NCOA4 mRNA induction by DFO is blocked by combined HIF1A and HIF2A knockdown. (A) HIF1A, (B) HIF2A, and (C) NCOA4 expression relative to ACTB in Hep3B cells that were or were not treated with DFO after transfection with the indicated siRNAs. In this experiment, Hep3B cells (2.5 × 105 cells per well in 6-well plates) were reverse-transfected with scrambled siRNA, HIF1A siRNA, and/or HIF2A siRNA in CM without antibiotics. Control cells were transfected with 10 nM scrambled siRNA. To deplete cells of either HIF1A or HIF2A, 5 nM scrambled siRNA plus 5 nM of the specific siRNA were used to control for total siRNA concentration. To deplete cells of both HIF1A and HIF2A, 5 nM of each specific siRNA was used. Twenty-four hours after transfection, some wells received additional DFO-supplemented CM without antibiotics to achieve a final DFO concentration of 100 μM (+DFO), whereas other wells received additional CM without antibiotics or DFO (−DFO). RNA was harvested for quantitative reverse transcriptase-polymerase chain reaction analysis 18 hours later. For all panels, the mean mRNA ratio from control cells that were not treated with DFO was normalized to 1. n = 3-6 per group. For all bar graphs, data represent mean ± SD. ***P < .001 and ****P < .0001 by 2-way ANOVA with Tukey’s post hoc test.