Figure 5.

Peroxide increases HIF target gene expression in an FIH-dependent manner. (A) RCC4 cells were incubated at 1% O₂ for 3 h before bolus T-hydro treatment (at doses indicated) for 4 h. Extracts were analysed by IB. (B) IB of extracts from U2OS cells incubated for 4 h either in normoxia or 1% O₂ and treated with −/+T-hydro (10 μM, added every hour). (C) Reverse transcription–quantitative polymerase chain reaction showing PHD3, adrenomedullin (ADM), CA9 and FIH mRNA levels after treatment of U2OS cells with control or FIH siRNA and incubations as in B. mRNA levels normalized to cyclophilin are expressed as fold change relative to normoxic control siRNA levels (n=3 biological replicates). Data are mean±s.d. (D) IB of extracts from U2OS cells incubated for 4 h either in normoxia, graded hypoxia, T-hydro (at concentrations indicated) or with MG132. The position of NRF2 is marked with an arrow, and a nonspecific band is marked as ^*. At 25 μM, T-hydro cells showed morphological change. CON, control; Dox, doxycycline; FIH, factor inhibiting hypoxia-inducible factor; HIF, hypoxia-inducible factor; IB, immunoblotting; NRF2, NF-E2-related factor 2; OH, hydroxylation; siRNA, short interfering RNA; T-hydro, tert-butyl hydroperoxide.