TfR1 knockdown causes iron deficiency and decreases responses to iron modulations in MDA-MB-231 cells. A: TfR1 levels in the parental MDA-MB-231 cells and TfR1-silenced transfectants. Lanes 1-2 represent stable transfectants, while lanes 4-5 illustrate TfR1 levels in cells collected after 24-h transient transfection. Lane 1: shRNA#1 (L1); lane 2: shRNA #3 (L3); lane 3: parental MDA-MB-231cells (control); lane 4: a mixture of shRNAs # 1, 2, & 3 (L1,2,3); lane 5: nonsense shRNA control (L-). B: Means of two densitometric analyses based on two independent transfection experiments. C: nonsense (L-) and TfR1 knockdown (L3) MDA-MB-231 cells were subjected to different treatments: DFO (0.5 mM; 6 h); hypoxia (1% O2, 6 h); iron (50 μM FAC, 6 h), and ferritin was quantitated by ELISA. Bars represent means ± SD of three independent experiments (n = 3). +Significantly different from L- nonsense controls (p < 0.05). * Significantly different from L3 normoxic controls (p < 0.05).