goat anti epha2  (R&D Systems)

Journal: JCI Insight

Article Title: MyD88/CD40 signaling retains CAR T cells in a less differentiated state

doi: 10.1172/jci.insight.136093

Figure Lengend Snippet: ( A ) Scheme of EphA2-CAR constructs. ( B ) Summary plot of %tCD19 + T cells ( n = 5, mean ± SEM, 1-way ANOVA with Tukey’s test for multiple comparisons). ( C ) Summary plot of %F(ab′) 2 -positive T cells ( n = 5, mean ± SEM, 1-way ANOVA with Tukey’s test for multiple comparisons). ( D ) CAR T cell production of Th1 (IFN-γ and IL-2) and Th2 (IL-4 and IL-10) cytokines after 24-hour coculture at a 2:1 ratio against EphA2-positive (LM7, U373 WT) and EphA2-negative (BV173, U373 EphA2 KO) cell lines or in media alone ( n = 5, mean ± SEM, 2-way ANOVA with Dunnett’s test for multiple comparisons, all statistical analysis is in comparison with NT cells). Dot colors: black, media; light gray, BV173 (EphA2 negative); dark gray, U373 EphA2 KO (EphA2 negative); dark blue: U373 (EphA2 positive); light blue, LM7 (EphA2 positive). ( E ) Summary plots of Th1 and Th2 cytokine production against EphA2-positive cell lines U373 and LM7 ( n = 5, mean ± SEM, values were log transformed before 2-way ANOVA with Tukey’s test for multiple comparisons). ( F ) CAR T cells were incubated with increasing amounts of tumor cells for 24 hours, and the remaining live tumor cells were quantified with an MTS assay (2-way ANOVA with Tukey’s test for multiple comparisons, mean ± SEM, LM7: n = 4, U373 WT and U373 EphA2 KO: n = 9). For LM7 and U373, asterisks refer to statistical comparison of MC-CAR with NT and CD28-CAR with MC-CAR. For U373 EphA2 KO, asterisks refer to statistical comparison of 41BB-CAR with NT and CD28-CAR with NT. # P < 0.1; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: After SDS-PAGE and wet transfer, the membrane was blocked with 5% milk in TBS-Tween (TBST), then incubated with primary antibodies: goat anti-human EphA2 (R&D Systems, Bio-Techne, AF3035), rabbit anti-human Bcl-2 (clone D55G8, Cell Signaling Technology), rabbit anti-human Myb (clone D2R4Y, Cell Signaling Technology), or mouse anti-human GAPDH (clone 0411, Santa Cruz Biotechnology).

Techniques: Construct, Comparison, Transformation Assay, Incubation, MTS Assay

Journal: JCI Insight

Article Title: MyD88/CD40 signaling retains CAR T cells in a less differentiated state

doi: 10.1172/jci.insight.136093

Figure Lengend Snippet: T cells were cocultured with tumor cells at a 2:1 ratio with weekly restimulation against fresh tumor cells until they lost their effector function and no longer killed all the tumor cells. ( A ) Average expansion of CAR T cells against EphA2-positive (U373 and LM7) and U373 EphA2 KO cell line (mean ± SEM, LM7: n = 4; U373: n = 8 [NT, CD28, MC], n = 4 [41BB]; U373 KO: n = 8 [NT, CD28, MC], n = 6 [41BB]). ( B ) Summary of the maximum expansion CAR T cells from individual donors achieved against EphA2-positive tumor cells and the maximum number of times CAR T cells were able to kill fresh EphA2-positive tumor cells ( n = 12 [NT, CD28, and MC], n = 8 [41BB]; median and quartiles, 1-way ANOVA with Tukey’s test for multiple comparisons). T cells were phenotyped 7 days after stimulation with U373. ( C ) Summary plot of CD4/CD8 composition after stimulation with U373 ( n = 3, mean ± SEM). ( D ) Scheme for phenotyping T cells and representative flow cytometry plots of CCR7 and CD45RA expression on CAR T cells after stimulation with U373. ( E ) Summary plot of T cell phenotype after stimulation with U373 ( n = 4, mean ± SEM, 2-way ANOVA with Tukey’s test for multiple comparisons). All statistical tests were performed in comparison with MC-CAR T cells (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001).

Article Snippet: After SDS-PAGE and wet transfer, the membrane was blocked with 5% milk in TBS-Tween (TBST), then incubated with primary antibodies: goat anti-human EphA2 (R&D Systems, Bio-Techne, AF3035), rabbit anti-human Bcl-2 (clone D55G8, Cell Signaling Technology), rabbit anti-human Myb (clone D2R4Y, Cell Signaling Technology), or mouse anti-human GAPDH (clone 0411, Santa Cruz Biotechnology).

Techniques: Flow Cytometry, Expressing, Comparison

Journal: JCI Insight

Article Title: MyD88/CD40 signaling retains CAR T cells in a less differentiated state

doi: 10.1172/jci.insight.136093

Figure Lengend Snippet: ( A – D ) NSG mice were injected with 1 × 10 6 LM7-ffLuc i.p. One week later, mice were injected with 1 × 10 4 or 1 × 10 5 CD28-, 41BB-, or MC-CAR T cells. PBS and 1 × 10 5 Delta-CAR T cells were used as controls. ( A ) Total flux from tumor cells in all mice treated with 1 × 10 4 EphA2 CAR T cells (PBS: n = 5; CD28, 41BB, MC: n = 10). ( B ) Total flux from tumor cells in all mice treated with 1 × 10 5 EphA2 CAR T cells (Delta: n = 5, CD28: n = 8, 41BB: n = 9, MC: n = 10). ( C and D ) Kaplan-Meier survival analysis of mice treated with 1 × 10 4 ( C ) or 1 × 10 5 ( D ) EphA2 CAR T cells (log-rank Mantel-Cox test with Bonferroni’s correction for multiple comparisons; * P < 0.05; ** P < 0.01; *** P < 0.001). Experiments were repeated twice with CAR T cells generated from 2 different healthy donors.

Article Snippet: After SDS-PAGE and wet transfer, the membrane was blocked with 5% milk in TBS-Tween (TBST), then incubated with primary antibodies: goat anti-human EphA2 (R&D Systems, Bio-Techne, AF3035), rabbit anti-human Bcl-2 (clone D55G8, Cell Signaling Technology), rabbit anti-human Myb (clone D2R4Y, Cell Signaling Technology), or mouse anti-human GAPDH (clone 0411, Santa Cruz Biotechnology).

Techniques: Injection, Generated

Journal: Investigative Ophthalmology & Visual Science

Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

doi: 10.1167/iovs.17-22941

Figure Lengend Snippet: Reciprocal regulation of ephrin-A1 and EphA2 expression in human and mouse cornea. Frozen corneal tissue sections from human cadavers (A) and wild-type Balb/C mice (B) were immunostained with antibodies against EphA2 or ephrin-A1 (red, bottom). DAPI (blue) was used to highlight nuclei. (A) Arrowheads indicate the limbus–cornea junction where the limbus ends and the cornea begins. (B) Mouse eyelids are marked as a reference point for limbal tissue orientation. Arrows show concentrated ephrin-A1 staining and paucity of EphA2 staining in the limbus. White dotted lines demarcate the basement membrane region. CC, central cornea; L, limbus. n = 3. Scale bar denotes 100 μm.

Article Snippet: Frozen sections (5 μm) of optimal cutting temperature compound (OCT)-embedded human and mouse corneas were fixed in 4% paraformaldehyde, blocked in 10% donkey serum in PBS, and incubated overnight with the following primary antibodies: a goat anti-human EphA2 (AF3035) or a goat anti-mouse EphA2 (AF639; R&D Systems), a rabbit anti-ephrin-A1 (V18), a rabbit polyclonal antibody against ADAM10 (Abcam), or a mouse monoclonal antibody against E-cadherin (HECD1; Abcam).

Techniques: Expressing, Staining, Membrane

Journal: Investigative Ophthalmology & Visual Science

Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

doi: 10.1167/iovs.17-22941

Figure Lengend Snippet: Ephrin-A1 is redistributed into the cornea of injured mouse eyes. Whole mounts of mouse anterior segmental epithelium in uninjured (Control) eyes and following central corneal wounding (24 hours after injury). Immunostaining was performed for (A) EphA2 (red) and (B) Ephrin-A1 (red). Scale bar denotes 80 μm. W, wound opening. Arrowheads show clusters of ephrin-A1–expressing cells in the cornea. Arrow represents EphA2-enriched areas near the wound edge. White dotted line marks the wound edge. n = 3. (C) Whole mounts of control (upper) and injured (lower) mouse corneas that were dual stained for EphA2 (red) and ephrin-A1 (green). Confocal stitched images show the entire cornea. L, limbus; PC, peripheral cornea; CC, central cornea; W, wound opening. White dotted line marks the wound edge. (D) Immunoblotting of EphA2, pS897-EphA2, or ephrin-A1 in samples isolated from wounded corneas. ERK1/2 was used as a loading control. (E) Densitometry results of Western blots represented in D are shown as fold changes over levels present in uninjured, control corneas. *P < 0.05, n = 3. (F) Real time-quantitative PCR measurement of mRNA transcript levels for EphA2 and ephrin-A1 in samples isolated from wounded corneas. *P < 0.05, n = 4–6.

Article Snippet: Frozen sections (5 μm) of optimal cutting temperature compound (OCT)-embedded human and mouse corneas were fixed in 4% paraformaldehyde, blocked in 10% donkey serum in PBS, and incubated overnight with the following primary antibodies: a goat anti-human EphA2 (AF3035) or a goat anti-mouse EphA2 (AF639; R&D Systems), a rabbit anti-ephrin-A1 (V18), a rabbit polyclonal antibody against ADAM10 (Abcam), or a mouse monoclonal antibody against E-cadherin (HECD1; Abcam).

Techniques: Control, Immunostaining, Expressing, Staining, Western Blot, Isolation, Real-time Polymerase Chain Reaction

Journal: Investigative Ophthalmology & Visual Science

Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

doi: 10.1167/iovs.17-22941

Figure Lengend Snippet: Reciprocal regulation of ephrin-A1 and EphA2 expression in corneal epithelial cell cultures. (A) Immunostaining of EphA2 (green), ephrin-A1 (red), and E-cadherin (magenta) in mono-cultures of hTCEPi cells transduced with an empty control or an ephrin-A1 cDNA construct. Representative images from n = 3. (B) Immunoblotting for total EphA2, ephrin-A1, or E-cadherin in hTCEPi cells overexpressing ephrin-A1 (EFNA1). GAPDH was used as a protein loading control. Representative blots from n = 4. (C) Immunostaining of EphA2 (green), ephrin-A1 (red), and E-cadherin (magenta) in hTCEPi cells knocked down for ephrin-A1 (siEphrin-A1), EphA2 (siEphA2), or both proteins (siEphA2+siEphrin-A1). Scale bar denotes 100 μm. Representative images from n = 3. (D) Immunoblotting for total EphA2, ephrin-A1, or E-cadherin in hTCEPi cells with siRNA targeted knockdown of ephrin-A1, EphA2, or double knockdown. GAPDH was used as a protein loading control. Representative blots from n = 3.

Article Snippet: Frozen sections (5 μm) of optimal cutting temperature compound (OCT)-embedded human and mouse corneas were fixed in 4% paraformaldehyde, blocked in 10% donkey serum in PBS, and incubated overnight with the following primary antibodies: a goat anti-human EphA2 (AF3035) or a goat anti-mouse EphA2 (AF639; R&D Systems), a rabbit anti-ephrin-A1 (V18), a rabbit polyclonal antibody against ADAM10 (Abcam), or a mouse monoclonal antibody against E-cadherin (HECD1; Abcam).

Techniques: Expressing, Immunostaining, Transduction, Control, Construct, Western Blot, Knockdown

Journal: Investigative Ophthalmology & Visual Science

Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

doi: 10.1167/iovs.17-22941

Figure Lengend Snippet: EphA2/Ephrin-A1 signaling complexes in a heterotypic cell confrontation coculture model. (A) hTCEpi cells were differentially labeled with fluorescent cell trackers (red and green) and seeded into discrete culture compartments using a silicone chamber confrontation apparatus. After removal of the silicone divider, live cell imaging was used to monitor cell confrontation for 48 hours. Snapshots of 0, 24, and 48 hours are shown. (B) EphA2-expressing control cells (Control, green) confronting “like” control cells (Control, red) are presented on the left, while control cells (Control, green) confronting “unlike” ephrin-A1–overexpressing cells (EFNA1, red) are presented on the right. After removal of the silicone divider, time-lapse imaging was used to examine the formation and organization of the epithelial boundary between these two cell populations. White solid lines mark the midline where the silicone divider was present prior to removal. Dotted lines indicate the boundary between the two cell populations after initiation of confrontation. Snapshots of 0, 6, 12, 24, and 48 hours are shown. Scale bar denotes 1 mm. (C, D) Line graphs showing the migrating front of control cells (Control, green) confronting control (Control, red) (C) or ephrin-A1 (EFNA1, red) (D) overexpressing cells normalized with respect to the midline. Negative values on the y-axis represent the reversal of migration initiated by ephrin-A1. n = 3 (D). (E) Higher-magnification images from early time points (2, 3, 4, 6, 8, and 12 hours) of ephrin-A1–overexpressing cells (EFNA1, red) confronting control cells (CTRL, green). Arrowhead points to the initial confrontation area at 4 hours. Scale bar denotes 200 μm.

Article Snippet: Frozen sections (5 μm) of optimal cutting temperature compound (OCT)-embedded human and mouse corneas were fixed in 4% paraformaldehyde, blocked in 10% donkey serum in PBS, and incubated overnight with the following primary antibodies: a goat anti-human EphA2 (AF3035) or a goat anti-mouse EphA2 (AF639; R&D Systems), a rabbit anti-ephrin-A1 (V18), a rabbit polyclonal antibody against ADAM10 (Abcam), or a mouse monoclonal antibody against E-cadherin (HECD1; Abcam).

Techniques: Labeling, Live Cell Imaging, Expressing, Control, Imaging, Migration

Journal: Investigative Ophthalmology & Visual Science

Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

doi: 10.1167/iovs.17-22941

Figure Lengend Snippet: Cell–cell border localization of E-cadherin is reduced at EphA2/Ephrin-A1 boundaries. (A) Immunofluorescent staining of EphA2 (green), ephrin-A1 (red), and E-cadherin (magenta) in cells present at the boundary of Control:Control- or Control:Ephrin-A1–expressing cell cocultures 48 hours after removal of the silicone barrier. A magnified view of the boundary is shown below in control cells confronting ephrin-A1–expressing cells. Dotted lines indicate the boundary between the two different cell populations 48 hours after initiation of confrontation. n = 4. Scale bar denotes 80 μm. (B) Control or ephrin-A1–expressing cells were transduced to express mCherry (Control-mCherry or EFNA1-mCherry, respectively) to differentiate these cell populations from control cells transfected with siControl (siCTRL) or siEphA2. Immunostaining of E-cadherin (magenta) was performed in cocultures 48 hours after initiation of confrontation. Dotted lines indicate the boundary between the two different cell populations at 48 hours. n = 3.

Article Snippet: Frozen sections (5 μm) of optimal cutting temperature compound (OCT)-embedded human and mouse corneas were fixed in 4% paraformaldehyde, blocked in 10% donkey serum in PBS, and incubated overnight with the following primary antibodies: a goat anti-human EphA2 (AF3035) or a goat anti-mouse EphA2 (AF639; R&D Systems), a rabbit anti-ephrin-A1 (V18), a rabbit polyclonal antibody against ADAM10 (Abcam), or a mouse monoclonal antibody against E-cadherin (HECD1; Abcam).

Techniques: Staining, Control, Expressing, Transfection, Immunostaining

Journal: Investigative Ophthalmology & Visual Science

Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

doi: 10.1167/iovs.17-22941

Figure Lengend Snippet: Ephrin-A1–induced boundary formation requires EphA2. (A) Control (Control, red) or (B) ephrin-A1–expressing (EFNA1, red) cells are shown at 48 hours after initiation of confrontation with cells transfected with siControl (siCTRL, green), siEphA2 (green), siEphrin-A1 (siEFNA1, green), or double siRNA (siEphA2+siEFNA1, green) oligonucleotides. Solid white lines mark the midline where the silicone divider was present at the time of removal. White dotted lines indicate the boundary between two cell populations 48 hours after initiation of confrontation. (C) Quantification of confrontation response in A and B as measured by % deviation from migration front of red-labeled cells (Control, red or Ephrin-A1, red). *P < 0.05 versus siCTRL; #P < 0.05, Ephrin-A1, red versus Control, red, n = 3. (D) A representative Western blotting showing the levels of EphA2 or ephrin-A1 after siRNA depletion in lysates harvested at the end of the experiment (96 hours after siRNA transfection).

Article Snippet: Frozen sections (5 μm) of optimal cutting temperature compound (OCT)-embedded human and mouse corneas were fixed in 4% paraformaldehyde, blocked in 10% donkey serum in PBS, and incubated overnight with the following primary antibodies: a goat anti-human EphA2 (AF3035) or a goat anti-mouse EphA2 (AF639; R&D Systems), a rabbit anti-ephrin-A1 (V18), a rabbit polyclonal antibody against ADAM10 (Abcam), or a mouse monoclonal antibody against E-cadherin (HECD1; Abcam).

Techniques: Control, Expressing, Transfection, Migration, Labeling, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

doi: 10.1167/iovs.17-22941

Figure Lengend Snippet: ADAM10 mediates Ephrin-A1/EphA2 boundary organization via EGFR signaling. (A) E-cadherin (E-cad; top) and ADAM10 (bottom) immunofluorescence staining in human anterior segmental epithelium. Scale bar denotes 100 μm. (B) E-cadherin staining of control cells (Control, green) confronted by “like” control cells (Control, red) or ephrin-A1–expressing cells (EFNA1, red) confronted by “unlike” control cells (Control, green; bottom) in the presence of general MMP inhibitor, TAPI, or a specific ADAM10 inhibitor, GI254023X (GIX). Red dotted lines indicate the boundary between the two cell populations 48 hours after initiation of confrontation. Scale bar denotes 80 μm. (C) Quantification of confrontation experiments at 48 hours in cocultures treated with DMSO, GIX, LY294002 (LY), Y-27632 (Y), or U0126 (U). * P < 0.05, n = 3–4. (D) Quantification of confrontation experiments at 48 hours using ephrin-A1–expressing cells (EFNA1) in contact with “unlike” control cells that had been treated with DMSO or the EGFR inhibitor, AG1478 (AG). Cells were either pretreated before the initiation of confrontation for 1 hour (AG pretreat) or treated with inhibitor 5 (AG @ 5 hrs) or 24 hours (AG @ 24 hrs) after initiation of confrontation. (E, F) Various concentrations of EGF (0.1, 1, 10, or 100 ng/mL) were added to the culture medium of these ephrin-A1 and control cell cocultures after pretreatment with GIX for 5 hours. Images (E) and quantification (F) are shown 48 hours after confrontation. Solid white lines mark the midline where the silicone divider was present at the time of its removal. White dotted lines indicate the boundary between the two different cell populations 48 hours after initiation of confrontation. n = 3–4.

Article Snippet: Frozen sections (5 μm) of optimal cutting temperature compound (OCT)-embedded human and mouse corneas were fixed in 4% paraformaldehyde, blocked in 10% donkey serum in PBS, and incubated overnight with the following primary antibodies: a goat anti-human EphA2 (AF3035) or a goat anti-mouse EphA2 (AF639; R&D Systems), a rabbit anti-ephrin-A1 (V18), a rabbit polyclonal antibody against ADAM10 (Abcam), or a mouse monoclonal antibody against E-cadherin (HECD1; Abcam).

Techniques: Immunofluorescence, Staining, Control, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

doi: 10.1167/iovs.17-22941

Figure Lengend Snippet: Reciprocal regulation of ephrin-A1 and EphA2 expression in human and mouse cornea. Frozen corneal tissue sections from human cadavers (A) and wild-type Balb/C mice (B) were immunostained with antibodies against EphA2 or ephrin-A1 (red, bottom). DAPI (blue) was used to highlight nuclei. (A) Arrowheads indicate the limbus–cornea junction where the limbus ends and the cornea begins. (B) Mouse eyelids are marked as a reference point for limbal tissue orientation. Arrows show concentrated ephrin-A1 staining and paucity of EphA2 staining in the limbus. White dotted lines demarcate the basement membrane region. CC, central cornea; L, limbus. n = 3. Scale bar denotes 100 μm.

Article Snippet: The cells on glass coverslips were then permeabilized in 0.1% Triton X-100 for 5 minutes and processed for immunofluorescence staining as previously described using a goat antibody against EphA2 (AF3035; R&D Systems, Minneapolis, MN, USA), rabbit polyclonal antibody against ephrin-A1 (V18; Santa Cruz Biotechnologies), or a mouse monoclonal antibody against E-cadherin (HECD1; Abcam) with detection using an Alexa Fluor-488 or -555 or 647-nm conjugated donkey anti-goat, anti-rabbit or anti-mouse antibody (Invitrogen).

Techniques: Expressing, Staining, Membrane

Journal: Investigative Ophthalmology & Visual Science

Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

doi: 10.1167/iovs.17-22941

Figure Lengend Snippet: Ephrin-A1 is redistributed into the cornea of injured mouse eyes. Whole mounts of mouse anterior segmental epithelium in uninjured (Control) eyes and following central corneal wounding (24 hours after injury). Immunostaining was performed for (A) EphA2 (red) and (B) Ephrin-A1 (red). Scale bar denotes 80 μm. W, wound opening. Arrowheads show clusters of ephrin-A1–expressing cells in the cornea. Arrow represents EphA2-enriched areas near the wound edge. White dotted line marks the wound edge. n = 3. (C) Whole mounts of control (upper) and injured (lower) mouse corneas that were dual stained for EphA2 (red) and ephrin-A1 (green). Confocal stitched images show the entire cornea. L, limbus; PC, peripheral cornea; CC, central cornea; W, wound opening. White dotted line marks the wound edge. (D) Immunoblotting of EphA2, pS897-EphA2, or ephrin-A1 in samples isolated from wounded corneas. ERK1/2 was used as a loading control. (E) Densitometry results of Western blots represented in D are shown as fold changes over levels present in uninjured, control corneas. *P < 0.05, n = 3. (F) Real time-quantitative PCR measurement of mRNA transcript levels for EphA2 and ephrin-A1 in samples isolated from wounded corneas. *P < 0.05, n = 4–6.

Article Snippet: The cells on glass coverslips were then permeabilized in 0.1% Triton X-100 for 5 minutes and processed for immunofluorescence staining as previously described using a goat antibody against EphA2 (AF3035; R&D Systems, Minneapolis, MN, USA), rabbit polyclonal antibody against ephrin-A1 (V18; Santa Cruz Biotechnologies), or a mouse monoclonal antibody against E-cadherin (HECD1; Abcam) with detection using an Alexa Fluor-488 or -555 or 647-nm conjugated donkey anti-goat, anti-rabbit or anti-mouse antibody (Invitrogen).

Techniques: Control, Immunostaining, Expressing, Staining, Western Blot, Isolation, Real-time Polymerase Chain Reaction

Journal: Investigative Ophthalmology & Visual Science

Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

doi: 10.1167/iovs.17-22941

Figure Lengend Snippet: Reciprocal regulation of ephrin-A1 and EphA2 expression in corneal epithelial cell cultures. (A) Immunostaining of EphA2 (green), ephrin-A1 (red), and E-cadherin (magenta) in mono-cultures of hTCEPi cells transduced with an empty control or an ephrin-A1 cDNA construct. Representative images from n = 3. (B) Immunoblotting for total EphA2, ephrin-A1, or E-cadherin in hTCEPi cells overexpressing ephrin-A1 (EFNA1). GAPDH was used as a protein loading control. Representative blots from n = 4. (C) Immunostaining of EphA2 (green), ephrin-A1 (red), and E-cadherin (magenta) in hTCEPi cells knocked down for ephrin-A1 (siEphrin-A1), EphA2 (siEphA2), or both proteins (siEphA2+siEphrin-A1). Scale bar denotes 100 μm. Representative images from n = 3. (D) Immunoblotting for total EphA2, ephrin-A1, or E-cadherin in hTCEPi cells with siRNA targeted knockdown of ephrin-A1, EphA2, or double knockdown. GAPDH was used as a protein loading control. Representative blots from n = 3.

Article Snippet: The cells on glass coverslips were then permeabilized in 0.1% Triton X-100 for 5 minutes and processed for immunofluorescence staining as previously described using a goat antibody against EphA2 (AF3035; R&D Systems, Minneapolis, MN, USA), rabbit polyclonal antibody against ephrin-A1 (V18; Santa Cruz Biotechnologies), or a mouse monoclonal antibody against E-cadherin (HECD1; Abcam) with detection using an Alexa Fluor-488 or -555 or 647-nm conjugated donkey anti-goat, anti-rabbit or anti-mouse antibody (Invitrogen).

Techniques: Expressing, Immunostaining, Transduction, Control, Construct, Western Blot, Knockdown

Journal: Investigative Ophthalmology & Visual Science

Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

doi: 10.1167/iovs.17-22941

Figure Lengend Snippet: EphA2/Ephrin-A1 signaling complexes in a heterotypic cell confrontation coculture model. (A) hTCEpi cells were differentially labeled with fluorescent cell trackers (red and green) and seeded into discrete culture compartments using a silicone chamber confrontation apparatus. After removal of the silicone divider, live cell imaging was used to monitor cell confrontation for 48 hours. Snapshots of 0, 24, and 48 hours are shown. (B) EphA2-expressing control cells (Control, green) confronting “like” control cells (Control, red) are presented on the left, while control cells (Control, green) confronting “unlike” ephrin-A1–overexpressing cells (EFNA1, red) are presented on the right. After removal of the silicone divider, time-lapse imaging was used to examine the formation and organization of the epithelial boundary between these two cell populations. White solid lines mark the midline where the silicone divider was present prior to removal. Dotted lines indicate the boundary between the two cell populations after initiation of confrontation. Snapshots of 0, 6, 12, 24, and 48 hours are shown. Scale bar denotes 1 mm. (C, D) Line graphs showing the migrating front of control cells (Control, green) confronting control (Control, red) (C) or ephrin-A1 (EFNA1, red) (D) overexpressing cells normalized with respect to the midline. Negative values on the y-axis represent the reversal of migration initiated by ephrin-A1. n = 3 (D). (E) Higher-magnification images from early time points (2, 3, 4, 6, 8, and 12 hours) of ephrin-A1–overexpressing cells (EFNA1, red) confronting control cells (CTRL, green). Arrowhead points to the initial confrontation area at 4 hours. Scale bar denotes 200 μm.

Article Snippet: The cells on glass coverslips were then permeabilized in 0.1% Triton X-100 for 5 minutes and processed for immunofluorescence staining as previously described using a goat antibody against EphA2 (AF3035; R&D Systems, Minneapolis, MN, USA), rabbit polyclonal antibody against ephrin-A1 (V18; Santa Cruz Biotechnologies), or a mouse monoclonal antibody against E-cadherin (HECD1; Abcam) with detection using an Alexa Fluor-488 or -555 or 647-nm conjugated donkey anti-goat, anti-rabbit or anti-mouse antibody (Invitrogen).

Techniques: Labeling, Live Cell Imaging, Expressing, Control, Imaging, Migration

Journal: Investigative Ophthalmology & Visual Science

Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

doi: 10.1167/iovs.17-22941

Figure Lengend Snippet: Cell–cell border localization of E-cadherin is reduced at EphA2/Ephrin-A1 boundaries. (A) Immunofluorescent staining of EphA2 (green), ephrin-A1 (red), and E-cadherin (magenta) in cells present at the boundary of Control:Control- or Control:Ephrin-A1–expressing cell cocultures 48 hours after removal of the silicone barrier. A magnified view of the boundary is shown below in control cells confronting ephrin-A1–expressing cells. Dotted lines indicate the boundary between the two different cell populations 48 hours after initiation of confrontation. n = 4. Scale bar denotes 80 μm. (B) Control or ephrin-A1–expressing cells were transduced to express mCherry (Control-mCherry or EFNA1-mCherry, respectively) to differentiate these cell populations from control cells transfected with siControl (siCTRL) or siEphA2. Immunostaining of E-cadherin (magenta) was performed in cocultures 48 hours after initiation of confrontation. Dotted lines indicate the boundary between the two different cell populations at 48 hours. n = 3.

Article Snippet: The cells on glass coverslips were then permeabilized in 0.1% Triton X-100 for 5 minutes and processed for immunofluorescence staining as previously described using a goat antibody against EphA2 (AF3035; R&D Systems, Minneapolis, MN, USA), rabbit polyclonal antibody against ephrin-A1 (V18; Santa Cruz Biotechnologies), or a mouse monoclonal antibody against E-cadherin (HECD1; Abcam) with detection using an Alexa Fluor-488 or -555 or 647-nm conjugated donkey anti-goat, anti-rabbit or anti-mouse antibody (Invitrogen).

Techniques: Staining, Control, Expressing, Transfection, Immunostaining

Journal: Investigative Ophthalmology & Visual Science

Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

doi: 10.1167/iovs.17-22941

Figure Lengend Snippet: Ephrin-A1–induced boundary formation requires EphA2. (A) Control (Control, red) or (B) ephrin-A1–expressing (EFNA1, red) cells are shown at 48 hours after initiation of confrontation with cells transfected with siControl (siCTRL, green), siEphA2 (green), siEphrin-A1 (siEFNA1, green), or double siRNA (siEphA2+siEFNA1, green) oligonucleotides. Solid white lines mark the midline where the silicone divider was present at the time of removal. White dotted lines indicate the boundary between two cell populations 48 hours after initiation of confrontation. (C) Quantification of confrontation response in A and B as measured by % deviation from migration front of red-labeled cells (Control, red or Ephrin-A1, red). *P < 0.05 versus siCTRL; #P < 0.05, Ephrin-A1, red versus Control, red, n = 3. (D) A representative Western blotting showing the levels of EphA2 or ephrin-A1 after siRNA depletion in lysates harvested at the end of the experiment (96 hours after siRNA transfection).

Article Snippet: The cells on glass coverslips were then permeabilized in 0.1% Triton X-100 for 5 minutes and processed for immunofluorescence staining as previously described using a goat antibody against EphA2 (AF3035; R&D Systems, Minneapolis, MN, USA), rabbit polyclonal antibody against ephrin-A1 (V18; Santa Cruz Biotechnologies), or a mouse monoclonal antibody against E-cadherin (HECD1; Abcam) with detection using an Alexa Fluor-488 or -555 or 647-nm conjugated donkey anti-goat, anti-rabbit or anti-mouse antibody (Invitrogen).

Techniques: Control, Expressing, Transfection, Migration, Labeling, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

doi: 10.1167/iovs.17-22941

Figure Lengend Snippet: ADAM10 mediates Ephrin-A1/EphA2 boundary organization via EGFR signaling. (A) E-cadherin (E-cad; top) and ADAM10 (bottom) immunofluorescence staining in human anterior segmental epithelium. Scale bar denotes 100 μm. (B) E-cadherin staining of control cells (Control, green) confronted by “like” control cells (Control, red) or ephrin-A1–expressing cells (EFNA1, red) confronted by “unlike” control cells (Control, green; bottom) in the presence of general MMP inhibitor, TAPI, or a specific ADAM10 inhibitor, GI254023X (GIX). Red dotted lines indicate the boundary between the two cell populations 48 hours after initiation of confrontation. Scale bar denotes 80 μm. (C) Quantification of confrontation experiments at 48 hours in cocultures treated with DMSO, GIX, LY294002 (LY), Y-27632 (Y), or U0126 (U). * P < 0.05, n = 3–4. (D) Quantification of confrontation experiments at 48 hours using ephrin-A1–expressing cells (EFNA1) in contact with “unlike” control cells that had been treated with DMSO or the EGFR inhibitor, AG1478 (AG). Cells were either pretreated before the initiation of confrontation for 1 hour (AG pretreat) or treated with inhibitor 5 (AG @ 5 hrs) or 24 hours (AG @ 24 hrs) after initiation of confrontation. (E, F) Various concentrations of EGF (0.1, 1, 10, or 100 ng/mL) were added to the culture medium of these ephrin-A1 and control cell cocultures after pretreatment with GIX for 5 hours. Images (E) and quantification (F) are shown 48 hours after confrontation. Solid white lines mark the midline where the silicone divider was present at the time of its removal. White dotted lines indicate the boundary between the two different cell populations 48 hours after initiation of confrontation. n = 3–4.

Article Snippet: The cells on glass coverslips were then permeabilized in 0.1% Triton X-100 for 5 minutes and processed for immunofluorescence staining as previously described using a goat antibody against EphA2 (AF3035; R&D Systems, Minneapolis, MN, USA), rabbit polyclonal antibody against ephrin-A1 (V18; Santa Cruz Biotechnologies), or a mouse monoclonal antibody against E-cadherin (HECD1; Abcam) with detection using an Alexa Fluor-488 or -555 or 647-nm conjugated donkey anti-goat, anti-rabbit or anti-mouse antibody (Invitrogen).

Techniques: Immunofluorescence, Staining, Control, Expressing

Journal: Scientific Reports

Article Title: A role of the SAM domain in EphA2 receptor activation

doi: 10.1038/srep45084

Figure Lengend Snippet: ( A ) Schematic illustration of EpA2 truncation constructs. The constructs were cloned into the LZRS IRES retroviral vector. ( B ) Total cell lysates from PC3 cells expressing the indicated EphA2 truncation mutants were immunoblotted with the indicated antibodies. The pY-EphA/B antibody was raised against the phosphorylated di-tyrosine motif in the juxtamembrane motif conserved in both EphA and EphB receptors. Total EphA2 levels were detected with an antibody against the ectodomain of EphA2. ( C ) Immunoblot of total cell lysate from DU145 cells expressing different truncation constructs. ( D , E ) Immunoblot of PC3 ( D ) and DU145 ( E ) cell lysates following stimulation with 3 μg/ml ephrinA1-Fc for 15 min. Ponceau Red staining was used as loading control. Except for the loading controls, full gels containing all bands are included; long and short exposures are also included to show the differences in EphA2 phosphorylation levels.

Article Snippet: Antibodies used include goat anti-EphA2 ectodomain antibody (R&D, AF3035) and mouse anti-tubulin (Sigma Aldrich, T5168).

Techniques: Construct, Clone Assay, Plasmid Preparation, Expressing, Western Blot, Staining

Journal: Scientific Reports

Article Title: A role of the SAM domain in EphA2 receptor activation

doi: 10.1038/srep45084

Figure Lengend Snippet: ( A ) Constitutively active EphA2 after SAM deletion caused compaction of DU145 epithelial colonies. DU145 cells were seeded at low density (1,000 cells/well) on 6-well cluster plates and culture for 10 days to allow the emergence of cellular colonies. ( B ) The constitutively active EphA2-ΔS-GFP rendered DU145 cells partially resistant to the HGF-induced cell scattering. Note the Bright field images of DU145 cells with control vector (top row) and EphA2ΔS-GFP (bottom row) treated with Fc (first column), EA1-Fc (second column), HGF + Fc (third column) and HGF + EA1-Fc (forth column). HGF-induced DU145 cell scattering was reduced in vector control cells by EA1-Fc. EphA2-ΔS-GFP expressing cells became more resistant to the HGF-induced scattering as some of the cells still remained attach to each other in the colonies (**).

Article Snippet: Antibodies used include goat anti-EphA2 ectodomain antibody (R&D, AF3035) and mouse anti-tubulin (Sigma Aldrich, T5168).

Techniques: Plasmid Preparation, Expressing

Journal: Scientific Reports

Article Title: A role of the SAM domain in EphA2 receptor activation

doi: 10.1038/srep45084

Figure Lengend Snippet: ( A ) Schematic diagram of the FCS experiment. 488 nm pulsed laser beam was focused onto the peripheral membrane of cultured cell to excite the diffusive receptors. The emitted photons were collected by the same objective and directed to a single-photon detector so that the fluorescence fluctuation caused by receptor diffusion can be recorded. Auto-correlation of the fluorescence intensity trace was performed to obtain the auto-correlation function curve (AFC). Insert: epi-fluorescence image of DU145 cell expressing EphA2FL-GFP; the red dot represents the position of the laser beam which is always placed on a flat membrane area. Scale bar is 5 μm. ( B ) Representative AFCs of FCS measurements on DU145 cells with expression of truncation mutant of EphA2 constructs. τ D reports on mobility of the diffusive receptors and is used to calculate the diffusion coefficients. G(0) reports on concentration of the diffusive receptors and is used to calculate molecular brightness.

Article Snippet: Antibodies used include goat anti-EphA2 ectodomain antibody (R&D, AF3035) and mouse anti-tubulin (Sigma Aldrich, T5168).

Techniques: Cell Culture, Fluorescence, Diffusion-based Assay, Expressing, Mutagenesis, Construct, Concentration Assay

Journal: Scientific Reports

Article Title: A role of the SAM domain in EphA2 receptor activation

doi: 10.1038/srep45084

Figure Lengend Snippet: Molecular brightness ( A , green) and diffusion coefficients ( B , black) of truncation mutant constructs of EphA2 in DU145 cells. And molecular brightness ( C , dark green) of Src16-GFP (monomer control) and Myr-GCN4-GFP (dimer control). The median values were reported next to the box plots. Each data point was the average of five 15 s FCS measurements performed on one cell. The grey numbers on top the plots are the total number of cells used. The one-way ANOVA test was performed to obtain the p values (****p < 0.0001, ns: p = 0.9098). The molecular brightness of EphA2ΔS-GFP and EphA2ΔKS-GFP are larger than that of EphA2FL-GFP. EphA2FL-GFP also has the larger diffusion coefficient compared to EphA2ΔKS-GFP and EphA2ΔS-GFP in DU145 cell lines. The results suggest that EphA2ΔS-GFP and EphA2ΔKS-GFP underwent oligomerization beyond that of EphA2FL-GFP in DU145 cell lines.

Article Snippet: Antibodies used include goat anti-EphA2 ectodomain antibody (R&D, AF3035) and mouse anti-tubulin (Sigma Aldrich, T5168).

Techniques: Diffusion-based Assay, Mutagenesis, Construct

Journal: Scientific Reports

Article Title: A role of the SAM domain in EphA2 receptor activation

doi: 10.1038/srep45084

Figure Lengend Snippet: Molecular brightness ( A , green) and diffusion coefficients ( B , black) of truncation mutant constructs of EphA2 in 728 cells. And molecular brightness ( C , dark green) of Src16-GFP (monomer control) and Myr-GCN4-GFP (dimer control). The median values were reported next to the box plots. Each data point was the average of five 15 s FCS measurements performed on one cell. The grey numbers on top the plots are the total number of cells used. The one-way ANOVA test was performed to obtain the p values (****p < 0.0001, ns: p = 0.6643). The molecular brightness of EphA2ΔS-GFP and EphA2ΔKS-GFP are larger than that of EphA2FL-GFP. EphA2FL-GFP also has the larger diffusion coefficient compared to EphA2ΔKS-GFP and EphA2ΔS-GFP in 728 cell lines. The results suggest that EphA2ΔS-GFP and EphA2ΔKS-GFP underwent oligomerization beyond that of EphA2FL-GFP in 728 cell lines.

Article Snippet: Antibodies used include goat anti-EphA2 ectodomain antibody (R&D, AF3035) and mouse anti-tubulin (Sigma Aldrich, T5168).

Techniques: Diffusion-based Assay, Mutagenesis, Construct

Journal: Scientific Reports

Article Title: A role of the SAM domain in EphA2 receptor activation

doi: 10.1038/srep45084

Figure Lengend Snippet: Molecular brightness ( A , green) and diffusion coefficients ( B , black) of truncation mutant constructs of EphA2 in 728 cells with EA1-Fc treatment. And molecular brightness ( C , dark green) of Src16-GFP (monomer control) and Myr-GCN4-GFP (dimer control). The median values were reported next to the box plots. Each data point was the average of five 15 s FCS measurements performed on one cell. The blue circles and values are the diffusion coefficients and molecular brightness of EphA2 constructs before Fc-EA1 treatment. The grey numbers on top the plots are the total number of cells used. The one-way ANOVA test was performed to obtain the p values (****p < 0.0001, ns : p = 0.0617, ns: p = 0.6878). The molecular brightness of the three constructs all increased upon Fc-EA1 binding. The diffusion coefficients of the three EphA2 constructs all decreased ~50% indicating the formation of larger assemblies of EphA2 receptors, which agrees with the increase of molecular brightness as larger clusters are brighter particles with slower motion. These observations demonstrated EphA2 constructs undergo further oligomerization upon Fc-EA1 binding.

Article Snippet: Antibodies used include goat anti-EphA2 ectodomain antibody (R&D, AF3035) and mouse anti-tubulin (Sigma Aldrich, T5168).

Techniques: Diffusion-based Assay, Mutagenesis, Construct, Binding Assay