Nuclear entry of FIH is mainly HIF1α-dependent, and requires inhibition of FIH enzymatic activity. (A) U2OS cells were transfected with the indicated siRNAs for 3 days, followed by culture in normoxia (20% O2) or hypoxia (1% O2, 3h). Images show immunofluorescence staining of FIH (green) in U2OS cells after the indicated treatments. TO-PRO-3 (blue) was used to stain nuclei. (B) Immunofluorescence staining of FIH (green), HIF1α N803OH (green) and HIF1α (red) in U2OS cells treated with DMSO, DM-NOFD (1 mM), IOX2 (0.25 mM) or DM-NOFD (1 mM) plus IOX2 (0.25 mM) for 3 h. TO-PRO-3 (blue) was used to stain nuclei. (C) Protein levels of FIH, HIF1α and HIF1α N803OH in U2OS cells after the indicated treatments. β-tubulin was used as a loading control. Total cell lysates from the treated U2OS cells were immunoprecipitated with an anti-HIF1α antibody. Scale bars: 20 µm.