Volume 39, Issue 5, Pages 710-715 (November 2003) The role of the iron responsive element in the control of ferroportin1/IREG1/MTP1 gene expression  Athina Lymboussaki, Elisa Pignatti, Giuliana Montosi, Cinzia Garuti, David J. Haile, Antonello Pietrangelo  Journal of Hepatology  Volume 39, Issue 5, Pages 710-715 (November 2003) DOI: 10.1016/S0168-8278(03)00408-2

Fig. 1 Expression of the SLC40A1 promoter/5′ UTR driven luciferase reporter gene is regulated by iron in three cell lines. HepG2 and CaCO2 cells were transiently transfected by lipofection and U937 cells by electroporation with the SLC40A1-2534-Luc construct. Twenty-four hours later, cells were exposed to various agents for 16–20 h, then harvested and cell lysates were assayed for luciferase activity. Promoter activity is expressed as arbitrary luciferase light units after correction for the background activity conferred by the promoterless control plasmid pGL3-Control. The reported values of Firefly luciferase have been normalized for the Renilla luciferase light units obtained by cotransfecting the phRL-CMV plasmid. Results are expressed as mean of triplicate wells±S.D. in five separate experiments. (A) HepG2, (B) CaCO2 and (C) U937 cells. 1: untreated; 2: iron; 3: DFO (desferrioxamine). Journal of Hepatology 2003 39, 710-715DOI: (10.1016/S0168-8278(03)00408-2)

Fig. 2 Iron-dependent regulation of the SLC40A1 promoter/5′ UTR driven luciferase reporter is independent of promoter sequences. HepG2 and CaCO2 cells were transiently transfected by lipofection with three 5′–deletion constructs of the SLC40A1-2534-Luc promoter linked to the luciferase reporter gene in the pGL3-Control vector. A schematic structure of the promoter constructs is shown at the top of the figure: the numbers indicate the 5′ end of the construct and the start of the luciferase gene (+297); the arrow indicates the transcription start site. Gray box, luciferase reporter gene; black box, iron-responsive element; striped box, NF-kB consensus sequence; white box, consensus binding site for interferon responsive element. The luciferase activity was assayed and calculated as specified in the legend of Fig. 1. A, B, C: HepG2 cells; D, E, F: CaCO2 cells. A, D: SLC40A1-Δ1418-Luc. B, E: SLC40A1-Δ303-Luc C, F: SLC40A1-Δ28-Luc. 1: untreated; 2: iron; 3: DFO. Journal of Hepatology 2003 39, 710-715DOI: (10.1016/S0168-8278(03)00408-2)

Fig. 3 mRNA levels of the SLC40A1 promoter/5′ UTR driven luciferase gene and the native SLC40A1 gene are not altered by iron status of cells. (A) The exogenously transferred luciferase reporter gene was analyzed for expression by reverse transcriptase-PCR. RNA was extracted from SLC40A1-2534-Luc transfected HepG2 cells either untreated (lane 2) or treated for 16–20 h with iron (lane 3), DFO (lane 4). Lane 1 is a negative control for PCR of luciferase gene using untransfected cells. In each test tube beta-actin mRNA was also analyzed by specific primers. (B) Representative Northern-blot analysis of endogenous genes. RNA obtained from HepG2 cells treated for 16–20 h with several agents was subjected to Northern blot and hybridized with the cDNA probes indicated: TfR: transferrin receptor; l-ferritin: ferritin L chain. 1: untreated; 2: iron; 3: DFO. Journal of Hepatology 2003 39, 710-715DOI: (10.1016/S0168-8278(03)00408-2)

Fig. 4 Deletion of the IRE in the SLC40A1 promoter/5′ UTR driven luciferase reporter results in loss of iron-dependent regulation. (A) Radiolabeled RNA transcribed from the wild-type IRE of SLC40A1 5′ UTR (lane 1) and mutant ΔIRE 5′ UTR (lane 2) was assayed for total IRP binding by electrophoretic mobility shift assay (EMSA) as described in Materials and methods. (B) HepG2 and (C) CaCO2 cells were transiently transfected with the SLC40A1-ΔIRE-Luc construct and subsequently treated for 16–20 h with iron or DFO. The luciferase activity was assayed and calculated as specified in the legend of Fig. 1. (D) EMSA was done using whole cytoplasmic HepG2 extracts from cells treated with various agents and radiolabeled ferritin H-chain IRE as described above. 1: untreated cells; 2: iron; 3: DFO. Journal of Hepatology 2003 39, 710-715DOI: (10.1016/S0168-8278(03)00408-2)