Journal: Journal for Immunotherapy of Cancer

Article Title: CD55-expressing myeloid-derived suppressor cells (MDSCs) drive cancer immunoevasion

doi: 10.1136/jitc-2025-012980

Figure Lengend Snippet: CD55 is highly and consistently on MDSCs in patients with cancer. ( A–H ) UMAP plots showing MDSCs score, CD55 expression, and colocalization of MDSCs score with CD55 expression in myeloid cells from scRNA-seq datasets of various cancer types, including ( A ) colorectal (n=30), ( B ) gastric (n=55), ( C ) liver (n=66), ( D ) lung (n=54), ( E ) prostate (n=34), ( F ) breast (n=70), ( G ) cervical (n=11), and ( H ) ovarian cancer (n=12). ( I–K ) Flow cytometric analysis of CD55 expression (MFI) and the percentage of CD55-positive cells in ( I ) total MDSCs, ( J ) PMN-MDSCs, and ( K ) M-MDSCs isolated from peripheral blood of healthy donors (n=10) and patients with different cancer types (n=10 per cancer type), as indicated. ( L ) Representative immunofluorescence images showing CD55 (green) and CD33 (red) expression in adjacent normal tissues and tumor tissues from patients with colorectal cancer (n=10). Nuclei were stained with DAPI (blue). Insets show higher magnification of the boxed areas. The quantification of CD33 + CD55 + cells/mm² is presented on the right. Data are presented as mean± SEM. Data of ( I ), ( J ), and ( K ) were analyzed using one-way ANOVA followed by Bonferroni’s post hoc test. Data of ( L ) was analyzed using Student’s t-test. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, and ns for non-significant. ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; MDSCs, myeloid-derived suppressor cells; MFI, mean fluorescence intensity; M-MDSCs, monocytic myeloid-derived suppressor cells; PMN-MDSCs, polymorphonuclear myeloid-derived suppressor cells; scRNA-seq, single-cell RNA sequencing; UMAP, uniform manifold approximation and projection.

Article Snippet: Purified murine CD55 protein (TargetMol) at various concentrations (320, 160, 80, 40, 20, 10, and 5 nM) was preincubated with each test compound (10 μM, dissolved in DMSO, Sigma) and then injected over a sensor chip surface on which murine PYGL protein had been immobilized using standard amine coupling procedures.

Techniques: Expressing, Isolation, Immunofluorescence, Staining, Derivative Assay, Fluorescence, Single Cell, RNA Sequencing

Journal: Journal for Immunotherapy of Cancer

Article Title: CD55-expressing myeloid-derived suppressor cells (MDSCs) drive cancer immunoevasion

doi: 10.1136/jitc-2025-012980

Figure Lengend Snippet: Global CD55 knockout inhibits tumor progression and alleviates the immunosuppressive tumor microenvironment in MC38-bearing mice. ( A ) Representative images of tumors from WT and Cd55 −/− mice at endpoint. ( B ) Tumor growth curves in WT and Cd55 −/− mice over time. ( C ) Tumor weight at endpoint in WT and Cd55 −/− mice. ( D ) Flow cytometry analysis of immune cell subsets in the spleens of WT and Cd55 −/− tumor-bearing mice, including MDSCs, M-MDSCs, PMN-MDSCs, TAMs, DCs, CD3 + T cells, CD4 + T cells, CD8 + T cells, NK cells, and B cells. ( E ) Flow cytometry analysis of the same immune cell subsets within tumors from WT and Cd55 −/− mice. ( F ) Representative immunofluorescence staining of tumor sections showing CD4 + T cells (yellow), CD8 + T cells (green) and Gr-1 + MDSCs (red) in WT and Cd55 −/− mice. Nuclei were counterstained with DAPI (blue). Quantification of CD4 + T cells, CD8 + T cells and MDSCs per field is shown in the right panels. Data are presented as mean± SEM. All data are presented from n=5 biological replicates per group and were analyzed using Student’s t-test. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, and ns for non-significant. DAPI, 4′,6-diamidino-2-phenylindole; DCs, dendritic cells; MDSCs, myeloid-derived suppressor cells; M-MDSCs, monocytic myeloid-derived suppressor cells; NK, natural killer; PMN-MDSCs, polymorphonuclear myeloid-derived suppressor cells; TAMs, tumor-associated macrophages; WT, wild-type.

Article Snippet: Purified murine CD55 protein (TargetMol) at various concentrations (320, 160, 80, 40, 20, 10, and 5 nM) was preincubated with each test compound (10 μM, dissolved in DMSO, Sigma) and then injected over a sensor chip surface on which murine PYGL protein had been immobilized using standard amine coupling procedures.

Techniques: Knock-Out, Flow Cytometry, Immunofluorescence, Staining, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: CD55-expressing myeloid-derived suppressor cells (MDSCs) drive cancer immunoevasion

doi: 10.1136/jitc-2025-012980

Figure Lengend Snippet: MDSC-specific deletion of CD55 contributes to tumor suppression and immunosuppressive tumor microenvironment alleviation in MC38-bearing mice. ( A ) Tumor growth in WT mice that received bone marrow transplantation from either WT or Cd55 − / − donors. Representative tumor images and tumor volume curves are shown. ( B ) Tumor growth in WT and Cd55 − / − mice treated with either isotype control or anti-Gr-1 antibody (MDSCs depletion). Representative tumor images and tumor volume curves are shown. ( C ) Tumor growth in myeloid-specific Cd55 knockout mice ( Cd55 fl/fl Lyz2-cre ) and littermate controls ( Cd55 fl/fl ). Representative tumor images and tumor volume curves are shown. ( D ) Flow cytometric analysis and quantification of MDSCs, PMN-MDSCs, and M-MDSCs in tumors from Cd55 fl/fl and Cd55 fl/fl Lyz2-cre mice. For quantification, the percentages of PMN-MDSCs and M-MDSCs were calculated by multiplying their proportions within the CD11b + gate by the percentage of CD11b + cells within the CD45 + gate. ( E ) Flow cytometric analysis and quantification of T cells, including CD3 + T cells, CD4 + T cells, and CD8 + T cells, in tumors from Cd55 fl/fl and Cd55 fl/fl Lyz2-cre mice. For quantification, the percentages of CD4 + and CD8 + T cells were calculated by multiplying their proportions within the CD3 + gate by the percentage of CD3 + cells within the CD45 + gate. ( F ) Representative immunofluorescence staining of tumor sections from Cd55 fl/fl and Cd55 fl/fl Lyz2-cre mice. CD4 + and CD8 + T cells were stained with anti-CD4 (yellow) and anti-CD8 (green), respectively; MDSCs were stained with anti-Gr-1 (red); nuclei were counterstained with DAPI (blue). Quantification of CD4 + T cells, CD8 + T cells, and MDSCs per field is shown in the right panels. Data are presented as mean±SEM. Data in ( A ), ( C ), ( D ), ( E ), and ( F ) were analyzed using Student’s t-test. Data in ( B ) were analyzed using two-way ANOVA followed by Bonferroni’s post hoc test. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, and ns for non-significant. ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; MDSCs, myeloid-derived suppressor cells; M-MDSCs, monocytic myeloid-derived suppressor cells; PMN-MDSCs, polymorphonuclear myeloid-derived suppressor cells; WT, wild-type.

Article Snippet: Purified murine CD55 protein (TargetMol) at various concentrations (320, 160, 80, 40, 20, 10, and 5 nM) was preincubated with each test compound (10 μM, dissolved in DMSO, Sigma) and then injected over a sensor chip surface on which murine PYGL protein had been immobilized using standard amine coupling procedures.

Techniques: Transplantation Assay, Control, Knock-Out, Immunofluorescence, Staining, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: CD55-expressing myeloid-derived suppressor cells (MDSCs) drive cancer immunoevasion

doi: 10.1136/jitc-2025-012980

Figure Lengend Snippet: CD55 deficiency suppresses the formation and immunosuppressive activity of MDSCs. ( A ) Schematic of BM chimera generation: BM cells from CD45.1 (WT) and CD45.2 ( Cd55 − / − ) donor mice were intravenously transplanted into lethally irradiated syngeneic CD45.2 WT recipient mice. 6 weeks post-transplantation, recipient mice were subcutaneously inoculated with the MC38 cell line. Representative flow cytometry plots show the proportion of CD45.1 + and CD45.2 + cells in the peripheral blood. ( B ) Flow cytometric analysis of MDSCs, M-MDSCs, and PMN-MDSCs in the spleen and tumor of chimeric mice gated on CD45.1 (WT donor-derived) and CD45.2 ( Cd55 − / − donor-derived) cells. Quantification of each MDSC subset is shown. ( C ) Flow cytometric analysis of CD55 expression in BM-derived MDSCs from WT and Cd55 − / − mice. MFI is quantified. ( D ) Flow cytometric analysis and quantification of BM-derived MDSC populations (total MDSCs, M-MDSCs, and PMN-MDSCs) from WT and Cd55 − / − mice. For quantification, the percentages of PMN-MDSCs and M-MDSCs were calculated by multiplying their proportions within the CD11b + gate by the percentage of CD11b + cells within the CD45 + gate. ( E ) qPCR analysis of immunosuppressive genes ( Arg1, S100a8, S100a9, Il10, and Tgfb1 ) in freshly isolated BM cells and BM-derived MDSCs from WT and Cd55 − / − mice. ( F ) Proliferation of CFSE-labeled CD8 + T cells co-cultured with BM-derived MDSCs from WT or Cd55 − / − mice at different ratios (2:1, 4:1, 8:1). Proliferation was measured by CFSE dilution; representative histograms and quantification are shown. ( G ) Flow cytometric analysis of IFN-γ and TNF-α production by CD8 + T cells co-cultured with BM-derived MDSCs from WT or Cd55 − / − mice, with or without stimulation. Quantification of cytokine-producing CD8 + T cells is shown. Data are presented as mean±SEM. All groups had n=3 biological replicates. Data in ( B ), ( C ), ( D ), ( F ), and ( G ) were analyzed using Student’s t-test. Data in ( E ) were analyzed using two-way ANOVA followed by Bonferroni’s post hoc test. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, and ns for non-significant. ANOVA, analysis of variance; ARG-1, arginase-1; BM, bone marrow; CFSE, carboxyfluorescein succinimidyl ester; IFN-γ, interferon-gamma; MDSCs, myeloid-derived suppressor cells; MFI, mean fluorescence intensity; M-MDSCs, monocytic myeloid-derived suppressor cells; PMN-MDSCs, polymorphonuclear myeloid-derived suppressor cells; qPCR, quantitative PCR; TNF-α, tumor necrosis factor-alpha; WT, wild-type.

Article Snippet: Purified murine CD55 protein (TargetMol) at various concentrations (320, 160, 80, 40, 20, 10, and 5 nM) was preincubated with each test compound (10 μM, dissolved in DMSO, Sigma) and then injected over a sensor chip surface on which murine PYGL protein had been immobilized using standard amine coupling procedures.

Techniques: Activity Assay, Irradiation, Transplantation Assay, Flow Cytometry, Derivative Assay, Expressing, Isolation, Labeling, Cell Culture, Fluorescence, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: CD55-expressing myeloid-derived suppressor cells (MDSCs) drive cancer immunoevasion

doi: 10.1136/jitc-2025-012980

Figure Lengend Snippet: CD55 interacts with PYGL to promote glycolysis and thereby enhance the immunosuppressive activity of MDSCs. ( A ) Volcano plot showing differentially expressed proteins between CD55-immunoprecipitated (CD55-IP) and IgG control groups in BM-derived MDSCs. ( B ) Volcano plot comparing mRNA profiles of BM-derived MDSCs from Cd55 − / − and WT mice. ( C ) Venn diagram showing overlap of significantly upregulated proteins in the CD55-IP group and mRNA upregulated in WT MDSCs. PYGL was identified as a common target. ( D ) Co-IP analysis demonstrating the interaction between CD55 and PYGL in BM-derived MDSCs. ( E ) Western blot analysis and quantification of CD55, total PYGL, p-PYGL, and the p-PYGL/PYGL ratio in freshly isolated BM cells and BM-derived MDSCs from WT and Cd55 − / − mice. ( F ) Seahorse analysis showing PER and glycolytic function (basal and compensatory glycolysis) in fresh BM and BM-derived MDSCs from WT and Cd55 − / − mice. ( G ) Western blot and quantification of STAT3 and p-STAT3 in fresh BM and BM-derived MDSCs from WT and Cd55 − / − mice. ( H ) Western blot analysis and quantification of CD55, PYGL, p-PYGL and the p-PYGL/PYGL ratio in WT or Cd55 − / − MDSCs transduced with shRNA against Pygl. ( I ) Seahorse analysis showing PER and glycolytic function in shRNA-treated WT and Cd55 − / − MDSCs, with or without Pygl knockdown. ( J ) Western blot and quantification of STAT3 and p-STAT3 in MDSCs from WT and Cd55 − / − mice transduced with control or Pygl-targeting shRNA. ( K ) qPCR analysis of immunosuppressive gene expression ( Arg1, S100a8, S100a9, Il10, and Tgfb1 ) in MDSCs from the indicated groups. Data are presented as mean±SEM. All groups had n=3 biological replicates. Data in ( E ), ( F ), ( G ), ( H ), ( I ), ( J ), and ( K ) were analyzed using two-way ANOVA followed by Bonferroni’s post hoc test. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, and ns for non-significant. ANOVA, analysis of variance; ARG-1, arginase-1; BM, bone marrow; Co-IP, Co-immunoprecipitation; 2-DG, 2-deoxyglucose; IL, interleukin; IP, immunoprecipitation; MDSCs, myeloid-derived suppressor cells; mRNA, messenger RNA; PER, proton efflux rate; p-STAT3, phosphorylated STAT3; PYGL, glycogen phosphorylase; qPCR, quantitative PCR; Rot/AA, rotenone/antimycin A; shRNA, short hairpin RNA; WT, wild-type.

Article Snippet: Purified murine CD55 protein (TargetMol) at various concentrations (320, 160, 80, 40, 20, 10, and 5 nM) was preincubated with each test compound (10 μM, dissolved in DMSO, Sigma) and then injected over a sensor chip surface on which murine PYGL protein had been immobilized using standard amine coupling procedures.

Techniques: Activity Assay, Immunoprecipitation, Control, Derivative Assay, Co-Immunoprecipitation Assay, Western Blot, Isolation, Transduction, shRNA, Knockdown, Gene Expression, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: CD55-expressing myeloid-derived suppressor cells (MDSCs) drive cancer immunoevasion

doi: 10.1136/jitc-2025-012980

Figure Lengend Snippet: Tomatidine inhibits CD55-mediated phosphorylation of PYGL in MDSCs to suppress glycolysis and immunosuppressive. ( A ) Molecular docking model illustrating the predicted interaction interface between CD55 and PYGL. ( B ) SPR sensorgrams showing the binding kinetics between CD55 and PYGL under the potential drug interventions. ( C ) Western blot analysis of CD55, total PYGL, and p-PYGL, as well as the p-PYGL/PYGL ratio, in BM-derived MDSCs treated with increasing concentrations of tomatidine (0–25 μM). Quantification of protein expression levels, including the phosphorylation ratio, is shown below. ( D ) Flow cytometric analysis of MDSC populations (total MDSCs, PMN-MDSCs, and M-MDSCs) in vitro treated with tomatidine. In vitro treatment with tomatidine. A representative gating strategy and quantification of each subset are shown. ( E ) Seahorse analysis was conducted to measure the PER in MDSCs exposed to increasing concentrations of tomatidine. Quantification of both basal and compensatory glycolysis is provided. ( F ) Western blot analysis of total STAT3 and p-STAT3 in BM-derived MDSCs following tomatidine treatment. Protein quantification is shown on the right. ( G ) qPCR analysis of immunosuppressive gene expression ( Arg1, S100a8, S100a9, Il10, and Tgfb1 ) in BM-derived MDSCs treated with tomatidine at indicated concentrations. Data are presented as mean±SEM. All groups had n=3 biological replicates. Data in ( C ), ( D ), ( E ), ( F ), and ( G ) were analyzed using one-way ANOVA followed by Bonferroni’s post hoc test. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, and ns for non-significant. ANOVA, analysis of variance; ARG-1, arginase-1; BM, bone marrow; 2-DG, 2-deoxyglucose; IL, interleukin; MDSCs, myeloid-derived suppressor cells; M-MDSCs, monocytic myeloid-derived suppressor cells; mRNA, messenger RNA; PER, proton efflux rate; PMN-MDSCs, polymorphonuclear myeloid-derived suppressor cells; p-STAT3, phosphorylated STAT3; PYGL, glycogen phosphorylase; qPCR, quantitative PCR; Rot/AA, rotenone/antimycin A; SPR, surface plasmon resonance.

Article Snippet: Purified murine CD55 protein (TargetMol) at various concentrations (320, 160, 80, 40, 20, 10, and 5 nM) was preincubated with each test compound (10 μM, dissolved in DMSO, Sigma) and then injected over a sensor chip surface on which murine PYGL protein had been immobilized using standard amine coupling procedures.

Techniques: Phospho-proteomics, Binding Assay, Western Blot, Derivative Assay, Expressing, In Vitro, Gene Expression, Real-time Polymerase Chain Reaction, SPR Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: CD55-expressing myeloid-derived suppressor cells (MDSCs) drive cancer immunoevasion

doi: 10.1136/jitc-2025-012980

Figure Lengend Snippet: GM-CSF and IL-6 induce CD55 expression in MDSCs via C/EBPβ signaling. ( A ) Flow cytometry analysis showing increased CD55 expression (MFI) on MDSCs after GM-CSF and IL-6 stimulation. ( B ) Schematic of the DNA pulldown assay using a CD55 promoter probe and mass spectrometry analysis. A Venn diagram shows the intersection of identified transcription factors from DNA pulldown, UCSC, Remap, and TF database. ( C ) Luciferase reporter constructs containing WT or mutated CD55 promoter regions at BS1–BS3. ( D ) Relative luciferase activity in BM-derived MDSCs transfected with C/EBPβ and indicated promoter constructs. ( E ) Schematic of the CD55 promoter region with three putative C/EBPβ binding motifs. ( F ) ChIP confirming C/EBPβ binding at CD55 promoter sites. Quantification of enrichment (% of input) shown on the right. ( G ) C/EBPβ expression in BM-derived MDSCs under GM-CSF and IL-6 stimulation, with or without CEBPB knockdown. ( H ) CD55 expression in BM-derived MDSCs under GM-CSF and IL-6 stimulation with or without C/EBPβ knockdown. ( I ) Percentage of BM-derived MDSCs under GM-CSF and IL-6 stimulation among CD45 + cells after C/EBPβ knockdown. Data are presented as mean±SEM. All groups had n=3 biological replicates. Data in ( A ), ( D ), and ( E ) were analyzed using Student’s t-test. Data in ( G ), ( H ), and ( I ) were analyzed using one-way ANOVA followed by Bonferroni’s post hoc test. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, and ns for non-significant. ANOVA, analysis of variance; BM, bone marrow; ChIP, chromatin immunoprecipitation; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; LC, liquid chromatography; MDSCs, myeloid-derived suppressor cells; MFI, mean fluorescence intensity; MS, mass spectrometry; WT, wild-type.

Article Snippet: Purified murine CD55 protein (TargetMol) at various concentrations (320, 160, 80, 40, 20, 10, and 5 nM) was preincubated with each test compound (10 μM, dissolved in DMSO, Sigma) and then injected over a sensor chip surface on which murine PYGL protein had been immobilized using standard amine coupling procedures.

Techniques: Expressing, Flow Cytometry, Mass Spectrometry, Luciferase, Construct, Activity Assay, Derivative Assay, Transfection, Binding Assay, Knockdown, Chromatin Immunoprecipitation, Liquid Chromatography, Fluorescence

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet: Receptor-ligand CRISPR-Cas9 activation screen reveals that CD55 interacts with HLA-C∗07:01-VRIG tetramers (A) Schematic of the receptor ligand CRISPR-Cas9 activation screen. K562 cells transduced with a genome-wide activation library were stained with a pool of three HLA tetramers (HLA-A∗02:01-NLVP, HLA-B∗07:02-TPRV, and HLA-C∗07:01-VRIG), and enriched gRNAs in stained cells were identified using NGS. (B) SigmaFC scores of genes from two replicate screens. SigmaFC scores were calculated using PinAplPy, and top hits are annotated. (C) K562 cells stably expressing dCas9 and transduced with a gRNA upregulating CD55 or a control guide were stained with the HLA-A, -B, -C, or tetramers as in (A) or with HLA-E∗01:01-VMAP tetramers and analyzed by flow cytometry. (D) In vitro co-immunoprecipitation of recombinant CD55-Fc with HLA-A∗02:01-NLVP, HLA-B∗07:02-TPRV, HLA-C∗07:01-VRIG, or HLA-E∗01:01-VMAP tetramers. (E) Three different cell lines (HeLa, PC-3M, or SiHa) that express CD55 endogenously were stained for CD55 (top) or with HLA-C∗07:01-VRIG tetramers (bottom) and analyzed by flow cytometry. (F) HeLa wild-type or HeLa CD55 KO cells were stained with αCD55 or HLA-C∗07:01-VRIG tetramers and analyzed by flow cytometry. All data except (B) represent at least three independent experiments. CRISPRa, CRISPR activation screen; TMs, tetramers; WT, wild-type; KO, knockout. Related to Figure S1 and Table S1 .

Article Snippet: CD55-Fc , Sino Biological , Cat: 10101-H02H.

Techniques: CRISPR, Activation Assay, Transduction, Genome Wide, Staining, Stable Transfection, Expressing, Control, Flow Cytometry, In Vitro, Immunoprecipitation, Recombinant, Knock-Out

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet: Interaction of CD55 with HLA-C∗07:01-VRIG tetramers is allotype and peptide specific (A) HEK293T cells were transfected with a plasmid containing GFP and a truncation mutant of CD55 and analyzed by flow cytometry. GFP+ positive cells were analyzed for staining with HLA-C∗07:01-VRIG. Each mutant removes an additional SCR domain from CD55. Data are represented as mean ± SD. (B) HeLa cells were stained with HLA-C∗07:01-VRIG tetramers after pre-incubation with CD55 blocking antibodies targeting different SCR domains on CD55 and analyzed by flow cytometry. (C) HeLa cells were stained with either HLA-C∗07:01 or HLA-C∗07:02 tetramers loaded with the VRIG peptide and analyzed by flow cytometry. (D) HeLa cells were stained with HLA-C∗07:01 tetramers loaded with different alanine mutants of the VRIGHLYIL peptide and analyzed by flow cytometry. (E) CD55-Fc was immobilized on a Prot-G chip for SPR data using HLA-C∗07:01-VRIG tetramers as analyte to determine interaction on and off rates and K D . Response units were measured with increasing concentrations of HLA-C∗07:01-VRIG tetramers. All data represent at least three independent experiments, except (E), which represents a biological duplicate. FL, full length. Related to Figure S2 , Tables S2 and .

Article Snippet: CD55-Fc , Sino Biological , Cat: 10101-H02H.

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Flow Cytometry, Staining, Incubation, Blocking Assay

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet:

Article Snippet: CD55-Fc , Sino Biological , Cat: 10101-H02H.

Techniques: Virus, Recombinant, Blocking Assay, Genome Wide, Activation Assay, CRISPR, Knock-Out, Mutagenesis, Plasmid Preparation, Software, Imaging

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet:

Article Snippet: CD55-Fc , Sino Biological , Cat: 10101-H02H.

Techniques: Virus, Recombinant, Blocking Assay, Genome Wide, Activation Assay, CRISPR, Knock-Out, Mutagenesis, Plasmid Preparation, Software, Imaging

Journal: Cell reports

Article Title: The expression profile and tumorigenic mechanisms of CD97 (ADGRE5) in glioblastoma render it a targetable vulnerability

doi: 10.1016/j.celrep.2023.113374

Figure Lengend Snippet: (A) Schematic of possible CD97 signaling mechanisms tested. Listed in red are the methods for testing. (B) Phosphoproteomic data collected from GBM samples identify five phosphorylation sites on the CD97 cytosolic C terminus. The heatmap depicts the percentage of GBM samples with the detected phosphorylation site. (C) Homogenous time-resolved fluorescence (HTRF) ratios from a β-arrestin recruitment assay performed after overexpression of WT CD97 or the ΔPS mutant. Two PDGCs (n = 2 for each) are compiled (n = 4 per condition; unpaired t test; ns p > 0.05; *p < 0.05; **p < 0.01). (D) Immunoblot for p-ERK1/ERK2 after overexpression of WT CD97 or the ΔPS mutant. (E) Bar graphs displaying densitometry ratios from (D) (n = 7; paired t test; ns p > 0.05; *p < 0.05). Two PDGCs (n = 3–4 for each) are compiled. (F) Bar graphs depicting the number of tumorspheres in a tumorsphere formation assay after CD97 knockdown followed by overexpression of shRNA-resistant forms of WT CD97 or the ΔPS mutant. The SCR shRNA groups used for normalization are not shown (n = 4 per PDGC; ANOVA F 5,12 = 80.79, p < 0.0001; Tukey’s multiple comparisons test: EV vs. WT: ***p < 0.001; EV vs. ΔPS: ns p > 0.05; WT vs. ΔPS: *p < 0.05). (G‒I) Single-cell RNA-/ATAC-seq data from showing expression of CD97 , CD55 , and THY1/CD90 . (J) A bar graph depicting the percentage of positive cells measured by flow cytometry after surface staining of PDGCs with fluorescein isothiocyanate (FITC)-conjugated antibodies against CD55, THY1/CD90, and an IgG control (n = 3 for each PDGC; two-way ANOVA F 2,20 = 95.11, p < 0.0001; Tukey’s multiple comparisons test: IgG vs. CD55: **p < 0.01; IgG vs. CD90: ****p < 0.00001; CD55 vs. CD90: ****p < 0.01). (K) Immunoblot for p-ERK1/ERK2 from CD97-overexpressing PDGCs plated on laminin or recombinant forms of putative ligands CD55 and THY1/CD90. (L) Quantification of densitometry ratios from immunoblots in (K) (n = 2–4 per PDGC; EV p-ERK/ERK: two-way ANOVA F 2,6 = 0.9450, p > 0.05; EV ERK/GAPDH: two-way ANOVA F 2,6 = 1.047, p > 0.05; CD97 overexpression p-ERK/ERK: two-way ANOVA F 2,16 = 12.48, p < 0.001; Tukey’s multiple comparisons test: laminin vs. CD55: ns, p < 0.05; laminin vs. CD90: ***p < 0.001; CD97 overexpression ERK/GAPDH: two-way ANOVA F 2,16 = 2.257, ns p > 0.05). Error bars indicate SEM.

Article Snippet: Wells were then additionally coated with recombinant human CD55 (Cat# 2009-CD-050, R&D) or recombinant human THY1/CD90 (Cat# 16897-HCCH, Sino Biological) at a concentration of 24 μg/mL overnight.

Techniques: Phospho-proteomics, Fluorescence, Over Expression, Mutagenesis, Western Blot, Tube Formation Assay, Knockdown, shRNA, Expressing, Flow Cytometry, Staining, Control, Recombinant

Journal: Cell reports

Article Title: The expression profile and tumorigenic mechanisms of CD97 (ADGRE5) in glioblastoma render it a targetable vulnerability

doi: 10.1016/j.celrep.2023.113374

Figure Lengend Snippet:

Article Snippet: Wells were then additionally coated with recombinant human CD55 (Cat# 2009-CD-050, R&D) or recombinant human THY1/CD90 (Cat# 16897-HCCH, Sino Biological) at a concentration of 24 μg/mL overnight.

Techniques: Recombinant, Modification, Disruption, Activation Assay, Knock-Out, shRNA