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.