(A) Diagram of IRP binding to the IRE in FTL mRNA (Anderson and McLaren, 2012). Hyperferritinemia mutations are highlighted in orange (Cazzola et al., 1997), green (Camaschella et al., 2000), and blue (Luscieti et al., 2013) with their corresponding serum ferritin levels listed. Normal serum ferritin levels are under 300 µg/L. The 3RE is indicated in purple. Nucleotides that directly interact with the IRP are also identified (i.e. A15, G16, U17). (B) Luciferase activity of HepG2 cells transfected with mRNAs encoding the WT FTL 5ʹ-UTR or various hyperferritinemia mutations (G51C, G52C, or Loop mutant (A15G/G16C)), normalized to WT FTL reporter luciferase luminescence. The results are from three biological replicates, with error bars representing the standard deviation of the mean. (C) Binding of eIF3 to luciferase reporter mRNAs with WT or mutant forms of the FTL 5ʹ-UTR, using EIF3B immunoprecipitation (IP), followed by RNA extraction and RT-qPCR. Cells were harvested 8 hr post-transfection. Data are shown as the percent in the IP, compared to input levels. Error bars are the standard deviation of the mean of duplicate qPCR measurements from a representative IP. (D) Dose-response curve of RNA competition assays, based on gel shifts of NIR-labeled WT IRE-containing RNA, with WT, G51C, G52C, or Loop mutant (A15G/G16C) RNAs serving as cold competitors. Fold excess of competitor WT extended up to 100,000x. Recombinant IRP1 was used. Error bars represent standard deviations for each concentration of competitor. (E) The calculated IC50 values of the various competitor RNAs, based on the data in (D), with error bars representing the standard deviation from the mean IC50 value. N.A., the IC50 value could not be determined for the Loop mutant due to lack of any detectable competition. Note that the data for ∆3RE in panels (D) and (E) are from Figure 2B and C, measured in duplicate. The remaining experiments in (D) and (E) were carried out in triplicate.