Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”). Scale bars: (A–F) 500 μm; (G) 50 μm; (H–P) 500 μm.

Article Snippet: EphA2 antibody, anti-mouse, APC, REAfinity , Miltenyi Biotec B.V. & Co. KG , Cat# 130-109-187 RRID: AB_2651638.

Techniques: Imaging, Comparison, Staining, Fluorescence

Journal: Journal of Virology

Article Title: A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1128/JVI.00803-18

Figure Lengend Snippet: CRISPR-Cas9 guide RNA sequences used to target the indicated genes

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio; integrin α3 antibody (P1B5) was purchased from Calbiochem; integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) were obtained from R&D Systems; integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) were obtained from Novus Biologicals; integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) were obtained from BioLegend; xCT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ab37185 and ab181602, respectively) were obtained from Abcam; DC-SIGN antibody (DCN47.5) was obtained from Miltenyi Biotec; EphA4 antibody (4C8H5) was obtained from ThermoFisher; EphB2 antibody (2D12C6) was obtained from Santa Cruz Biotech; and Flag antibody (M2) was obtained from Sigma-Aldrich.

Techniques: CRISPR, Sequencing

Journal: Journal of Virology

Article Title: A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1128/JVI.00803-18

Figure Lengend Snippet: EphA4 and EphB2 are dispensable for infection of Caki-1 cells. (A, E, F, and H) A total of 120 μg (A, E, and H) or 50 μg (F) of the indicated whole-cell lysate proteins was run on a 10% SDS-PAGE gel and blotted for EphA4 (A and E) or EphB2 (F and H) and GAPDH as a loading control. (B and C) WT and EPHA4 KO Caki-1 cells (B) or WT, EPHA2 KO, and EPHA4/EPHA2 DKO Caki-1 cells (C) were infected with KSHV in triplicate, and the infection percentage was measured by flow cytometry at 2 days postinfection. The infection percentages of KO cell lines were normalized to the average infection percentage of WT cells, and data from a representative experiment are shown. (D) EPHA4 KO and EPHA4/EPHA2 DKO Caki-1 cells were immunostained for surface EphA2 expression. Gray histograms represent isotype controls. (G) WT and EPHB2 KO Caki-1 cells were infected with KSHV in triplicate, and infection percentages were quantified by flow cytometry at 2 days postinfection. The rates of infection of the KO line were normalized to the average WT infection rate, and data from a representative experiment are shown. *, P < 0.05.

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio; integrin α3 antibody (P1B5) was purchased from Calbiochem; integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) were obtained from R&D Systems; integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) were obtained from Novus Biologicals; integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) were obtained from BioLegend; xCT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ab37185 and ab181602, respectively) were obtained from Abcam; DC-SIGN antibody (DCN47.5) was obtained from Miltenyi Biotec; EphA4 antibody (4C8H5) was obtained from ThermoFisher; EphB2 antibody (2D12C6) was obtained from Santa Cruz Biotech; and Flag antibody (M2) was obtained from Sigma-Aldrich.

Techniques: Infection, SDS Page, Control, Flow Cytometry, Expressing

Journal: Journal of Virology

Article Title: A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1128/JVI.00803-18

Figure Lengend Snippet: Perturbations in KSHV receptor expression do not unexpectedly affect other known receptors. WT, EPHA2 KO, ITGB1 KO, and ITGAV/ITGA3 DKO Caki-1 cells were concurrently immunostained for surface expression of nontargeted, known KSHV receptors and analyzed by flow cytometry. Gray histograms represent isotype controls.

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio; integrin α3 antibody (P1B5) was purchased from Calbiochem; integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) were obtained from R&D Systems; integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) were obtained from Novus Biologicals; integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) were obtained from BioLegend; xCT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ab37185 and ab181602, respectively) were obtained from Abcam; DC-SIGN antibody (DCN47.5) was obtained from Miltenyi Biotec; EphA4 antibody (4C8H5) was obtained from ThermoFisher; EphB2 antibody (2D12C6) was obtained from Santa Cruz Biotech; and Flag antibody (M2) was obtained from Sigma-Aldrich.

Techniques: Expressing, Flow Cytometry

Journal: Journal of Virology

Article Title: A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1128/JVI.00803-18

Figure Lengend Snippet: EphA2 is required for infection of Caki-1 and HeLa cells. (A) WT and EPHA2 KO Caki-1 cells were immunostained for surface EphA2 expression with the SHM16 antibody and analyzed by flow cytometry. The gray histogram represents the isotype control. (B) WT and EPHA2 KO Caki-1 cells were infected with KSHV in duplicate, and infection rates were quantified by flow cytometry. The rate of infection of the EPHA2 KO pool was normalized to the average WT infection rate, and data were pooled from multiple experiments. (C) WT Caki-1 and EPHA2 KO clone A1 cells were immunostained for surface EphA2 with the antibody AF3035 and analyzed by flow cytometry. The gray histogram represents the isotype control. (D) Fifteen micrograms of whole-cell lysate protein from WT Caki-1 cells and EPHA2 KO clone A1 was run on an SDS-PAGE gel and blotted for EphA2 with AF3035 and GAPDH. (E) EPHA2 KO clone A1 Caki-1 cells were preblocked with EGFR-Fc or ephrin-A4–Fc at 10 μg/ml at 4°C and then infected in triplicate in the presence of EGFR-Fc or ephrin-A4–Fc at 5 μg/ml at 37°C. The infection percentage was measured by flow cytometry at 2 days postinfection, and percent infection was normalized to the average EPHA2 KO infection rate. (D) Mixed EPHA2 KO HeLa cells were immunostained for surface EphA2 expression with the SHM16 antibody and analyzed by flow cytometry. The gray histogram represents the isotype control. (E) Mixed EPHA2 KO HeLa cells were infected with KSHV in triplicate, and the infection percentage was measured by flow cytometry at 2 days postinfection. The cells were also immunostained for surface EphA2 and gated on EphA2-high or -low cells, as indicated in panel D. The rates of infection of EphA2-low cells were normalized to those of EphA2-high cells in each well, and data from a representative experiment are shown. *, P < 0.05.

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio; integrin α3 antibody (P1B5) was purchased from Calbiochem; integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) were obtained from R&D Systems; integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) were obtained from Novus Biologicals; integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) were obtained from BioLegend; xCT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ab37185 and ab181602, respectively) were obtained from Abcam; DC-SIGN antibody (DCN47.5) was obtained from Miltenyi Biotec; EphA4 antibody (4C8H5) was obtained from ThermoFisher; EphB2 antibody (2D12C6) was obtained from Santa Cruz Biotech; and Flag antibody (M2) was obtained from Sigma-Aldrich.

Techniques: Infection, Expressing, Flow Cytometry, Control, SDS Page

Journal: Journal of Virology

Article Title: A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1128/JVI.00803-18

Figure Lengend Snippet: EphA2, EphA4, and EphA5 rescue KSHV infection in EPHA2 KO Caki-1 cells. (A) Diagram of generalized full-length and PPT-3×Flag-mature ephrin receptor constructs. SAM, sterile alpha motif. (B and C) Live (B) or fixed and permeabilized (C) 3×Flag-tagged ephrin receptor-transduced EPHA2 KO cells and a vector control were immunostained for surface (B) or intracellular (C) 3×Flag expression and analyzed by flow cytometry. Gray histograms represent isotype controls. (D) The indicated cell lysates were run on 10% SDS-PAGE gels and blotted for 3×Flag, EphA4, and EphA5 with matched GAPDH as a loading control. For the Flag and EphA5 blots, 15 μg of whole-cell lysate protein was loaded. For the EphA4 blot, 120 μg of whole-cell lysate protein was loaded. (E) The indicated cell lines were immunostained for surface EphA2 or EphA5 expression and analyzed by flow cytometry. Gray histograms represent isotype controls. (F) The indicated cell lines were infected with KSHV in triplicate, and the infection rate was quantified by flow cytometry at 2 days postinfection. Data from a representative experiment are shown. (G) The 3×Flag expression histograms of infected 3×Flag-tagged ephrin receptor-transduced cell lines were divided into five successive gates, as shown. The infection rate within each gate was plotted against the fold MFI over the isotype of each gate. *, P < 0.05.

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio; integrin α3 antibody (P1B5) was purchased from Calbiochem; integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) were obtained from R&D Systems; integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) were obtained from Novus Biologicals; integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) were obtained from BioLegend; xCT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ab37185 and ab181602, respectively) were obtained from Abcam; DC-SIGN antibody (DCN47.5) was obtained from Miltenyi Biotec; EphA4 antibody (4C8H5) was obtained from ThermoFisher; EphB2 antibody (2D12C6) was obtained from Santa Cruz Biotech; and Flag antibody (M2) was obtained from Sigma-Aldrich.

Techniques: Infection, Construct, Sterility, Plasmid Preparation, Control, Expressing, Flow Cytometry, SDS Page

Journal: Journal of Virology

Article Title: A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1128/JVI.00803-18

Figure Lengend Snippet: The EphA2 ectodomain is sufficient to rescue the rate of infection of EPHA2 KO cells. (A) Diagram of EphA2 truncation and domain swap constructs. (B and C) WT, EPHA2 KO, and EPHA2 KO cells transduced with the EphA2 constructs indicated in panel A were infected with KSHV in triplicate, and the infection rate was quantified by flow cytometry at 2 days postinfection. The infection rates were normalized to the average rate of infection of WT cells, and data from a representative experiment are shown. (D) WT, EPHA2 KO, and the indicated transduced EPHA2 KO Caki-1 cells were immunostained for surface EphA2 expression and analyzed by flow cytometry. Gray histograms represent the isotype controls. *, P < 0.05.

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio; integrin α3 antibody (P1B5) was purchased from Calbiochem; integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) were obtained from R&D Systems; integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) were obtained from Novus Biologicals; integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) were obtained from BioLegend; xCT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ab37185 and ab181602, respectively) were obtained from Abcam; DC-SIGN antibody (DCN47.5) was obtained from Miltenyi Biotec; EphA4 antibody (4C8H5) was obtained from ThermoFisher; EphB2 antibody (2D12C6) was obtained from Santa Cruz Biotech; and Flag antibody (M2) was obtained from Sigma-Aldrich.

Techniques: Infection, Construct, Transduction, Flow Cytometry, Expressing

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: EPHA2 receptor consists of the ligand-binding domain, a cysteine-rich domain, two fibronectin-III domains, a tyrosine kinase (TK) domain, a sterile alpha motif (SAM), and a PDZ-binding motif. Novel mutations (yellow) and known SNPs were found in ligand-binding domain and TK domain by us recently

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Ligand Binding Assay, Sterility, Binding Assay

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: Thirty five SCC, 39 MPM tumor samples, and six cell lines have been used to determine EPHA2 gene amplification. Fold change relative to reference gene LINE-1

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Amplification

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: a Lysates of six MPM cell lines and the Met-5A, a mesothelial control cell line, were immunoblotted with EPHA2 antibody. b Immunohistochemistry representative pictures of 65 MPM tumor and nine normal mesothelium samples were used in MPM TMA. c Protein expression quantity of 65 MPM tumor and nine normal mesothelium samples were used in MPM TMA. d Protein expression quantity of 48 NSCLC SQ and 24 adjacent normal samples were used in SSC TMA. H&E, EPHA2, phospho-(p-)EPHA2, and ephrin A1 were stained and scored. N: normal, T: Tumor

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Control, Immunohistochemistry, Expressing, Staining

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: BEAS2B EPHA2 isogenic cells were used to treat a Taxol, b cisplatin, c SU11274, and d rapamycin. Mutation G391R cells showed resistant to cisplatin inhibition but sensitive to MET inhibitor SU11274 and mTOR inhibitor Rapamycin. EV: empty vector

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Mutagenesis, Inhibition, Plasmid Preparation

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: a MPM cell lines H28, H513, H2052, H2373, H2461, and H2596 were treated with cisplatin with 1, 5, and 10 μM for 48 h. b HEK293 EPHA2 isogenic cells treatment with doxazosin for 48 h

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques:

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: a The crystal structure of EphA2 in the auto-inhibited conformation. The distance between Y772 and K702 shown by dotted line is 20.3 Å, b The EphA2 conformation after MD simulations showing the minimum distance between Y772 and K702 which is 3.4 Å, c The distribution of the distance between Y772 and K702 in the wild type and mutants A859D and T647M, d The surface showing the ATP binding site in EphA2, and e The distribution of the volume in (Å 3 ) of the ATP-binding site for the wild type and the two mutants calculated from the snapshots of the MD simulations

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Binding Assay