Hypoxia does not change Tf-dependent iron uptake. (A-B) HEK293 cells were grown for the indicated times under hypoxic conditions or under normoxia with Df (16 hours) or FAC (5 hours). (A) Northern analysis of poly(A)⁺ mRNA. RNAs were transferred to a nylon membrane and hybridized with a ³²P-labeled TfR or actin probe as a control. Df, 200 μM; FAC, 150 μg/mL. Experiment was performed 4 times with a representative blot shown. (B) Immunoblot analysis of cytosolic cell extracts with antihuman TfR antibodies. Df, 100 μM; FAC, 50 μg/mL. Experiment was performed 5 times with a representative blot shown. (C) HEK293 cells were grown for the indicated times under hypoxic conditions or under normoxia with 1000 × cold Fe-Tf. Cells were incubated with ⁵⁵Fe-Tf in serum-free DMEM for the last one hour of treatment, washed, harvested, and radioactivity quantified by counting cell extracts. The bar graph shows the mean percent radioactivity ± SEM, n = 9 (0, 1, 7, and 21 hours hypoxia) or n = 3 (1000 × cold Fe-Tf) of hypoxia-treated cell extracts. The ^* indicates point that differs from the control group with P < .005 using a Student t test.