(A) Expression levels of Bnip3, p27Kip1, cleaved caspase 3, and Sirt1 and formation of LC3II in retrovirally mediated Sirt1-knockdown cells under hypoxia (1% O2, 24 hours) in CR serum condition. To detect LC3I and LC3II bands, cells were preincubated with lysosomal inhibitor (E64d and pepstatin A). (B) Quantitative analysis of the ratio of LC3II to LC3I (n = 4). (C) Acetylation of Foxo3 in Sirt1-knockdown cells under hypoxia in CR serum. (D) ChIP analysis to determine Foxo3 binding to promoters of Bnip3, p27Kip1, and Bim in Sirt1-knockdown cells under hypoxia in CR serum. (E) Expression levels of Bnip3, p27Kip1, cleaved caspase 3, and Sirt1 and formation of LC3II in retrovirally mediated Sirt1-overexpressing cells transfected with siRNA control or siRNA for Foxo3 under hypoxia in AL serum. To detect LC3I and LC3II bands, cells were preincubated as in A. (F) Quantitative analysis of the ratio of LC3II to LC3I (n = 4). (G) ChIP analysis to determine Foxo3 binding to promoters of Bnip3, p27Kip1, and Bim in Sirt1-overexpressing cells transfected with siRNA control or siRNA for Foxo3 under hypoxia in AL serum. LY294002 was used as a PI3K inhibitor at 20 μM. Data are mean ± SEM. *P < 0.05.