Figure 5.

Autophagy is dysfunctional in AMD RPE. (a and c) Analysis of autophagy dynamics in normal RPE (a), and AMD RPE (c), n=5. LC3 immunoblots of control and AMD RPE under three different conditions, nutrient, starvation in the presence of IGF-1, and starvation in the absence of IGF-1 are shown. Beta actin is used as a normalization control. Spliced membranes are indicated by the vertical lines. (b and d) The ratios of the LC3-II/LC3-I levels as determined by densitometry are illustrated in the graphs, showing that an increase in autophagy dynamics in the absence of IGF-1 under starvation conditions is observed only in normal, but not in AMD RPE. Densitometry was performed on three repeats of the experiment for each sample in five normal and five AMD RPE (b and d). Asterisks in (b) represent P-value <0.0001 of LC3-II/LC3-I ratios as determined by one-way Anova followed by Tukey's test. (e and f) Autophagic flux is lower in AMD RPE as compared with normal RPE. (e) Immunoblot of p62, demonstrating lower autophagic flux in AMD RPE as shown by inability of AMD RPE to downregulate p62 levels during starvation in the absence of IGF-1. Beta actin is used as a normalization control. (f) Relative expression of p62 in control and AMD RPE in the presence and absence of IGF-1, as determined by densitometry analysis of the immunoblot in (e), n=5. The asterisks (*) indicate statistical significance determined by ANOVA followed by Tukey's test (P<0.05)