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 Molecular Cell Biology

Article Title: Inhibition of EphA2 by syndecan-4 in wounded skin regulates clustering of fibroblasts

doi: 10.1093/jmcb/mjae054

Figure Lengend Snippet: Expression of EphRs and cell repulsion are increased in the absence of SDC4. ( A ) Representative collisions between MEFs seeded on 5-μm fibronectin stripes (see and ). ( B ) Collisions were scored as ‘following’ if cells moved in the same direction after collision or ‘repulsion’ if cells moved in opposite directions ( n = 29). ( C ) Collisions, in the presence of 20 ng/ml TGFβ, were scored ( n = 34). ( D – F ) qPCR analysis of EphR and ephrin expression levels. Histograms depict fold changes relative to Sdc4 +/+ MEFs from a representative experiment, with n = 3 and experiments repeated up to 5 times. ( G – I ) Western blot analysis of EphA2 ( G , n = 3), EphA3 ( H , n = 3), and EphA4 ( I , n = 4) protein levels. ( J and K ) qPCR analysis of SDC4 and EphA2 mRNA levels ( J , n = 8) and western blot analysis of EphA2 protein level ( K , n = 3) in MEFs transfected with non-targeting (Control) or SDC4-targeting (SDC4) siRNA. Error bars represent standard error; significance tested by analysis of variance (ANOVA); * P < 0.05, ** P < 0.005, *** P < 0.0005.

Article Snippet: Protein was either immunoprecipitated with anti-EphA2 antibody (R&D Systems, AF639) and DynabeadsTM Protein G (ThermoFisher) before western blotting with DyLight800-conjugated streptavidin (ThermoFisher), for phosphotyrosine (4g10, Millipore) or pY596/pY602 EphA2 , or precipitated with DynabeadsTM Streptavidin (ThermoFisher) before western blotting for EphA2.

Techniques: Expressing, IF-cells, Western Blot, Transfection, Control

Journal: Journal of Molecular Cell Biology

Article Title: Inhibition of EphA2 by syndecan-4 in wounded skin regulates clustering of fibroblasts

doi: 10.1093/jmcb/mjae054

Figure Lengend Snippet: Reciprocal relationship between expression of SDC4 and surface expression of EphA2. ( A and B ) MEFs were surface-labelled with biotin before immunoprecipitating EphA2 and probing with fluorophore-conjugated streptavidin ( A , n = 3) or precipitating biotinylated proteins and blotting for EphA2 ( B , n = 7). ( C – E ) SDC4 was overexpressed in MEFs via retroviral infection with the SDC4 cDNA, using empty vector as a negative control. ( C ) SDC4 expression in infected MEFs by flow cytometry. ( D ) Western blot analysis of EphA2 protein levels in cell lysates ( n = 10). ( E ) Surface EphA2 protein levels by immunoprecipitating EphA2 from MEFs surface-labelled with biotin and probing with fluorophore-conjugated streptavidin ( n = 5). Error bars represent standard error; significance tested by ANOVA; * P < 0.05, ** P < 0.005, *** P < 0.0005.

Article Snippet: Protein was either immunoprecipitated with anti-EphA2 antibody (R&D Systems, AF639) and DynabeadsTM Protein G (ThermoFisher) before western blotting with DyLight800-conjugated streptavidin (ThermoFisher), for phosphotyrosine (4g10, Millipore) or pY596/pY602 EphA2 , or precipitated with DynabeadsTM Streptavidin (ThermoFisher) before western blotting for EphA2.

Techniques: Expressing, Retroviral, Infection, Plasmid Preparation, Negative Control, Flow Cytometry, Western Blot

Journal: Journal of Molecular Cell Biology

Article Title: Inhibition of EphA2 by syndecan-4 in wounded skin regulates clustering of fibroblasts

doi: 10.1093/jmcb/mjae054

Figure Lengend Snippet: Phosphorylation and endocytosis of EphA2 increase proportionally to expression. ( A and B ) MEFs were stimulated with 1 μg/ml clustered ephrinA1-Fc, followed by western blot analysis using antibody 4G10 for total phosphotyrosine ( A , n = 4) or an antibody against EphA2 residues Y596 and Y602 ( B , n = 4). Histograms represent phosphorylation levels normalised to EphA2. ( C and D ) EphA2 endocytosis in fibroblasts transfected with non-targeting (Control) or SDC4-targeting (SDC4) siRNA. ( C ) Flow cytometry analysis of SDC4 expression. ( D ) Fibroblasts were surface-labelled with biotin and stimulated with 1 μg/ml clustered ephrinA1-Fc. The internalised proteins were precipitated with streptavidin before blotting for EphA2 ( n = 6). ( E and F ) MEFs were stimulated with 1 μg/ml clustered ephrinA1-Fc before fixation and staining (see also ). ( E ) Representative images of MEFs stained for EEA1 (green), EphA2 (red), and counterstained with phalloidin (blue). Scale bar, 10 μm. ( F ) Pearson correlation analysis for colocalization between EphA2 and EEA1 ( n = 10). Error bars represent standard error; significance tested by ANOVA; * P < 0.05, ** P < 0.005, *** P < 0.0005.

Article Snippet: Protein was either immunoprecipitated with anti-EphA2 antibody (R&D Systems, AF639) and DynabeadsTM Protein G (ThermoFisher) before western blotting with DyLight800-conjugated streptavidin (ThermoFisher), for phosphotyrosine (4g10, Millipore) or pY596/pY602 EphA2 , or precipitated with DynabeadsTM Streptavidin (ThermoFisher) before western blotting for EphA2.

Techniques: Phospho-proteomics, Expressing, Western Blot, Transfection, Control, Flow Cytometry, Staining

Journal: Journal of Molecular Cell Biology

Article Title: Inhibition of EphA2 by syndecan-4 in wounded skin regulates clustering of fibroblasts

doi: 10.1093/jmcb/mjae054

Figure Lengend Snippet: Sdc4 –/– MEFs contract in response to ephrinA1. Sdc4 +/+ and Sdc4 –/– MEFs transfected with either non-targeting (Control) or EphA2-targeting (EphA2) siRNA were spread on 5 μg/ml fibronectin-coated dishes and stimulated with 1 μg/ml clustered ephrinA1-Fc. ( A ) Representative frames before and after the addition of ephrinA1-Fc (see – ). Scale bar, 20 μm. ( B ) Percentage of cells that contracted upon the addition of ephrinA1-Fc. The histogram represents the average of 3 experiments, with a total of 47 cells of each type scored. ( C ) Western blot analysis of EphA2 expression in cells used for the contraction assay and subsequent migration assay. Error bars indicate standard error; significance tested by ANOVA; *** P < 0.0005.

Article Snippet: Protein was either immunoprecipitated with anti-EphA2 antibody (R&D Systems, AF639) and DynabeadsTM Protein G (ThermoFisher) before western blotting with DyLight800-conjugated streptavidin (ThermoFisher), for phosphotyrosine (4g10, Millipore) or pY596/pY602 EphA2 , or precipitated with DynabeadsTM Streptavidin (ThermoFisher) before western blotting for EphA2.

Techniques: Transfection, Control, Western Blot, Expressing, Contraction Assay, Migration

Journal: Journal of Molecular Cell Biology

Article Title: Inhibition of EphA2 by syndecan-4 in wounded skin regulates clustering of fibroblasts

doi: 10.1093/jmcb/mjae054

Figure Lengend Snippet: SDC4 regulates EphA2 expression by activation of PKCα. ( A ) SDC4 cytodomain indicating the functions of residues to be tested. ( B – F ) Western blot analysis of EphA2 protein levels in Sdc4 +/+ MEFs, Sdc4 –/– MEFs, and Sdc4 –/– MEFs stably rescued with the indicated SDC4 cDNAs ( B , n = 5), Sdc4 +/+ ( C , n = 10) and Sdc4 –/– ( D , n = 4) MEFs treated daily with indicated concentrations of PMA for 5 days, Sdc4 +/+ MEFs treated daily with indicated concentrations of the PKCα inhibitor, BIM-1, for 5 days ( E , n = 6), and Sdc4 +/+ MEFs transfected with control or PKCα-targeting siRNA ( F , n = 6). Error bars represent standard error; significance tested by ANOVA; * P < 0.05, ** P < 0.005, *** P < 0.0005.

Article Snippet: Protein was either immunoprecipitated with anti-EphA2 antibody (R&D Systems, AF639) and DynabeadsTM Protein G (ThermoFisher) before western blotting with DyLight800-conjugated streptavidin (ThermoFisher), for phosphotyrosine (4g10, Millipore) or pY596/pY602 EphA2 , or precipitated with DynabeadsTM Streptavidin (ThermoFisher) before western blotting for EphA2.

Techniques: Expressing, Activation Assay, Western Blot, Stable Transfection, Transfection, Control

Journal: Journal of Molecular Cell Biology

Article Title: Inhibition of EphA2 by syndecan-4 in wounded skin regulates clustering of fibroblasts

doi: 10.1093/jmcb/mjae054

Figure Lengend Snippet: Homotypic repulsion of Sdc4 –/– MEFs is due to high EphA2 expression. ( A ) Representative collisions between Sdc4 +/+ ( n = 93) and Sdc4 –/– MEFs transfected with either non-targeting (Control, n = 78) or EphA2-targeting (EphA2, n = 14) siRNA (see – ). ( B ) Collisions were scored as ‘following’ or ‘repulsion’. ( C ) Time before both nuclei made a retrograde step post-collision. Boxes indicate median and 1st and 3rd quartiles and whiskers indicate data range. Significance tested by ANOVA; * P < 0.05.

Article Snippet: Protein was either immunoprecipitated with anti-EphA2 antibody (R&D Systems, AF639) and DynabeadsTM Protein G (ThermoFisher) before western blotting with DyLight800-conjugated streptavidin (ThermoFisher), for phosphotyrosine (4g10, Millipore) or pY596/pY602 EphA2 , or precipitated with DynabeadsTM Streptavidin (ThermoFisher) before western blotting for EphA2.

Techniques: Expressing, Transfection, Control

Journal: Journal of Molecular Cell Biology

Article Title: Inhibition of EphA2 by syndecan-4 in wounded skin regulates clustering of fibroblasts

doi: 10.1093/jmcb/mjae054

Figure Lengend Snippet: EphA2 expression in the fibroblasts of wounded skin is regulated by SDC4. ( A and B ) In mouse wound healing model, 4-mm full-thickness wounds were created on the backs of mice, and wound areas were recorded macroscopically. ( C – E ) qPCR analysis of EphA2 mRNA expression levels, relative to 18S internal control, in the bed of full-thickness skin wounds. Shown are relative expression changes over time in Sdc4 +/+ ( C ) and Sdc4 –/– ( D ) mice and direct comparison between two genotypes at 72 h ( E ) or 0 h ( F ) post-wounding ( n = 9). Error bars represent standard error; significance tested by ANOVA; * P < 0.05.

Article Snippet: Protein was either immunoprecipitated with anti-EphA2 antibody (R&D Systems, AF639) and DynabeadsTM Protein G (ThermoFisher) before western blotting with DyLight800-conjugated streptavidin (ThermoFisher), for phosphotyrosine (4g10, Millipore) or pY596/pY602 EphA2 , or precipitated with DynabeadsTM Streptavidin (ThermoFisher) before western blotting for EphA2.

Techniques: Expressing, Control, Comparison

Journal: Cell Adhesion & Migration

Article Title: EphA2 promotes cell adhesion and spreading of monocyte and monocyte/macrophage cell lines on integrin ligand-coated surfaces

doi: 10.1080/19336918.2015.1107693

Figure Lengend Snippet: Expression of endogenous and exogenous/dominant negative EphA2 in U937, EphA2ΔC-EGFP-U937, J774.1, and EphA2ΔC-EGFP-J774.1 cells. ( A ) Typical phase contrast and fluorescence micrographs highlighting the expression of the EphA2ΔC-EGFP protein. ( B ) EphA2 mRNAs amplified from the intracellular and extracellular regions by RT-PCR showing the expression of endogenous and total (endogenous plus exogenous) EphA2, respectively, in U937 and its subline. Densitometric quantification of the RT-PCR amplification levels of EphA2 from 3 independent experiments normalized to the levels of GAPDH. Data is presented as the mean ± SD. ** P < 0.01. ( C ) Amplification of endogenous and exogenous EphA2 in J774.1 cells and their subline cells by RT-PCR. Densitometric quantification of the RT-PCR amplification levels were determined from 3 independent experiments and normalized to the levels of GAPDH. Data is presented as the mean ± SD.

Article Snippet: The rat monoclonal PE-conjugated antibody against mouse EphA2 used in this study was from R&D Systems (233720).

Techniques: Expressing, Dominant Negative Mutation, Fluorescence, Amplification, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Adhesion & Migration

Article Title: EphA2 promotes cell adhesion and spreading of monocyte and monocyte/macrophage cell lines on integrin ligand-coated surfaces

doi: 10.1080/19336918.2015.1107693

Figure Lengend Snippet: Representative histograms showing EphA2 and/or EGFP expression in U937 and EphA2ΔC-EGFP-U937 cells ( A ) as well as J774.1 and EphA2ΔC-EGFP-J774.1 cells ( B ).

Article Snippet: The rat monoclonal PE-conjugated antibody against mouse EphA2 used in this study was from R&D Systems (233720).

Techniques: Expressing

Journal: Cell Adhesion & Migration

Article Title: EphA2 promotes cell adhesion and spreading of monocyte and monocyte/macrophage cell lines on integrin ligand-coated surfaces

doi: 10.1080/19336918.2015.1107693

Figure Lengend Snippet: Molecular association of EphA2 with the β2 integrin/ICAM1 ( A ) and β2 integrin/VCAM1 complexes ( B ) in U937 and EphA2ΔC-EGFP-U937 cells.

Article Snippet: The rat monoclonal PE-conjugated antibody against mouse EphA2 used in this study was from R&D Systems (233720).

Techniques:

Journal: Cell Adhesion & Migration

Article Title: EphA2 promotes cell adhesion and spreading of monocyte and monocyte/macrophage cell lines on integrin ligand-coated surfaces

doi: 10.1080/19336918.2015.1107693

Figure Lengend Snippet: Primers and cycle numbers for PCR amplification used in U937 cells

Article Snippet: The rat monoclonal PE-conjugated antibody against mouse EphA2 used in this study was from R&D Systems (233720).

Techniques: Amplification

Journal: Cell Adhesion & Migration

Article Title: EphA2 promotes cell adhesion and spreading of monocyte and monocyte/macrophage cell lines on integrin ligand-coated surfaces

doi: 10.1080/19336918.2015.1107693

Figure Lengend Snippet: Primers and cycle numbers for PCR amplification used in J774.1 cells

Article Snippet: The rat monoclonal PE-conjugated antibody against mouse EphA2 used in this study was from R&D Systems (233720).

Techniques: Amplification