Fig. 5. HIF-1α but not HIF-2α is required for hypoxia-induced autophagy in 661W photoreceptor cells.

(A-D) 661W cells were transfected with a pool of HIF-1α targeting siRNAs or a non-targeting siRNA control pool, and incubated in hypoxic conditions for 16 or 24 hours post confluence. (A) Transcript expression for Hif-1α, Hif-2α, and Gapdh evaluated using RT-PCR. (B) Protein levels of HIF-1α, HIF-2α, BNIP3, LC3, and GAPDH evaluated by western blot analysis of 661W cell homogenates lysed after 16 hours of hypoxia. Ratios of HIF-1α/GAPDH show a significant decrease of HIF-1α expression following treatment with siHIF-1α (p*<0.05). Ratios of LC3-II/GAPDH show a significant decrease in LC3-II in the presence of siHIF-1α (p*<0.05). Blots are representative of 3 independent experiments. Comparison of LC3-II to GAPDH was utilized due to the variability of LC3-I levels in cultured cells. (C) Immunohistochemical analysis of representative fields of 661W cells with LC3B punctae labeled in green and DAPI-stained nuclei shown in blue. The number of LC3+ punctae/cell was quantified after hypoxic incubation for 24 hours (bar graphs). Error bars represent mean ± SEM, n=3 independent experiments using 9 non-overlapping fields of 100 µm for each condition (p***<0.0005). (D) Caspase 8 activation and cell viability was evaluated following hypoxia for times indicated, n = 3 independent experiments using 9 non-overlapping fields of 100 µm for each condition (p**<0.005, p*** <0.0005, Student’s t-test). (E-H) 661W cells were transfected with a pool of HIF-2α targeting siRNAs or a non-targeting siRNA control pool, incubated in hypoxic conditions for 16 or 24 hours post confluence and evaluated as in A-D.