Journal: Frontiers in Immunology

Article Title: Relevance of PSGL-1 Expression in B Cell Development and Activation

doi: 10.3389/fimmu.2020.588212

Figure Lengend Snippet: Flow Cytometry Antibodies for characterization of murine immune cells.

Article Snippet: CD135 (FLT3) , APC , 1:25 , Miltenyi , 130-111-152.

Techniques: Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Relevance of PSGL-1 Expression in B Cell Development and Activation

doi: 10.3389/fimmu.2020.588212

Figure Lengend Snippet: Flow cytometry patterns used to define murine immune populations.

Article Snippet: CD135 (FLT3) , APC , 1:25 , Miltenyi , 130-111-152.

Techniques: Flow Cytometry, Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: IFNβ-Induced CXCL10 Chemokine Expression Is Regulated by Pellino3 Ligase in Monocytes and Macrophages

doi: 10.3390/ijms232314915

Figure Lengend Snippet: INFβ-dependent CXCL10 induction is downregulated in Pellino3 deficient THP-1 cell line. ( A ) Pellino3 knockout in THP-1 cells was generated using Guide-it CRISPR/Cas9 Gesicle Production System (Takara Bio) according to the manufacturer’s instruction. Cell lysates from WT and PELI3 −/− THP-1 were subjected to SDS-PAGE followed by Western blotting. Protein detection was performed using specific antibodies and appropriate secondary antibodies conjugated to the fluorescent dye in the infrared range. Visualization was performed using the Odyssey CLx Imaging System LI-COR. Wild type (WT) and PELI3 −/− THP-1 cells were treated with IFNβ (1000 U/mL) or IFNγ (15 ng/mL) for 4 h ( B , C ) or 16 h ( D , E ). ( B , C ) Thereafter, total RNA was isolated and reverse transcribed. Quantitative real-time PCR (2 –(ΔΔCT) ) was used to assay the expression levels of CXCL10 and CXCL11 . Relative expression values were normalized to the HPRT1 reference gene and non-treated cells were assigned an arbitrary value of 1. ( B , D ) CXCL10 and CXCL11 level was measured by ELISA. ( F ) Total RNA was isolated from WT and PELI3 −/− THP-1 cells and converted to first-strand cDNA. This was used as a template for PCR amplifying genes as indicated. Products were resolved on 1.5% ( w/v ) agarose gel electrophoresis. All results presented are representative of at least three independent experiments. * p ≤ 0.01 (unpaired Student’s t -test).

Article Snippet: Protein detection was performed using rabbit anti-human PELI3 antibody (BioRad, Hercules, CA, USA) and appropriate secondary antibodies conjugated to the fluorescent dye in the infrared range (IRDye 800CW Goat anti-Rabbit IgG (H + L) antibody, LI-COR, Lincoln, NE, USA).

Techniques: Knock-Out, Generated, CRISPR, SDS Page, Western Blot, Imaging, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Agarose Gel Electrophoresis

Journal: International Journal of Molecular Sciences

Article Title: IFNβ-Induced CXCL10 Chemokine Expression Is Regulated by Pellino3 Ligase in Monocytes and Macrophages

doi: 10.3390/ijms232314915

Figure Lengend Snippet: Pellino3 regulates IFNβ-induced Tyk2 phosphorylation. ( A , B ) Wild type (WT) and PELI3 −/− THP-1 cells were treated with IFNβ (1000 U/mL) for indicated time periods. Whole-cell lysates were subjected to SDS-PAGE followed by Western blotting. Protein detection was performed using specific antibodies and appropriate secondary antibodies conjugated to the fluorescent dye in the infrared range. Visualization was performed using the Odyssey CLx Imaging System LI-COR. The results presented are representative of at least three independent experiments.

Article Snippet: Protein detection was performed using rabbit anti-human PELI3 antibody (BioRad, Hercules, CA, USA) and appropriate secondary antibodies conjugated to the fluorescent dye in the infrared range (IRDye 800CW Goat anti-Rabbit IgG (H + L) antibody, LI-COR, Lincoln, NE, USA).

Techniques: Phospho-proteomics, SDS Page, Western Blot, Imaging

Journal: International Journal of Molecular Sciences

Article Title: IFNβ-Induced CXCL10 Chemokine Expression Is Regulated by Pellino3 Ligase in Monocytes and Macrophages

doi: 10.3390/ijms232314915

Figure Lengend Snippet: IFNβ-dependent production of CXCL10 is mediated by NF-κB, but in Pellino3 independent mechanism. Wild type (WT) and PELI3 −/− THP-1 cells were pretreated with DMSO (−/−) or JSH-23 (5 µM) for 1 h. Next, the cells were treated with IFNβ (1000 U/mL) for 4 h ( A ) or 16 h ( B ). ( A ) Thereafter, total RNA was isolated and reverse-transcribed. Quantitative real-time PCR (2 −(ΔΔCT) ) was used to assay the expression levels of CXCL10. Relative expression values were normalized to the HPRT1 reference gene, and non-treated cells were assigned an arbitrary value of 1. ( B ) CXCL10 level was measured by ELISA. All results presented are representative of at least three independent experiments. * p ≤ 0.01 (unpaired Student’s t -test).

Article Snippet: Protein detection was performed using rabbit anti-human PELI3 antibody (BioRad, Hercules, CA, USA) and appropriate secondary antibodies conjugated to the fluorescent dye in the infrared range (IRDye 800CW Goat anti-Rabbit IgG (H + L) antibody, LI-COR, Lincoln, NE, USA).

Techniques: Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: IFNβ-Induced CXCL10 Chemokine Expression Is Regulated by Pellino3 Ligase in Monocytes and Macrophages

doi: 10.3390/ijms232314915

Figure Lengend Snippet: IFNβ/Pellino3-dependent activation of CXCL10 is regulated by IRF9. ( A – C ) Wild type (WT) and PELI3 −/− THP-1 cells were treated with IFNβ (1000 U/mL) for indicated time periods. Whole-cell lysates ( A ) or nuclear fraction ( B ) were subjected to SDS-PAGE followed by Western blotting. Protein detection was performed using specific antibodies and appropriate secondary antibodies conjugated to the fluorescent dye in the infrared range. Visualization was performed using the Odyssey CLx Imaging System LI-COR. The results presented are representative of at least three independent experiments. ( C ) Cells were fixed with formaldehyde, followed by nuclei isolation and sonication. Sonicated nuclear lysates were immunoprecipitated with an anti-IRF9 or rabbit IgG control antibody. Input DNA (prior to immunoprecipitation) and immunoprecipitated chromatin were analyzed by quantitative real-time PCR (2 –(ΔΔCT) ) (top) and 35 cycles of standard PCR (bottom) with primers designed to amplify an ISRE binding site in the human CXCL10 gene promoter region. Products of standard PCR were resolved on 1.5% ( w/v ) agarose gel electrophoresis. All results presented are representative of at least three independent experiments. * p ≤ 0.01 (unpaired Student’s t -test).

Article Snippet: Protein detection was performed using rabbit anti-human PELI3 antibody (BioRad, Hercules, CA, USA) and appropriate secondary antibodies conjugated to the fluorescent dye in the infrared range (IRDye 800CW Goat anti-Rabbit IgG (H + L) antibody, LI-COR, Lincoln, NE, USA).

Techniques: Activation Assay, SDS Page, Western Blot, Imaging, Isolation, Sonication, Immunoprecipitation, Control, Real-time Polymerase Chain Reaction, Binding Assay, Agarose Gel Electrophoresis

Journal: International Journal of Molecular Sciences

Article Title: IFNβ-Induced CXCL10 Chemokine Expression Is Regulated by Pellino3 Ligase in Monocytes and Macrophages

doi: 10.3390/ijms232314915

Figure Lengend Snippet: Pellino3 regulates IFNβ-induced Cxcl10 production in murine macrophages cell line, BMDM. Wild type (WT) and Peli3 −/− BMDM cells were treated with mIFNβ (mouse IFNβ) (50 ng/mL) for 4 h ( A ) or 16 h ( B ). ( A ) Thereafter, total RNA was isolated and reverse-transcribed. Quantitative real-time PCR (2 –(ΔΔCT) ) was used to assay the expression levels of Cxcl10 . Relative expression values were normalized to the Hprt1 reference gene and non-treated cells were assigned an arbitrary value of 1. ( B ) Cxcl10 level was measured by ELISA. ( C ) Total RNA was isolated from WT and Peli3 −/− BMDM cells and converted to first-strand cDNA. This was used as a template for PCR amplifying Ifnar1 and Ifnar2 . Products were resolved on 1.5% ( w/v ) agarose gel electrophoresis. ( D ) Whole cell lysates were subjected to SDS-PAGE followed by Western blotting. Protein detection was performed using specific antibodies and appropriate secondary antibodies conjugated to the fluorescent dye in the infrared range. Visualization was performed using the Odyssey CLx Imaging System LI-COR. The results presented are representative of at least three independent experiments. All results presented are representative of at least three independent experiments. * p ≤ 0.01 (unpaired Student’s t -test).

Article Snippet: Protein detection was performed using rabbit anti-human PELI3 antibody (BioRad, Hercules, CA, USA) and appropriate secondary antibodies conjugated to the fluorescent dye in the infrared range (IRDye 800CW Goat anti-Rabbit IgG (H + L) antibody, LI-COR, Lincoln, NE, USA).

Techniques: Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Agarose Gel Electrophoresis, SDS Page, Western Blot, Imaging

Journal: International Journal of Molecular Sciences

Article Title: IFNβ-Induced CXCL10 Chemokine Expression Is Regulated by Pellino3 Ligase in Monocytes and Macrophages

doi: 10.3390/ijms232314915

Figure Lengend Snippet: Pellino3 promotes mIFNβ-induced production Cxcl10 via IRF9-dependent pathway in BMDM cell line. Wild type (WT) and Peli3 −/− BMDM cells were pretreated with DMSO (−/−) or JSH-23 (5 µM) ( A , B ) for 1 hr. Next, the cells were treated with mIFNβ (mouse IFNβ) (50 ng/mL) for 4 h ( A ) or 16 h ( B ). ( A ) Thereafter, total RNA was isolated and reverse-transcribed. Quantitative real-time PCR (2 –(ΔΔCT) ) was used to assay the expression levels of Cxcl10 . Relative expression values were normalized to the Hprt1 reference gene and non-treated cells were assigned an arbitrary value of 1. ( B ) Cxcl10 level was measured by ELISA. ( C , D ) Wild type (WT) and Peli3 −/− BMDM cells were treated with mIFNβ (50 ng/mL) for indicated time periods. Whole-cell lysates ( C ) or nuclear fraction ( D ) were subjected to SDS-PAGE followed by Western blotting. Protein detection was performed using specific antibodies and appropriate secondary antibodies conjugated to the fluorescent dye in the infrared range. Visualization was performed using the Odyssey CLx Imaging System LI-COR. The results presented are representative of at least three independent experiments. All results presented are representative of at least three independent experiments. * p ≤ 0.01 (unpaired Student’s t -test).

Article Snippet: Protein detection was performed using rabbit anti-human PELI3 antibody (BioRad, Hercules, CA, USA) and appropriate secondary antibodies conjugated to the fluorescent dye in the infrared range (IRDye 800CW Goat anti-Rabbit IgG (H + L) antibody, LI-COR, Lincoln, NE, USA).

Techniques: Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, SDS Page, Western Blot, Imaging

Journal: Molecular Cancer

Article Title: New frontiers in immune checkpoint B7-H3 (CD276) research and drug development

doi: 10.1186/s12943-023-01751-9

Figure Lengend Snippet: Summary of selected drugs in testing and clinical trials targeting B7-H3 ( ClinicalTrials.gov ) and their mechanisms

Article Snippet: TAA06 Injection , B7-H3-targeted CAR-T therapy , FDA approved drug (Orphan drug) , Neuroblastoma , PersonGen BioTherapeutics (Suzhou) Co., Ltd.

Techniques: Clinical Proteomics, Injection

Journal: Cells

Article Title: Extracellular Vesicles Derived from Bone Marrow in an Early Stage of Ionizing Radiation Damage Are Able to Induce Bystander Responses in the Bone Marrow

doi: 10.3390/cells11010155

Figure Lengend Snippet: Gating Strategy for the Identification of Different Bone Marrow Cell Populations by Flow Cytometry. ( A ) Lymphoid progenitor cells were identified as CD45 and CD90.2 double positive cells in bone marrow cells. ( B ) Granulocytes/monocytes were characterized as Gr1 and CD11b double positive cells in bone marrow cells. ( C ) Hematopoietic stem and progenitor cells were identified as Sca1 and cKit double positive cells in the Lineage negative bone marrow cells. ( D ) Hematopoietic stem cell subpopulation characterization was done by using the CD34 and CD135 markers in the hematopoietic stem and progenitor cell pool. Long-term hematopoietic stem cells were identified as CD34 and CD135 double negative cells, short-term hematopoietic stem cells were CD34+CD135- cells, multipotent progenitors were characterized as CD34 and CD135 double positive cells. ( E ) Mesenchymal stem and stromal cells were identified as Sca1 and CD44 double positive cells in the lineage-negative bone marrow cells.

Article Snippet: The following directly labelled anti-mouse monoclonal antibodies were used: CD90.2-PE and CD45-PE/Cy7 for lymphoid progenitors (LP), Gr1-AF647 and CD11b-PE for granulocytes/monocytes (GM), Lineage Cocktail (CD3, Gr1, CD11b, CD45R, Ter119)-PB, Sca1-PE, cKit (CD117)-BV785, CD34-AF647, CD135-PE/Cy5 for hematopoietic stem and progenitor cells (HSPCs) and Lineage Cocktail-PB, Sca1-PE and CD44-AF647 for mesenchymal stem/stromal cells (MSCs), all purchased from SONY (SONY Biotechnology, San Jose, CA, USA).

Techniques: Flow Cytometry

Journal: Cells

Article Title: Extracellular Vesicles Derived from Bone Marrow in an Early Stage of Ionizing Radiation Damage Are Able to Induce Bystander Responses in the Bone Marrow

doi: 10.3390/cells11010155

Figure Lengend Snippet: Both Direct Irradiation and Extracellular Vesicles-Mediated Bystander Effects Induce Persistent Redistribution between Multipotent Progenitors and Long-Term Hematopoietic Stem Cells in the Bone Marrow. Irradiation, extracellular vesicle treatment and bone marrow cell phenotyping was carried out as described in the Materials and Methods section. Cellular subpopulations were identified as Lin-Sca1+cKit+CD34-CD135- long-term hematopoietic stem cells (LT-HSC), Lin-Sca1+cKit+CD34+CD135- short-term hematopoietic stem cells (ST-HSC) and Lin-Sca1+cKit+CD34+CD135+ multipotent progenitor cells (MPP) [ , ] and were measured by flow cytometry. ( A ) multipotent progenitors; ( C ) short-term hematopoietic stem cells; ( E ) long-term hematopoietic stem cells. Relative changes in hematopoietic stem cell subpopulations were evaluated 24 h after treatment. Grey bars represent total-body irradiated mice; red bars represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 24 h after irradiation; yellow bars represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 3 months after irradiation. ( B ) multipotent progenitors; ( D ) short-term hematopoietic stem cells; ( F ) long-term hematopoietic stem cells. Relative changes in hematopoietic stem cell numbers were evaluated 3 months after treatment. Grey bars represent total-body irradiated mice; blue bars represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 24 h after irradiation. ( G ) Distribution of individual subpopulations within the hematopoietic stem and progenitor cells was evaluated 24 h after treatment. Left columns represent total-body irradiated mice; middle columns represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 24 h after irradiation; right columns represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 3 months after irradiation. ( H ) Distribution of individual subpopulations within the hematopoietic stem and progenitor cells was evaluated 3 months after treatment. Left columns represent total-body irradiated mice; right columns represent mice treated with bone marrow-derived extracellular vesicles, which were isolated from mice 24 h after irradiation. Bars represent mean fraction of subpopulations, dots show individual values, error bars represent SD. n = 6–8, significance tested for individual subtypes of hematopoietic stem cells by Student’s t -test, p * < 0.05, p ** < 0.01, p *** < 0.001.

Article Snippet: The following directly labelled anti-mouse monoclonal antibodies were used: CD90.2-PE and CD45-PE/Cy7 for lymphoid progenitors (LP), Gr1-AF647 and CD11b-PE for granulocytes/monocytes (GM), Lineage Cocktail (CD3, Gr1, CD11b, CD45R, Ter119)-PB, Sca1-PE, cKit (CD117)-BV785, CD34-AF647, CD135-PE/Cy5 for hematopoietic stem and progenitor cells (HSPCs) and Lineage Cocktail-PB, Sca1-PE and CD44-AF647 for mesenchymal stem/stromal cells (MSCs), all purchased from SONY (SONY Biotechnology, San Jose, CA, USA).

Techniques: Irradiation, Flow Cytometry, Derivative Assay, Isolation