Regulation of expression of murine transferrin receptor 2 by Hiroshi Kawabata, Rasha S. Germain, Takayuki Ikezoe, Xiangjun Tong, Eric M. Green, Adrian F. Gombart, and H. Phillip Koeffler Blood Volume 98(6):1949-1954 September 15, 2001 ©2001 by American Society of Hematology

DNA sequences of the promoter region of murine TfR2 aligned with those of human TfR2.Identical residues are shaded. DNA sequences of the promoter region of murine TfR2 aligned with those of human TfR2.Identical residues are shaded. Sequences for exon 1 of murine TfR2 and human TfR2-α are encircled by a box. Putative GATA-1 and C/EBP sites are underlined and double-underlined, respectively. The murine TfR2 promoter sequence up to −1150 base is available in GenBank (accession number AF207742). Hiroshi Kawabata et al. Blood 2001;98:1949-1954 ©2001 by American Society of Hematology

Alternative forms of murine TfR2 transcripts Alternative forms of murine TfR2 transcripts.Using primers for 5′ and 3′ ends of the murine TfR2 coding region, the transcripts were amplified by RT-PCR, cloned, sequenced, and compared with the genomic sequences. Alternative forms of murine TfR2 transcripts.Using primers for 5′ and 3′ ends of the murine TfR2 coding region, the transcripts were amplified by RT-PCR, cloned, sequenced, and compared with the genomic sequences. The sequences of the primers are 5′-CACAAGCATGGAGCAACGTTGG-3′ and 5′-AGGGAGAAAGGAGAATCACGTGG-3′. Type 1 is the representative form, which is identical to that cloned by 5′ and 3′ RACE. Type 2 is a longer form that has additional sequences (intron 5 in the type 1) between exons 5 and 6. Type 3 is a shorter form that lacks exon 2. Sequences after exon 6 were the same in all 3 types, so only the 5′ portion of this gene (exons 1-6) is shown in this figure. Hiroshi Kawabata et al. Blood 2001;98:1949-1954 ©2001 by American Society of Hematology

Tissue distribution of murine TfR2.(A) RT-PCR analyses. Tissue distribution of murine TfR2.(A) RT-PCR analyses. RT-PCR was performed with primers for murine TfR2 (32 cycles) as well as GAPDH (24 cycles). Mouse multiple cDNA panels were used as templates. Whole-mouse cDNA and dH2O were used as positive and negative controls, respectively (right 2 lanes). The products were subjected to electrophoresis, Southern blotting, hybridization with radiolabeled probes, and autoradiography. D indicates day. (B) Northern blot analysis of various murine tissues. Various murine tissues were prepared from C3H mice, and total RNA (5 μg/lane) was extracted and Northern blotted. (C) Expression profiles of TfR1 and TfR2 in the liver and spleen during development. Spleens and livers were excised from day 13 and day 18 embryos, from postnatal day 1 and day 4 mice, and adult mice. (B-C) Total RNA was extracted, and expression levels of TfR1, TfR2, and GAPDH were compared by Northern analysis. (D) Relative expression levels of TfRs in the liver and spleen during development are calculated by an image analyzer, standardized by the values of GAPDH, and shown as bar graphs (■, TfR1; , TfR2). The values of postnatal day 1 for spleen and day 13 embryo for liver are adjusted to 1.0. Hiroshi Kawabata et al. Blood 2001;98:1949-1954 ©2001 by American Society of Hematology

Expression of TfR2 mRNA in MEL cells during erythrocytic differentiation and in response to iron status.(A) Expression of TfR2 mRNA during differentiation of MEL cells. Expression of TfR2 mRNA in MEL cells during erythrocytic differentiation and in response to iron status.(A) Expression of TfR2 mRNA during differentiation of MEL cells. MEL cells were cultured in the presence of 2% DMSO, 75 μM hemin, 2% DMSO + 75 μM hemin, or 2% DMSO + 10−7 M dexamethasone (Dex) for 5 days. Upper panel shows the hemoglobin concentration in the cell lysates. Hemoglobin concentration was determined by the benzidine method and shown as a percentage of total cellular lysate. Lower panel shows the results of Northern blot analysis. Approximately 20 μg total RNA was loaded in each lane. The membrane was sequentially hybridized with32P-labeled murine TfR1, TfR2 (3′-part, 1.4 kb), and murine GAPDH cDNA probes. (B) Effects of cellular iron status on expression of murine Tf receptors in MEL cells. MEL cells were cultured with various concentrations of Fe2(NO3)3 or DFO for 2 days, and Northern blot analysis was performed. The membrane was sequentially hybridized with 32P-labeled murine TfR1, TfR2, and GAPDH cDNA probes. (A-B) Relative expression levels of TfRs are calculated by an image analyzer, standardized by the values of GAPDH, and shown as bar graphs (■, TfR1; , TfR2). Hiroshi Kawabata et al. Blood 2001;98:1949-1954 ©2001 by American Society of Hematology

Reporter assay for murine TfR2 promoter Reporter assay for murine TfR2 promoter.NIH-3T3 cells were transfected by Geneporter system (Gene Therapy Systems) with either empty pGL3-basic (C) or mTfR2 promoter reporter plasmids (P) pGL-mTfR2-pro-0.3 kb, -0.4 kb, -1.0 kb, or -2.1 kb together with expr... Reporter assay for murine TfR2 promoter.NIH-3T3 cells were transfected by Geneporter system (Gene Therapy Systems) with either empty pGL3-basic (C) or mTfR2 promoter reporter plasmids (P) pGL-mTfR2-pro-0.3 kb, -0.4 kb, -1.0 kb, or -2.1 kb together with expression plasmids for GATA-1 (G), FOG-1 (F), EKLF (E), and/or C/EBP-α (A). To normalize the transfection efficiency, pCMV  ·  SPORT-β-Gal (Life Technologies) was cotransfected. Cells were harvested after 60 hours, and luciferase and β-galactosidase activities were measured with a luminometer and with the β-galactosidase Enzyme Assay System (Promega), respectively. Experiments were performed in triplicate and were repeated twice, with very similar results. Mean values of relative luciferase activities and SDs of a representative experiment are shown. Hiroshi Kawabata et al. Blood 2001;98:1949-1954 ©2001 by American Society of Hematology