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: Cancer Research

Article Title: Combination Therapy with HSP90 Inhibitor 17-DMAG Reconditions the Tumor Microenvironment to Improve Recruitment of Therapeutic T cells

doi: 10.1158/0008-5472.can-12-0538

Figure Lengend Snippet: Figure 1. 17-DMAG promotes proteasome-dependent EphA2 protein degradation in MCA205 sarcoma cells and the enhanced recognition of tumor cells by anti-EphA2 CD8þ T cells in vitro. A, MCA205 tumor cells were treated with various doses of 17-DMAG for 24 hours in vitro and then lysed, with EphA2 and control b-actin protein expression subsequently monitored by Western blotting as described in Materials and Methods. NC, negative control lysate from EphA2neg B16 melanoma cells. B, proteasome inhibitor (MG-132), but not lysosome inhibitor chloroquine (CLQ), blocks 17-DMAG (500 nmol/L)-induced degradation of EphA2 protein in MCA205 tumor cells. C, treatment of MCA205 cells with 17-DMAG at the indicated doses for 24 hours (or 48 hours, data not shown) did not affect MHC class I expression on tumor cells. D, 17-DMAG–treated EphA2þ MCA205 cells were better recognized versus control, untreated tumor cells by anti-EphA2 CD8þ T cells (developed from EphA2/ mice, per Supplementary Fig. S1 and Materials and Methods) in CD107 translocation assays as described in Materials and Methods. All data are representative of those obtained in 3 independent experiments.

Article Snippet: The following primary antibodies were used for staining sections: rat anti-mouse CD31 (BD Biosciences), rabbit anti-mouse EphA2 (Santa Cruz Biotechnology), rat antimouse VCAM-1, goat anti-mouse CXCL10 (R&D Systems).

Techniques: In Vitro, Control, Expressing, Western Blot, Negative Control, Translocation Assay

Journal: Cancer Research

Article Title: Combination Therapy with HSP90 Inhibitor 17-DMAG Reconditions the Tumor Microenvironment to Improve Recruitment of Therapeutic T cells

doi: 10.1158/0008-5472.can-12-0538

Figure Lengend Snippet: Figure 2. Treatment of mice bearing established MCA205 tumors with oral 17-DMAG transiently promotes a therapeutically preferred immunophenotype in the TME and is optimally effective in a 5-day regimen. A, C57BL/6 mice bearing established MCA205 tumors (day 18; 100 mm2 mean tumor size) were left untreated or they were administered 17-DMAG (10, 15, or 25 mg/kg/d for up to 10 days via oral gavage) and tumor size (mean SD, 5 animals per group) monitored longitudinally. , P < 0.05; , P < 0.01 (ANOVA) for 15 or 25 versus 10 mg/kg/d or untreated; not significant (ANOVA) for 15 versus 25 mg/kg/d. B, tumors were excised on the indicated day after initiating treatment, and single-cell suspensions of enzymatic tumor digests analyzed for immune cell infiltrates by flow cytometry as described in Materials and Methods. Tumor cells isolated from enzymatic digests (per B) were also analyzed as target cells for anti-EphA2 CD8þ T effector cells generated from EphA2/ mice (see Supplementary Fig. S1) as monitored using CD107 translocation (C) and IFN-g secretion (D) assays as described in Materials and Methods. All data are representative of those obtained in 3 independent experiments. For B–D, , P < 0.05; , P < 0.01 (ANOVA) versus all other determinations.

Article Snippet: The following primary antibodies were used for staining sections: rat anti-mouse CD31 (BD Biosciences), rabbit anti-mouse EphA2 (Santa Cruz Biotechnology), rat antimouse VCAM-1, goat anti-mouse CXCL10 (R&D Systems).

Techniques: Cytometry, Isolation, Generated, Translocation Assay

Journal: Cancer Research

Article Title: Combination Therapy with HSP90 Inhibitor 17-DMAG Reconditions the Tumor Microenvironment to Improve Recruitment of Therapeutic T cells

doi: 10.1158/0008-5472.can-12-0538

Figure Lengend Snippet: Figure 3. The impact of 17-DMAG–based therapy for 5 days persists after discontinuation of drug delivery. A, MCA205 tumor–bearing mice (5 mice/group) were left untreated or they were treated for 5 days with orally administered 17-DMAG (15 mg/kg/d), with tumor growth then monitored over a 4-week period. B, EphA2 protein expression in tumors harvested from 17-DMAG–treated versus untreated mice was analyzed longitudinally by Western blotting as outlined in Materials and Methods. C, tumor cells from untreated or 17-DMAG–treated mice were analyzed at the indicated time points for their ability to be recognized by anti-EphA2 CD8þ T cells generated from EphA2/ mice (see Supplementary Fig. S1) in CD107 translocation and IFN-g secretion assays, as described in Materials and Methods. D, single-cell suspensions from harvested tumor digests were analyzed by flow cytometry for the indicated T-cell, DC, and MDSC phenotypes. All data are representative of those obtained in 3 independent experiments. , P < 0.05; , P < 0.01 (t test) for treated versus untreated controls.

Article Snippet: The following primary antibodies were used for staining sections: rat anti-mouse CD31 (BD Biosciences), rabbit anti-mouse EphA2 (Santa Cruz Biotechnology), rat antimouse VCAM-1, goat anti-mouse CXCL10 (R&D Systems).

Techniques: Expressing, Western Blot, Generated, Translocation Assay, Cytometry

Journal: Cancer Research

Article Title: Combination Therapy with HSP90 Inhibitor 17-DMAG Reconditions the Tumor Microenvironment to Improve Recruitment of Therapeutic T cells

doi: 10.1158/0008-5472.can-12-0538

Figure Lengend Snippet: Figure 4. 17-DMAG administration improves the immunogenicity and antitumor efficacy of an EphA2 peptide–based vaccine in the MCA205 tumor model. A, C57BL/6 mice bearing established EphA2þ MCA205 sarcomas (s.c. right flank) remained untreated or they were treated with DC-based vaccines (s.c., left flank on days 0 and 7 of the treatment regimen) that contained or lacked EphA2 peptide epitopes, alone or in combination with 17-DMAG (15 mg/kg/d on the first 5 days of the treatment regimen by oral gavage). Tumor size (mean SD) is reported in mm2. All the mice in the DC/EphA2 þ 17-DMAG–treated group rendered tumor-free (80%) were rechallenged (s.c., right flank) with MCA205 tumor cells on day 30 of the experiment (as indicated by arrow with R inset) and monitored through day 60 after treatment initiation. B, CD8þ TILs recovered from tumors on day 14 after treatment initiation were assessed for their ability to recognize syngenic control DCs pulsed with no peptide or DC pulsed with the EphA2671–679 þ EphA2682–689 peptides. After 48-hour incubation, cell-free supernatants were analyzed for IFN-g content by ELISA. Response to DC (no peptide) was <50 pg/mL in all instances. C, single-cell suspensions of enzymatically digested day 14 (posttreatment initiation) tumors were analyzed by flow cytometry for the indicated T-cell, DC, and MDSC phenotypes as described in Materials and Methods. Each filled circle represents data from an individual animal in a given control or treatment cohort, with the mean of data indicated by a gray bar for each cohort. All data are representative of those obtained in 3independent experiments. , P < 0.05; , P < 0.01 (ANOVA) versus all other cohorts.

Article Snippet: The following primary antibodies were used for staining sections: rat anti-mouse CD31 (BD Biosciences), rabbit anti-mouse EphA2 (Santa Cruz Biotechnology), rat antimouse VCAM-1, goat anti-mouse CXCL10 (R&D Systems).

Techniques: Immunopeptidomics, Vaccines, Control, Incubation, Enzyme-linked Immunosorbent Assay, Cytometry

Journal: Cancer Research

Article Title: Combination Therapy with HSP90 Inhibitor 17-DMAG Reconditions the Tumor Microenvironment to Improve Recruitment of Therapeutic T cells

doi: 10.1158/0008-5472.can-12-0538

Figure Lengend Snippet: Figure 5. 17-DMAG improves the antitumor efficacy of an EphA2 peptide–based vaccine in the EphA2neg B16 melanoma model based on immune targeting of EphA2þ VEC. A, C57BL/6 mice bearing established s.c. B16 melanomas (right flank) were left untreated or treated as outlined, with tumor size (mean SD) reported in mm2 followed for up to 30 days. , P < 0.001 (ANOVA) versus all other cohorts. B, day 14 (posttreatment initiation) tumors were harvested and tissue sections analyzed by immunofluorescence microscopy and MetaMorph quantitation for coexpression of CD31 (i.e., VEC) and EphA2 proteins as described in Materials and Methods. , P < 0.05; , P < 0.01 (ANOVA) versus all other cohorts. Anti-EphA2 CD8þ T cells isolated from the spleens of immune EphA2/ mice (C; as outlined in Supplementary Fig. S1), or TIL from B16 tumor–bearing animals treated with combined DC/EphA2 peptide vaccination þ 17-DMAG (D; per Supplementary Fig. 5A) were analyzed for reactivity against flow-sorted CD31þ VECs isolated from the tumors of B16-bearing animals left untreated or treated for 6 days with DC/EphA2 vaccine only, 17-DMAG or DC/EphA2 vaccine þ 17-DMAG. CD31þ kidney VECs were also flow-sorted from animals treated for 6 days with DC/EphA2 vaccine þ 17-DMAG to discern autoimmunity of T cells against tumor-uninvolved VECs. C, the MHC class I–restricted nature of VEC recognition by CD8þ T cells was assessed by inclusion of anti-class I or isotype control mAb per culture well, as described in Materials and Methods. C and D, , P < 0.05; , P < 0.01 (t test) versus control antibody treatment or untreated controls, respectively. All data are representative of those obtained in 3 independent experiments. HPF, high-power field.

Article Snippet: The following primary antibodies were used for staining sections: rat anti-mouse CD31 (BD Biosciences), rabbit anti-mouse EphA2 (Santa Cruz Biotechnology), rat antimouse VCAM-1, goat anti-mouse CXCL10 (R&D Systems).

Techniques: Microscopy, Quantitation Assay, Isolation, Control

Journal: Cancer Research

Article Title: Combination Therapy with HSP90 Inhibitor 17-DMAG Reconditions the Tumor Microenvironment to Improve Recruitment of Therapeutic T cells

doi: 10.1158/0008-5472.can-12-0538

Figure Lengend Snippet: Figure 6. 17-DMAG improves the antitumor efficacy of adoptively transferred anti-EphA2 CD8þ T cells in a combination therapy. A, C57BL/6 mice bearing established s.c. MCA205 sarcomas (right flank) were left untreated or they were treated with 17-DMAG (15 mg/kg/d provided orally on the first 5 days of the treatment regimen) adoptive transfer (i.v. tail vein on day 4 of the treatment regimen) of 5 106 CD8þ T cells isolated from EphA2/

Article Snippet: The following primary antibodies were used for staining sections: rat anti-mouse CD31 (BD Biosciences), rabbit anti-mouse EphA2 (Santa Cruz Biotechnology), rat antimouse VCAM-1, goat anti-mouse CXCL10 (R&D Systems).

Techniques: Adoptive Transfer Assay, Isolation

Journal: Cells

Article Title: The EphA2 Receptor Regulates Invasiveness and Drug Sensitivity in Canine and Human Osteosarcoma Cells

doi: 10.3390/cells13141201

Figure Lengend Snippet: EphA2 is overexpressed in osteosarcoma cells. ( A ) Canine osteosarcoma cells Abrams, Eva, McKinley, Gracie, D17, Payton, and Igler, as well as non-malignant canine osteoblast cells (CnOb), were lysed and EphA2 expression was analyzed by Western blotting using an anti-EphA2 antibody. Tubulin expression was used as a loading control. EphA2 expression was quantified by densitometry, normalized on corresponding tubulin controls, and presented as percentages relative to osteoblast control (100%). The graph summarizes three independent experiments. ( B ) EphA2 expression in human osteosarcoma cells U2OS, SAOS2, MG-63, SJSA1, and 143B, as well as in non-malignant human osteoblast cells (hOb), was analyzed as in A. The graph summarizes three independent experiments. ( C ) EphA2 expression in the indicated canine osteosarcoma cell lines, a commercially available canine osteoblast cell line (CnOb), and 3 in-house preparations of canine primary osteoblasts (cOb-1-3) isolated from femoral bone samples were assessed by Western blotting and quantified as in A. The graph presents EphA2 signal intensity as arbitrary units (AU). * p < 0.05, ** p < 0.01; n.s. statistically not significant.

Article Snippet: The primary antibody against EphA2 (#12927, Cell Signaling Technologies) was diluted in PBS containing 1% BSA and 0.3% Triton X-100 and added to sections, followed by incubation with the antibody for 1 h at room temperature.

Techniques: Expressing, Western Blot, Control, Isolation

Journal: Cells

Article Title: The EphA2 Receptor Regulates Invasiveness and Drug Sensitivity in Canine and Human Osteosarcoma Cells

doi: 10.3390/cells13141201

Figure Lengend Snippet: EphA2 promotes osteosarcoma cell proliferation. ( A ) Indicates that canine and human osteosarcoma cells were transduced with a specific shRNA targeting EphA2 (shA2) or with a non-silencing, scrambled shRNA (NS, control). EphA2 expression was then assessed by Western blotting using a specific anti-EphA2 antibody. ( B ) Abrams-NS and Abrams-shA2 cells were grown on glass coverslips to 70% confluency, fixed with 4% paraformaldehyde, and stained with anti-EphA2 (green), rhodamine phalloidin (cytoskeleton, red) and DAPI (cell nucleus, blue). Staining with a matching non-specific IgG was shown as specificity control. Fluorescent images were taken using an Olympus IX83 microscope at 200× magnification. Scale bar, 100 µm. ( C ) Indicates that cells were seeded in 96-well plates (2 × 10 3 cells per well, n ≥ 6) and cultured for 48 h. To assess cell viability, resazurin was added to the wells, and the fluorescence was measured after 4 h using a Varioskan LUX plate reader. Data are shown as mean ± SD. Experiments were repeated three times. *** p < 0.001.

Article Snippet: The primary antibody against EphA2 (#12927, Cell Signaling Technologies) was diluted in PBS containing 1% BSA and 0.3% Triton X-100 and added to sections, followed by incubation with the antibody for 1 h at room temperature.

Techniques: Transduction, shRNA, Control, Expressing, Western Blot, Staining, Microscopy, Cell Culture, Fluorescence

Journal: Cells

Article Title: The EphA2 Receptor Regulates Invasiveness and Drug Sensitivity in Canine and Human Osteosarcoma Cells

doi: 10.3390/cells13141201

Figure Lengend Snippet: EphA2 affects osteosarcoma cell motility and invasion. ( A ) Human 143B-NS and 143B-shA2, and canine Eva-NS and Eva-shA2 cells were seeded into 6-well plates (5 × 10 5 cells per well) in triplicates and cultured to form confluent monolayers. Using a 200 µL pipette tip, a wound/scratch was made in each monolayer, and wells were rinsed with medium to remove cell debris. Wound closure was monitored over time by imaging the same area of each wound (2 per scratch) using an inverted microscope at 40× magnification. Wound width in each image was measured using Microsoft PowerPoint. Graphs represent percentages of wounds closed at each timepoint relative to wound width at 0 h. Scale bar, 100 µm. ( B ) The indicated canine ( top ) and human ( bottom ) cells were seeded into transwell inserts (2 × 10 4 cells per insert) in serum-free media ( n = 3 per group). Cell culture media with 10% FBS was added to the lower chamber of each insert, and cells were incubated at 37 °C for 24 h. For assessing cell migration after 24 h, cells residing on the upper side of each insert porous membrane were removed by a cotton swab, and migrated cells at the lower surface of the membrane were fixed with methanol, stained with crystal violet, and counted. For cell counting, five selected fields of each insert were imaged at 100× magnification, and the average number of cells per field was calculated. Graphs represent percentages of migrated EphA2-silenced cells relative to that of matching non-silenced controls. ( C , D ) The indicated cells were serum-starved for 24 h and seeded into Matrigel-coated transwell inserts (2 × 10 4 cells per well) in serum-free media ( n = 3 per group). The lower chamber contained media with 10% FBS. After 48 h, the number of invaded cells at the lower surface of insert porous membranes were quantified and graphed as in ( B ). ( E ) Migration assays were conducted using canine Eva and human MG63 (3 × 10 4 cells per insert) OS cells as in ( B ). Soluble ephrinA1 ligand or IgG (1 µg/mL) was added to the upper chamber at cell seeding. Graphs summarize data from two independent experiments and represent the percentage of migrated cells relative to IgG controls. Data are shown as mean ± SD. Experiments were repeated three times unless otherwise indicated. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibody against EphA2 (#12927, Cell Signaling Technologies) was diluted in PBS containing 1% BSA and 0.3% Triton X-100 and added to sections, followed by incubation with the antibody for 1 h at room temperature.

Techniques: Cell Culture, Transferring, Imaging, Inverted Microscopy, Incubation, Migration, Membrane, Staining, Cell Counting

Journal: Cells

Article Title: The EphA2 Receptor Regulates Invasiveness and Drug Sensitivity in Canine and Human Osteosarcoma Cells

doi: 10.3390/cells13141201

Figure Lengend Snippet: EphA2 increases resistance to cisplatin. ( A ) The indicated canine OS cells were seeded into 96-well plates (4 × 10 3 cells per well) and allowed to adhere to the wells overnight. Culture media was replaced the next day with media containing increasing concentrations of cisplatin or media with PBS (as solvent control) at the volume matching the highest cisplatin dose as indicated ( n = 3 per group). The treated cells were cultured for 48 h. To assess cell survival, resazurin was added to the wells, and the fluorescence signal was quantified as in C. ( B ) The indicated human OS cells were tested for cisplatin sensitivity as described in A. Graphs represent cell survival as percentages relative to matching solvent controls. Data are shown as mean ± SD. Experiments were repeated three times. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibody against EphA2 (#12927, Cell Signaling Technologies) was diluted in PBS containing 1% BSA and 0.3% Triton X-100 and added to sections, followed by incubation with the antibody for 1 h at room temperature.

Techniques: Solvent, Control, Cell Culture, Fluorescence

Journal: Cells

Article Title: The EphA2 Receptor Regulates Invasiveness and Drug Sensitivity in Canine and Human Osteosarcoma Cells

doi: 10.3390/cells13141201

Figure Lengend Snippet: EphA2 affects osteosarcoma cell morphology. ( A , B ) The indicated canine and human OS cells were seeded into 6-well plates (50 cells per well, n = 3 per group) and allowed to grow into colonies for 7 days. Colonies were fixed with methanol and stained with crystal violet. The number of clustered, partially clustered, and spread colonies in each well were counted, and representative images were captured using a light microscope at 40× magnification. Graphs represent the proportion of each category of colonies relative to the total number of colonies. Representative images of these colonies are shown. ( C ) 143B-NS and 143B-shA2 cells were grown on glass coverslips to 50% confluency, fixed with 4% paraformaldehyde, and stained with rhodamine phalloidin (actin cytoskeleton, red) and DAPI (cell nucleus, blue). Fluorescent images were taken using an Olympus IX83 microscope at 200× magnification. Scale bar, 100 µm. Data are shown as mean ± SD. Experiments were repeated three times. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibody against EphA2 (#12927, Cell Signaling Technologies) was diluted in PBS containing 1% BSA and 0.3% Triton X-100 and added to sections, followed by incubation with the antibody for 1 h at room temperature.

Techniques: Staining, Light Microscopy, Microscopy

Journal: Cells

Article Title: The EphA2 Receptor Regulates Invasiveness and Drug Sensitivity in Canine and Human Osteosarcoma Cells

doi: 10.3390/cells13141201

Figure Lengend Snippet: EphA2 promotes osteosarcoma tumor growth. ( A ) Abrams-NS and Abrams-shA2 osteosarcoma cells were injected subcutaneously in the flank area of 4–8-week-old male immunodeficient NOD–SCID gamma mice (1.0 × 10 6 cells per mouse in 100 µL PBS, n = 4 per group). Tumor growth was monitored every 3–4 days, and tumor volume was calculated as a 2 × b/2, where a and b are the short and long diameters, respectively. The graph represents a summary of two independent experiments. ( B ) Eva-NS and Eva-shA2 cells were injected and tumor growth was monitored as in A ( n = 6 per group). The graph represents one of two independent experiments. Data are shown as mean ± SD. ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibody against EphA2 (#12927, Cell Signaling Technologies) was diluted in PBS containing 1% BSA and 0.3% Triton X-100 and added to sections, followed by incubation with the antibody for 1 h at room temperature.

Techniques: Injection

Journal: Cells

Article Title: The EphA2 Receptor Regulates Invasiveness and Drug Sensitivity in Canine and Human Osteosarcoma Cells

doi: 10.3390/cells13141201

Figure Lengend Snippet: EphA2 affects multiple signaling pathways in human and canine osteosarcoma cells. The indicated cells were cultured to approximately 70% confluency, serum-starved overnight, and the phosphorylation status of various proteins, as well as the expression of N-cadherin and Integrin β3, was assessed by Western blotting.

Article Snippet: The primary antibody against EphA2 (#12927, Cell Signaling Technologies) was diluted in PBS containing 1% BSA and 0.3% Triton X-100 and added to sections, followed by incubation with the antibody for 1 h at room temperature.

Techniques: Protein-Protein interactions, Cell Culture, Phospho-proteomics, Expressing, Western Blot

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