Tf-independent iron uptake displays a biphasic response during hypoxia. HEK293 cells were grown for the indicated times under hypoxic conditions or under normoxia with 1000 × cold Fe-NTA. Cells were incubated with ⁵⁵Fe-NTA in serum-free DMEM (pH 7.4) or HBSS (pH 6.0) 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 16 hours hypoxia) or n = 6 (1000 × cold Fe) of hypoxia-treated cell extracts. The ^* indicates points that differ from the control group with P < .005 using a Student t test. The ± refers to the presence or absence, respectively, of 20 μM ascorbic acid in serum-free DMEM or HBSS.