tatistical significance: p,0.05, p,0.01, p,0.001 as compared to the same uncomplexed chelator. The cells were cultured in 75 cm2 tissue culture flasks at 37uC in a humidified atmosphere containing 5% CO2. Sub-confluent cells were sub-cultured every 34 days. For proliferation experiments, the cells were seeded in 96-well plates at a density of 5,000 cells/well 24 h prior to the addition of each agent or their combinations. For flow cytometric analyses, the MCF-7 cells were seeded in 60-mm Petri dishes at a density of 210,000 cells/ well. For microscopic assessments, the MCF-7 cells were seeded in 12-well plates at a density of 35,000 cells/well. tration was 0.1%. At this concentration, DMSO had no effect on cellular proliferation. To assess the metal complexes, metal salts or Cu sulfate ) were added to cell culture medium before the addition of the chelator in either a 2:1 or 1:1 chelator-metal ratio, i.e., at a metal-binding equivalent ratio of 1. Cellular viability was determined using the neutral red uptake assay, which is based on the ability of viable cells to incorporate NR into lysosomes. The optical density of soluble NR was measured at l = 540 nm using a Tecan Infinite 200 M plate reader. Manual cell counts demonstrated that absorption was directly correlated to cell number. The proliferation of cells in the experimental groups was expressed as a percentage of the untreated control. Combination studies were designed according to the method described by Chou and Talalay. Briefly, the concentration of chelator or anti-neoplastic agent that induced a 50% decrease in proliferation was determined for each agent alone. Then, both the individual agent and the combinations were tested at concentrations corresponding to fractions and multiples of their IC50 values.All of the studied compounds and their combinations were MedChemExpress GSK1278863 incubated with MCF-7, T47D, or MDA-MB-231 cells for 72 h at 37uC at a concentration corresponding to their IC50 value. The combination index values were calculated using the Chou and Talalay method from n$4 experiments using CalcuSyn 2.0 software; CI,1, <1, or.1 indicate synergism, an additive effect, or antagonism, respectively. Statistical significance: p,0.05, p, 0.01, p,0.001 as compared to the same combination with uncomplexed parent chelator. doi:10.1371/journal.pone.0088754.t005 cytometer. A total of 10,000 events were collected per analysis. 6. Cell Cycle Analysis Following a 72 h/37uC incubation with each agent or the combinations, the MCF-7 cells were harvested by trypsinization, centrifuged at 300 x g, washed in PBS supplemented with 5% FBS, and suspended in a small amount of PBS+FBS. Icecold 70% ethanol was then added in a drop-wise fashion and the cells were fixed for 3 h/220uC. After fixation, the ethanol was removed by centrifugation and the cells were washed in PBS+FBS and resuspended PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19644140 in sodium citrate in PBS+FBS. The cells were then incubated with 200 mg/mL of RNAse A and 30 mg/mL of propidium iodide for 20 min/37uC. The cell cycle distributions were measured using an Accuri C6 flow grated on the bottom of the tissue culture plates provided by the manufacturer. The cell adhesion rate, which is expressed as the “cell index”and parallels the viability or proliferation of the cells, is monitored throughout the entire incubation period. This real-time analysis was performed for selected combinations of NHAPI 
with TMX. 4. Fluorescence Microscopy Assessments Changes in cellular morphology and the mitochondrial
