Gel filtration and anion exchange were utilized to remove trace contamination

tiated the increase in NDRG1 by iron depletion, while anti-sense ablation of eIF3a diminished this 22223206 response. These results show that eIF3a plays a role in NDRG1 expression. Hence, despite decreased eIF3a expression after iron depletion, this protein potentiates iron-regulated NDRG1 expression. In addition, ablation of eIF3a decreased proliferation, but increased cell motility and invasion, while its over-expression led to the opposite response. As these functional alterations correlated with changes in expression of NDRG1 and the eIF3a repressed target, p27kip1, these alterations may explain how eIF3a mediates its effects on metastasis and proliferation, respectively. Materials and Methods Reagents The chelator, 311, was synthesized and characterized as reported, while DFO was from Novartis. Ferric ammonium citrate, tunicamycin, hygromycin B from Streptomyces hygroscopicus, tetracycline hydrochloride and 3–2,5-diphenyl-2H-tetrazolium bromide were from Sigma-Aldrich. Cell Culture MCF7 breast cancer cells were from the American Type Culture Collection and cultured, as described. MCF7 TETOffH cells were obtained from Clontech. Murine embryonic fibroblasts from wild-type and homozygous HIF-1a knockout mice were obtained from Dr. R. Johnson . 9726632 Cells were cultured using standard conditions at 37uC in the presence of medium containing 10% fetal calf serum. Unless otherwise specified, cells were incubated under standard conditions in an atmosphere of 95% air/5% CO2. In several studies, cells were incubated with the well characterized iron chelators, DFO or 311, for 24 h/37uC. These ligands and experimental conditions have been clearly demonstrated to induce cellular iron depletion in these and other cell-types. The higher concentration of DFO, relative to 311, was implemented due to its limited ability to permeate cells. The iron-chelator, 311, was utilized at a lower concentration since this ligand shows far greater membrane permeability than DFO and demonstrates pronounced iron chelation efficacy. In some experiments, cells were incubated under hypoxic conditions in an atmosphere of 1% O2, 94% N2 and 5% CO2. eIF3a TET-Regulated Cells The pCbA-eIF3a and MedChemExpress INK-128 pCMV-eIF3a-AS plasmids were a kind gift from Prof. Jian-Ting Zhang. Transfections using these plasmids were carried out with MCF7 TET-OffH cells according to the manufacturer’s instructions. Briefly, eIF3a and eIF3a-AS fragments were subcloned into a pTRE2hyg/pur plasmid and transfected in TET-Off-MCF7 cells. Cells were then treated with hygromycin for 24 weeks in order to select those transfected with the pTRE plasmids to generate a stable transfection. Clones of hygromycin-resistant cells were obtained by limiting dilution and then tested with RT-PCR and westerns to assess expression of eIF3a mRNA and protein and examine the response to TET treatment. Immunofluorescence Immunofluorescence was performed according to standard procedures. Briefly, cells seeded on cover slips were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 for 5 min at room temperature. The cells were then incubated overnight at 4uC with the following primary antibodies: rabbit polyclonal anti-human eIF3a ”, Santa Cruz Biotechnology, Cat. # sc-30149, 1:150 1:250), rabbit polyclonal anti-human NDRG1 and mouse monoclonal antihuman eIF2a. This procedure was followed by an incubation with one or eIF3a Regulates NDRG1 during Iron Depletion both of the following secondary antibodies for 1 h at r