Polyfect reagent

Non-transferrin-bound iron (NTBI) uptake assays were performed as previously described [15 (link),25 (link)–27 (link)]. In brief, HEK293T cells were transfected with Dexras1-Myc or mutants using Polyfect reagent (Qiagen, Valencia, CA). After 48 hr, the cells were washed with phosphate-buffered saline (PBS) then resuspended into iron uptake buffer (25 mM Tris, 25 mM MES, 140 mM NaCl, 5.4 mM KCl, 5 mM glucose, 1.8 mM CaCl2 [pH 5.5]) and transferred to glass test tubes. Ascorbic acid was added to 1 mM FeSO₄ at a 44:1 ratio. ⁵⁵FeCl₃ (PerkinElmer Life Science, Waltham, MA) was added to the iron/ascorbic acid mixture, which was then added to the cells in iron uptake buffer to a final concentration of 20 μM. Cells were incubated at 37°C with shaking for 15 min. The cells were washed twice with cold PBS plus 0.5 mM EDTA and harvested. An aliquot of resuspended cells was taken for protein assay using the Bio-Rad Protein Assay Reagent; the protein concentrations of individual samples were used to quantitate ⁵⁵Fe incorporation (cpm/μg protein). Samples were normalized to control. Statistical comparisons of iron uptake were performed by student’s t-test. All NBTI uptake experiments were repeated at least three times, each sample in triplicate.

HEK293T cells were seeded in 24-well plates at a density of 5 × 104 cells/ well, and cultured at 37°C in 5% CO2 overnight. On the next day, 0.5 μg of DNA and 5 μl of PolyFect reagent (Qiagen, Valencia, CA) were suspended in serum-free, antibiotic-free DMEM to give a final volume of 30 μl, which was allowed to stand at room temperature for 10 min. Then, the mixture was introduced to the wells containing cultured cells and 375 μl of fresh medium. At 48 h post-transfection, supernatant containing the secreted antibody fragments was collected for subsequent experiments.

IGFBP-2 (1-163) (N-terminus) and IGFBP-2 (164-328) (C-terminus) were amplified using the primers and conditions listed in Supplementary Table S1 with the Herculase II polymerase (Stratagene). For the IGFBP-2 (1-328) (full length) construct, cDNA was made from RNA extracted from the human breast cancer cell line, MCF-7 (ATCC). The cDNA was amplified using primers and conditions listed in Supplementary Table S1. The insert and eukaryotic expression vector, pUMVC3 (National Gene Vector Biorepository), were cut with EcoRI and BamHI restriction enzymes and ligated using E. coli ligase (New England Biolabs). Transformation of XL1 Blue competent bacteria (Stratagene) allowed kanamycin resistant clone selection. Sequencing (www.agencourt.com) was performed on each clone and each large scale DNA prep (Qiagen) to confirm identity. All DNA plasmids were determined to express the correct sized protein in vitro by transfecting HEK-293 (ATCC) cells using Polyfect reagent (Qiagen) and Western blot probing with anti-IGFBP-2 polyclonal antibodies (Santa Cruz Biotechnology, Inc) (data not shown).