18 research outputs found

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

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    <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

    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.

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    <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.

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    <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

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

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    <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

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

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    <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.

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    <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

    Topographic distribution of Brn3a+ RGCs in excitotoxically injured retina after brimonidine pretreatment.

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    <p>Isodensity maps showing the topographic distribution of Brn3a+ RGCs in a representative retina after NMDA injury pretreated with saline or brimonidine (BMD) at 7 days (A,C,E,G,I) and 14 days (B,D,F,H,J) post lesion. Experimental groups were: (A, B) Injections of saline + saline, (C, D) saline + 5 μg NMDA, (E, F) brimonidine + 5 μg of NMDA, (G, H) saline + 10 μg of NMDA, and (I, J) brimonidine + 10 μg of NMDA. The total number of Brn3a<sup>+</sup>RGCs of each retina is indicated for each map. BMD; brimonidine, D; dorsal, N; nasal, V; ventral, T; temporal.</p
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