Equal volumes from each deproteinated sample was then analyzed for GSH and derivatized by 2-vinylpyridine, and measured for GSSG (Sigma Aldrich, 132292)

Equal volumes from each deproteinated sample was then analyzed for GSH and derivatized by 2-vinylpyridine, and measured for GSSG (Sigma Aldrich, 132292). in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD. < 0.05. (E) Autophagosome counts in ARPE-19 cells were obtained following immunostaining with LC3 antibody to identify autophagic puncta. A representative set of photomicrographs is usually shown for control cells and those exposed to H2O2. Autophagosome numbers were counted in a minimum of 3 experiments and statistical significance between the control group and each treatment group was determined by ANOVA. Differences between groups were considered statistically significant when < 0.05. Open in a separate window Physique 2. The effect of H2O2 around the expression of the autophagy-related proteins BECN1, ATG7, and ATG9 in ARPE-19 cells was determined by western blot. ACTB was used as an internal control. A representative protein gel blot is usually shown together with densitometric quantification from a mean of 3 experiments. Differences between groups were considered statistically significant when < 0.05. To confirm that the enhancement of autophagic flux is usually a common event in the RPE in response to acute oxidative stress, we examined the autophagic response following treatment with rotenone, which is an inhibitor of mitochondria complex I, and leads to increased A-381393 superoxide generation.19 ARPE-19 cells treated with 1 or 10?M rotenone for 24?h did not demonstrate any significant loss of cell viability by the crystal violet assay but did show a small (<14 %), but significant, reduction in mitochondrial respiration (Fig. 3A). ARPE-19 cells treated with rotenone exhibited a significant increase in LC3-II expression and the ratio of LC3-II/-I (Fig. 3B). A similar response was observed when cells were immunostained for endogenous LC3, with an increased number of autophagosomes in rotenone-treated cells compared to untreated control (Fig. 3C). Cell starvation, a common positive control in autophagy experiments, showed a dramatic increase in the LC3-II/-I ratio (Fig. 3B). Western blot analysis for expression of the autophagy proteins ATG7, ATG9, and BECN1 (Fig. 3D) A-381393 revealed comparable results to that achieved with H2O2, in that there were no significant changes in A-381393 protein levels. Open in a separate window Physique 3. Acute rotenone treatment increases autophagy flux in the RPE. ARPE-19 cells were exposed to 1 or 10?M rotenone for 6 or 24?h. (A) Cell viability and mitochondrial respiration following exposure to rotenone. (B) Autophagic flux was monitored by LC3-II/-I conversion using western blot with anti-LC3 antibody. ACTB was used as an internal control. A representative protein gel blot is usually shown together with densitometric quantification of the LC3-II/-I ratio from a mean of 3 experiments. Differences between groups were considered statistically significant when < 0.05. (C) Autophagosome counts A-381393 in ARPE-19 cells exposed to 0, 1, or 10 uM rotenone for 24?h were A-381393 obtained following immunostaining with LC3 antibody to identify autophagic puncta. Statistical significance between the control group and each treatment group was determined by ANOVA. (D) The effect of rotenone around the expression of the autophagy-related proteins BECN1, ATG7, and ATG9 was determined by western blot. ACTB was used as an internal control. A representative protein gel blot is usually shown together with densitometric quantification from a mean of 3 experiments. Differences between groups were considered statistically significant when < 0.05. Chronic H2O2 treatment reduces autophagic flux and the expression of autophagic factors in the RPE We next investigated whether chronic oxidative stress, which has been implicated in the pathogenesis of AMD and other neurogenerative diseases,11,20 affected EIF4EBP1 autophagy in cultured RPE cells in a similar fashion to a single acute exposure. ARPE-19 cells received repeated exposure to H2O2 (200?M or 400?M) every 24?h for up to 14 d. The level of damage as measured by protein carbonyl content in cells challenged by oxidative stress continued to be higher than in untreated control (Fig. 4A), but was lower than that for acute exposure (Fig. 1B). Chronic stress with 200 or 400?M H2O2 did not lead to a loss in cell viability.