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, Construct, 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: 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, 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: 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, Expressing