(A) Immunoblot analysis showing 24p3 in the CM of FL5.12 cells expressing either vector or Ec-24p3 in the presence or absence of ponasterone A.
(B) Iron binding assay. 24p3 was immunoprecipitated from the CM of cells described in (A) and analyzed for the presence of ⁵⁵Fe. CPM, counts per minute. Error bars indicate SD.
(C) (Top) GC-MS analysis on the TMS-derivatized, small molecule flow-through fraction eluted from immunopurified 24p3. (Bottom) Matching spectrum from the NIST database.
(D) GC-MS analysis on the eluted sample from immunopurified 24p3 and a set of DHBA standards.
(E) Chemical structures of 2,3-DHBA and 2,5-DHBA.
(F) Tryptophan fluorescence quenching assay. 2,3-DHBA (left) or 2,5-DHBA (right) was added to apo-24p3 at increasing concentrations, and tryptophan fluorescence was monitored. Error bars indicate SD.
(G) Iron binding assay. Purified apo-24p3 was incubated with ⁵⁵FeCl₃ and increasing concentrations of either 2,3-DHBA, 2,5-DHBA, iron-free enterobactin or desferrichrome, and radioactivity was measured in the 24p3 immunoprecipitate. Error bars indicate SD. See also Figure S1.

(A) Multiple sequence alignment of EntA/BDH2 homologues. Identical residues between all five proteins are indicated in yellow; conserved residues in green, and semi-conserved residues in blue.
(B) RT-PCR (left) and immunoblot (right) analysis monitoring murine Bdh2 expression in FL5.12 cells treated with one of two unrelated shRNAs targeting Bdh2 or a control non-silencing (NS), enhanced green fluorescent protein (EGFP) or luciferase (Luc) shRNA.
(C) GC-MS analysis of the eluted 24p3 sample prepared from parental FL5.12 cells or FL5.12 cells expressing a Bdh2 shRNA.
(D) Wild-type and mutant bacterial EntA and mouse BDH2 proteins were analyzed for their ability to convert 2,3-diDHBA to 2,3-DHBA (left) or NAD to NADH (right). Error bars indicate SD. Mutant EntA proteins were expressed at comparable levels to the wild-type proteins (Figure S2C). See also Figure S2.

(A) Iron binding assay. (Left) FL5.12 cells were treated in the presence or absence of IL-3, and with a Bdh2 or control shRNA, and the iron-binding ability of 24p3 in the CM was monitored as described in Figure 1B. Inset, immunoblot analysis monitoring 24p3 levels in the CM and tubulin levels in the whole cell extract (WCE). (Right) FL5.12/Ec-24p3 cells were treated in the presence or absence of ponasterone A. Error bars indicate SD.
(B) Iron exocytosis assay. ⁵⁵FeCl₃-loaded FL5.12 cells were either untreated (control) or treated with apo-24p3 or DFO, and the presence of ⁵⁵Fe in the medium was quantified.
(C) Apoptosis assay. FL5.12 cells cultured in the presence or absence of IL-3, with or without 2,5-DHBA, and expressing a NS or Bdh2 shRNA were analyzed for apoptosis by annexin V-FITC staining.

(A–E) FL5.12 cells expressing a Bdh2 or control shRNA were analyzed for total cytoplasmic iron levels by colorimetric analysis (A), for free cytoplasmic iron concentration by fluorescence calcein assay (B), for ferritin-L (C) and Tfr1 and IRP2 (D) levels by immunoblot analysis, and for cytosolic aconitase activity (E). Error bars indicate SD. See also Figures S3A–D and S4.

(A) FL5.12 cells expressing a NS or Bdh2 shRNA and treated in the presence or absence of hydrogen peroxide (H₂O₂) were analyzed for ROS by monitoring CDCF-DA fluorescence using flow cytometry. Error bars indicate SD.
(B) FL5.12 cells expressing a control or Bdh2 shRNA were analyzed for ROS by monitoring CDCF-DA fluorescence using fluorometry. Error bars indicate SD.
(C) FL5.12 cells expressing a control or Bdh2 shRNA were treated in the presence or absence of H₂O₂ and apoptosis was measured by annexin V-FITC staining. Error bars indicate SD. See also Figure S3E.

(A–F) FL5.12 cells expressing a Bdh2 or control shRNA were analyzed for mitochondrial iron levels by colorimetric analysis (A), for mitochondrial iron uptake (B), for RPA (C) and RPAC (D) fluorescence by fluorometry, for DHR 123 oxidation in the presence or absence of H₂O₂ by flow cytometry (E), and for activities of mitochondrial aconitase, succinate dehydrogenase and citrate synthase (F).
(G) In vitro mitochondrial iron import. (Left) Proteolytically treated cytosolic extracts from control or Bdh2 shRNA-treated FL5.12 cells were added to purified mitochondria, and iron import was determined by monitoring mitochondrial incorporation of ⁵⁵FeCl₃. (Right) Purified mitochondria were treated with 2,3-DHBA, 2,5-DHBA, enterobactin or DFO, and iron import was monitored. See also Figures S3F-J and S5.

(A) MEL cells expressing a NS or Bdh2 shRNA were differentiated. Following labeling with ⁵⁵Fe, cells were lysed and heme was extracted and analyzed for ⁵⁵Fe incorporation. Error bars indicate SD.
(B) MEL cells were differentiated as described in (A) and following cell lysis, hemoglobin levels were determined by benzidine reaction. Hemoglobin content was expressed relative to that in untreated MEL cells cultured in the absence of DMSO. Error bars indicate SD.
(C) MEL cells were differentiated as described in (A) and α- and β-globin mRNA levels monitored by RT-PCR. The levels of Bdh2 and Gapdh were monitored as controls.
(D) RT-PCR analysis. The bdh2 MO was injected into one-cell stage embryos and 24 hr later, RNA was extracted and analyzed by RT-PCR. Treatment with the bdh2 MO resulted in retention of the entire intron (upper band) as well as activation of a cryptic splice site that resulted in partial intron retention (lower band).
(E) Phenotypic analysis of control MO- and bdh2 MO-injected embryos at 72 hpf. Bdh2-depleted embryos lack hemoglobinized erythrocytes, as evidenced by the absence of red color (arrow).
(F) Whole-mount o-dianisidine staining of control MO and bdh2 MO-injected embryos at 72 hpf. Shown are lateral (top) and ventral (bottom) views of the anterior region of embryos. Control MO-injected embryos show positive o-dianisidine staining at the duct of Cuvier (DC) and the heart (H), which is markedly reduced in bdh2 MO-injected embryos. (Right) Quantification of the percent embryos with normal o-dianisidine staining.
(G) Blood cells were isolated from control or bdh2 MO-injected embryos and analyzed for hemoglobinization by o-dianisidine staining.
(H) Whole-mount o-dianisidine staining of embryos (72 hpf) injected with a control MO or bdh2 MO, or bdh2 MO-treated embryos co-injected with a bdh2 mRNA.
(I) RT-PCR (left) and in situ hybridization analysis (right) for hbae1 and hbae3 expression in control and bdh2 MO-injected embryos. See also Figures S4 and S6.