Relative mRNA levels of EDNs and EDNRs in E18 chicken retina, primary chicken Müller cells and the human MIO-M1 Müller cell line.

<p>qRT-PCR analysis of mRNA levels. Bar graphs showing the relative mRNA levels normalized to ß-actin for (A) EDNRA, EDNRB, EDNRB2 and SOX2, and for (B) EDN1, EDN2, EDN3 in chicken E18 retina (Chicken retina), primary chicken Müller cells (Chicken Müller cell) and the human MIO-M1 Müller cell line. Note that EDNRB2 is not found in human. For the MIO-M1 cells the relative mRNA levels of EDNRA and EDNRB are shown. Sox2 is included as an expression reference for the chicken cells. Bar graphs are mean ± SEM, n = 5 (*P < 0.01, ***P< 0.0001) analyzed by one-way ANOVA and Tukey’s post hoc test. Significance is only indicated for the comparisons: EDNRA-EDNRB, EDNRB-EDNRB2, EDN1-EDN2, and EDN2-EDN3.</p

P-ERK1/2 in primary chicken Müller cells and the human MIO-M1 cell line after stimulation with IRL1620.

<p>(A) Experimental outline. (B-G) Primary chick Müller cells and (H-M) human MIO-M1 cells. Serum-starved cells were treated with 5 μM IRL1620, and analyzed at 5 min up to 210 min. (B) Western blot analysis of P-ERK levels in IRL1620-treated primary chick Müller cells. (C) Bar graph with densitometry of P-ERK levels normalized by GAPDH levels. Normalization to total ERK showed similar results (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167778#pone.0167778.s005" target="_blank">S5 Fig</a>). Note that western blot analysis for ERK1/2 only shows one band in contrast to the two bands that are seen in mammals. (D–G) Fluorescence micrographs showing immunocytochemistry for P-ERK of IRL1620-treated primary chicken Müller cells at the indicated time points. 2M6 is a marker for chicken Müller cells. Cell nuclei were counter stained with DAPI. (H) Western blot analysis of P-ERK1/2 levels in IRL1620-treated MIO-M1 cells. (I) Bar graph with densitometry of P-ERK1/2 levels normalized by GAPDH levels. Normalization to total ERK1/2 showed similar results (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167778#pone.0167778.s006" target="_blank">S6 Fig</a>). (J-M) Fluorescence micrographs showing immunocytochemistry for P-ERK in IRL1620-treated MIO-M1 cells at the indicated time points. Glutamine Synthetase (GS) is a Müller cell marker. 2M6 does not label human cells. Bar graphs are mean ± SEM, n = 3, (*P < 0.01, **P < 0.001, ***P < 0.0001) analyzed by one-way ANOVA and Tukey’s post hoc test. Significance is indicated for comparisons IRL1620 0 min and IRL1620 10, 30, 180 and 210 min; and IRL1620 60 min with IRL1620 180 and 210 min. Scale bar in (G and M) is 30 μm; valid also for (D-F and J-L).</p

EDNRB agonist IRL1620 activates P-ERK1/2 in chicken retina.

<p>Immunohistochemistry and western blot analysis of P-ERK after intra-ocular injection of IRL1620 in E18 chicken embryo. (A) Experimental outline. (B–G) Fluorescence micrographs showing P-ERK and 2M6 (Müller cell marker) immunoreactivity in (B) normal untouched retina, retina after (C) 2 h, (D) 4 h, (E) 6 h, (F) 24 h IRL1620 treatment. (G) Vehicle-injected eye at 2 h (Ctrl). (H) Representative western blot analysis of P-ERK in retina 2, 4, 6, and 24 h after IRL1620 treatment. Note that western blot analysis for ERK1/2 in chicken only shows one band in contrast to the two bands that are seen in mammals (I) Bar graph with densitometry of P-ERK levels normalized by GAPDH levels. Normalization to total ERK gave similar results (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167778#pone.0167778.s001" target="_blank">S1 Fig</a>). Bar graph is mean ± SEM, n = 3 (**P < 0.001, ***P < 0.0001) analyzed by one-way ANOVA and Tukey’s post hoc test. Significance is only indicated for comparisons from control 2 h to IRL1620 2 h, 4 h and 6 h. Scale bar in (G) is 20 μm, also valid for (B–F).</p

Effects of EGFR kinase inhibitor AG1478 or EGFR-siRNA on IRL1620-induced P-ERK1/2 levels in Müller cells.

<p>Serum-starved primary chicken Müller cells and human MIO-M1 cells pretreated with 50 μM AG1478 or control (vehicle) for 30 min followed by treatment with 5 μM IRL1620 or vehicle for 10 and 180 min of chicken Müller cells, and for 10 min of human MIO-M1 cells. (A) Experimental outline. (B-G) Western blot analyses of P-ERK levels in (B, C) chicken Müller cells treated with IRL1620 and AG1478, and (D, E) EGF + AG1478. (C, E) Bar graphs with densitometry of P-ERK levels normalized by GAPDH levels. (F) Western blot analysis of P-ERK1/2 levels in human MIO-M1 cells treated with IRL1620 + AG1478. Note that western blot analysis for ERK1/2 shows two bands in human compared to one band in chicken. (G) Bar graph with densitometry of P-ERK1/2 levels normalized to GAPDH levels. (H) Human MIO-M1 cells transfected with EGFR-siRNA or non-targeted siRNA (NT-siRNA) in absence of serum for 48 h followed by treatment with 5 μM IRL1620 or vehicle for 10 min. (I) Western blot analysis of P-ERK 1/2 levels in human MIO-M1 cells treated with IRL1620+EGFR-siRNA or IRL1620+NT-siRNA. (J) Bar graph with densitometry of P-ERK1/2 levels normalized to GAPDH levels. Bar graphs are mean ±SEM, n = 3 (*P < 0.01, **P < 0.001, ***P < 0.0001) analyzed by one-way ANOVA and Tukey’s post hoc test. Significance is only indicated for the comparisons: IRL1620 10 min-IRL1620+AG1478 10 min, IRL1620 180 min-IRL1620+AG1478 180 min, IRL1620 10 min-IRL1620+NT-siRNA 10 min and IRL1620 10 min-IRL1620+EGFR-siRNA 10 min.</p

Effects of EDNRB blocker BQ-788 on IRL1620-induced P-ERK1/2 levels in primary chicken Müller cells and the human MIO-M1 cell line.

<p>Serum-starved primary chicken Müller cells and human MIO-M1 cells were pretreated with 50 μM EDNRB blocker BQ-788 or vehicle (control) for 30 min followed by 5 μM IRL1620 or vehicle for 10 and 180 min. (A) Experimental outline. (B, D) Representative western blot gels showing P-ERK levels in (B) primary chick Müller cells and (D) the human MIO-M1 cell line. (C, E) Bar graphs with densitometry of P-ERK levels normalized to GAPDH levels. Bar graphs are mean ± SEM, n = 3 (***P < 0.0001) analyzed by one-way ANOVA and Tukey’s post hoc test. Significance is indicated for the comparisons IRL1620 10 min-IRL1620+BQ-788 10 min and IRL1620 180 min-IRL1620+BQ-788 180 min.</p

Illustration summarizing the EDNR signaling leading to transactivation of EGFR in Müller cells.

<p>Diagram showing the signal transduction events and inhibitors with names within ellipses that were used to block the signaling steps. Stimulation of EDNRB in Müller cells with EDNRB-agonist IRL1620 that leads to activation of cytosolic Src kinase. The activated Src kinase triggers matrix-metalloproteinase (MMP), whose catalytic activity leads to release of membrane-bound heparin-binding epidermal growth factor (HB-EGFR) and consequently causes ligand-dependent transactivation of the epidermal growth factor receptor (EGFR). Activated Src kinase may also trigger ligand-independent EGFR phosphorylation of tyrosine residue (Y1173). This transactivation leads to MAPK/ERK signaling in Müller cells that could be blocked by the EDNRB antagonist BQ-788, Src-kinase inhibitors PP1 and PP2), EGFR-inhibitor AG1478, EGFR-small interfering RNA (EGFR-siRNA) or by the inhibitor GM6001, which inhibits extracellular matrix metalloproteinases.</p

Effect of MMPs inhibitor GM6001 on IRL1620- induced P-ERK1/2 in primary chicken Müller cells and the human MIO-M1 cell line.

<p>Serum-starved primary chicken Müller cells and human MIO-M1 cells pretreated with 50 μM GM6001 or control (vehicle) for 30 min followed by treatment with 5 μM IRL1620 or vehicles were analyzed. (A) Experimental outline. The MIO-M1 cells were only analyzed at 10 min because the peak at 180 min is not present. Western blot analyses of P-ERK levels in (B) chicken Müller cells treated with IRL1620 and/or GM6001 and (D) EGF and/or GM6001. EGF-treatment was only analyzed at time point 10 min. (C, E) Bar graphs with densitometry of P-ERK levels normalized by GAPDH levels. Western blot analysis of P-ERK1/2 levels in (F, G) human MIO-M1 cells treated with IRL1620 and/or GM6001. (G) Bar graph with densitometry of P-ERK1/2 levels normalized by GAPDH levels. Bar graphs are mean ±SEM, n = 3 (**P < 0.001) analyzed by one-way ANOVA and Tukey’s post hoc test. Significance is only indicated for the comparisons: IRL1620 10 min-IRL1620+GM6001 10 min, and IRL1620 180 min-IRL1620+GM6001 180 min.</p

Expression of HB-EGF in chicken Müller cells and in human MIO-M1 cell line.

<p>QRT-PCR analysis of heparin binding epidermal growth factor (HB-EGF), transcription factor SOX2 and cellular retinaldehyde-binding protein (CRALBP) mRNA levels in primary chicken Müller cell and in human MIO-M1 cell cultures. SOX2 and CRALBP were included as expression references known to be expressed in Müller cells. Bar graph showing the relative mRNA levels in relation to β-actin mRNA levels. Bar graph is mean ±SEM, n > 5.</p

Effects of Src-kinase inhibitors PP1 and PP2 on IRL1620-induced P-ERK1/2 and P-EGFR (Y1173) in primary chicken Müller cells and the human MIO-M1 cell line.

<p>Serum-starved primary chicken Müller cells and human MIO-M1 cells were pretreated with 5 μM PP1 or 5 μM PP2, or control (vehicle) for 20 min followed by treatment with 5 μM IRL1620 or vehicles and analyses. Western blot was used to analyze ERK-signaling with the different treatments. (A) Experimental outline. The MIO-M1 cells were only analyzed at 10 min because the peak at 180 min is not present. Western blot analyses of (B-E) P-ERK levels and (F, G) P-EGFR (Y1173) in chicken Müller cells. (C, E) Bar graphs with densitometry of P-ERK levels normalized by GAPDH and (G) P-EGFR (Y1173) levels normalized to total EGFR levels. Western blot analyses of (H) P-ERK1/2 levels and (J) P-EGFR (Y1173) levels in human MIO-M1 cells. Bar graphs with densitometry of (I) P-ERK1/2 levels normalized by GAPDH and (K) P-EGFR (Y1173) levels normalized to total EGFR levels. Bar graphs are mean ±SEM, n = 3 (***P < 0.0001) analyzed by one-way ANOVA and Tukey’s post hoc test. Significance is only indicated for the comparisons: IRL1620 10 min-IRL1620+PP1 10 min, IRL1620 10 min-IRL1620+PP2 10 min, IRL1620 180 min-IRL1620+PP1 180 min, and IRL1620 180 min-IRL1620+PP2 180 min.</p

Characterisation of the GABA_A receptor system in NPE cells.

<p>(A) Relative qRT PCR amplification levels of the 19 GABA_A receptor subunit mRNA in NPE cells. Grey columns for α subunits, red columns for β subunits, green columns for γ subunits, blue columns for δ, ε, π subunits and purple columns for ρ subunits. Error bars ±SD, n = 4 independent preparations each containing a pool of more than 10 NPE. (B) Electrophysiology of dissociated NPE cells. 1 µM GABA activated currents (−90 mV holding potential) that were inhibited by application of the GABA_A receptor antagonist SR-95531 (100 µM). n = 5. (C) Gaussian fits to all-points histograms derived from the current record shown in (B): solid line, currents after GABA application; broken line, currents after application of SR-95531. The difference between the two peaks in the presence of GABA equals the mean tonic current (−6.2 pA). (D) Relative qRT PCR amplification levels of NKCC1, KCC2, GAD65 and GAD67 mRNA in acute NPE cells compared to 6 months old retina (NKCC1 and KCC2) or cultured NPE cells (GAD65 and GAD67). Error bars ±SD, n = 4 as above.</p