Fig. 4. Hypoxia induces autophagy in 661W photoreceptor cells.

Western and immunohistochemical analysis of LC3 protein in 661W cells under hypoxic conditions. (A) LC3 and BNIP3 protein immunoreactivity on western blots of homogenates from 661W incubated in normoxic or hypoxic conditions for times indicated. C, control normoxic sample. Quantification of LC3-II/GAPDH ratios was based on densitometry measurements of western blots. The level of LC3-II has a significant peak 16 hours hypoxia (p **< 0.005). Images are representative of 9 independent experiments with cells between passage 4 and 6. (B) 661W cells were incubated in hypoxic conditions for 8 or 24 hours post confluence, fixed, and stained with antibody against LC3. DAPI-stained nuclei are shown in blue. The number of LC3+ punctae/cell was quantified at times indicated (bar graphs). Error bars represent mean ± SEM, n=3 independent experiments using 9 non-overlapping fields of 100 µm for each condition. p values were calculated using ANOVA followed by Bonferroni’s test, and are as shown: p**<0.005. (C) 661W cells were cultured under normoxic or hypoxic conditions for 24 hours in the presence or absence of Bafilomycin A, an inhibitor of autophagic vacuole maturation for the last 4 hours of the incubation and evaluated by western analysis with antibodies recognizing LC3 and p62. Ratios of p62/GAPDH are shown that are representative of 3 independent experiments.