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: 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: Fluorescence, Over Expression, Mutagenesis, Western Blot, Tube Formation Assay, shRNA, Expressing, Flow Cytometry, Staining, 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

Journal: The Journal of Experimental Medicine

Article Title: Loss of decay-accelerating factor triggers podocyte injury and glomerulosclerosis

doi: 10.1084/jem.20191699

Figure Lengend Snippet: ADR induces PLAD-dependent cleavage of DAF. (A and B) Representative images (A) and distribution of DAF expression (B) quantified in hiPod exposed to vehicle, ADR (0.3 µg/ml), PLADi (1 µM), or ADR + PLADi (0.3 µg/ml in 1 µM) for 24 h. DAF IF signal was normalized to actin expression pixel by pixel, and the MFI for each cell was computed. Results are representative of two independent experiments with similar results. (C and D) Representative blots (C) and densitometric analysis (D) of PLAD expression in hiPod cell lysates previously exposed to vehicle or ADR for 24 h. (E and F) Representative blots (E) and densitometric analysis (F) of DAF in the supernatants of hiPod exposed to ADR for 24 h with or without PLADi (WB). (G) Representative blot of DAF in the urine from BALB/c male mice at 2 wk after treatment with vehicle or ADR compared with recombinant mouse DAF (rDAF). In each group, we pooled and concentrated urine samples from eight mice (see Materials and methods). All experimental data were verified in at least three independent experiments. *P < 0.05; n.s., not significant. Scale bars: 50 µm. Error bars are SEM.

Article Snippet: The urine from mouse and human samples was separated on 4–20% precast Protean TGX gels (Bio-Rad Laboratories) with recombinant mouse (55 μg/ml, 5490-CD-050; R&D Systems) or human (15 μg/ml, SRP6437; Sigma-Aldrich) CD55 protein, respectively.

Techniques: Expressing, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Loss of decay-accelerating factor triggers podocyte injury and glomerulosclerosis

doi: 10.1084/jem.20191699

Figure Lengend Snippet: FSGS in humans is associated with DAF down-regulation and complement activation. (A–D) C3 (A), C3aR (B), C5aR (C), and DAF mRNA (D) expression in glomeruli of human biopsy specimens with pathological diagnosis of FSGS or diabetic kidney disease compared with normal kidneys. Data are from previously published microarray studies by and were subjected to further analysis using Nephroseq. (E–H) Representative renal staining and data quantification for C3d (IF; E and F) and DAF (immunohistochemistry; G and H) in patients with FSGS ( n = 18) and in kidneys from healthy renal donors ( n = 10). (I) Correlation between protein and C3a in urine samples from 27 patients with FSGS taken at the time of kidney biopsy (before therapy). (J and K) Differences in proteinuria (J) and urinary C3a (K) measured before versus 3–6 mo after steroid therapy in a subset of 13 patients with FSGS. (L) Correlation between the change in proteinuria and change in urinary C3a before and after therapy for each of the same 13 patients. (M) Representative blot of DAF in the urine from healthy control individuals and patients with FSGS compared with recombinant human DAF (rDAF). In each group, we pooled and concentrated urine samples from five and five subjects, respectively (see Materials and methods). *P ≤ 0.05. Scale bars: 25 μm. Error bars are SEM.

Article Snippet: The urine from mouse and human samples was separated on 4–20% precast Protean TGX gels (Bio-Rad Laboratories) with recombinant mouse (55 μg/ml, 5490-CD-050; R&D Systems) or human (15 μg/ml, SRP6437; Sigma-Aldrich) CD55 protein, respectively.

Techniques: Activation Assay, Expressing, Biomarker Discovery, Microarray, Staining, Immunohistochemistry, Control, Recombinant