrence . ITC: Invasive tumor cell, ITSC: Invasive tumor stem cell, CTSC: Circulating tumor stem cell.

Article Snippet: We isolated a candidate population of CD44-positive SK-LMS-1 subclone as human Ut-LMS stem-like cells from human Ut-LMS primary cells and SK-LMS-1 cells, using the CD44-positive selection method with MagCellect Plus Human CD44+ Cell Isolation Kit (R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: