ATF-4 expression is controlled by PHD3. (A) HeLa cells were transiently transfected with siRNA targeting PHD2 or PHD3 and exposed to 20% or 1% O₂. After 24 hours, the cells were lysed and analyzed by immunoblotting. One dish of HeLa cells was treated for 8 hours with 300 nM thapsigargin (t). Subsequently, cells were lysed and analyzed together with the protein extracts of the siRNA-transfected cells. (B) Hela cells were transiently transfected with siRNA targeting PHD3 or a control siRNA and exposed to 1% O₂ for 4 hours with subsequent reoxygenation. After the indicated time points, cells were lysed and analyzed by immunoblotting. (C) HeLa cells were transiently transfected with siRNA targeting PHD2 or PHD3 and exposed to 20% or 1% O₂. After 24 hours, the cells were lysed and total RNA was extracted and analyzed for ATF-4, PHD2, and PHD3 mRNA levels by real-time reverse transcriptase (RT)–PCR. Shown are mean values plus or minus SD of 3 independent experiments of ratios to the ribosomal protein L28 mRNA levels, which were used as housekeeping controls.