g Oxoplate-Oxoplate OP96F plates 
were purchased from PreSens. The Oxoplate system has been described in detail by Cook et al.. Briefly, wells containing 200 ml of air-saturated and oxygen-free water served as standards for high- and low-oxygen conditions, respectively. Oxygen-free water was prepared by the addition of sodium sulfite; the well was then overlaid with mineral oil to prevent diffusion of oxygen into the water during the course of the experiment. The remaining wells were open to the environment inside the plate reader so that atmospheric oxygen was able to diffuse into the media of the sample wells. Cells were grown in 10 cm culture dishes under normal culture conditions Laser Confocal Microscopy Unless otherwise noted, cells were maintained in a stagemounted atmospheric box at 37uC, 5% CO2, and 75% humidity during the course of the experiments. All samples were analyzed on an Olympus Fluoview FV1000 laser confocal microscope. Acetylacetonatobis iridium Mitochondria and Hypoxia Cell Line LNCaP LNr0-8 LNcyb C4-2 PC-3 MCF-7 MDAMB231 doi:10.1371/journal.pone.0088911.t001 Phenotype Well-differentiated Poorly-differentiated Moderately-differentiated Moderately-differentiated Poorly-differentiated Well-differentiated Poorly-differentiated Origin Lymph node metastasis mtDNA depleted LNCaP mtDNA replete LNr0-8 Castration resistant LNCaP from mouse bone Bone metastasis Pleural effusion Pleural effusion was utilized in all confocal experiments at a concentration of 5 mM. BTP has been described in detail by Zhang et al.. BTP is a phosphorescent compound that is phosphorescent in lowoxygen conditions and is quenched in the presence of oxygen. The extent of quenching is dependent upon intracellular oxygen concentration. Samples were incubated in the presence of BTP for 1 hour before imaging, hypoxic, or PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19640586 hyperoxic conditions as detailed for specific experiments). In all experiments, cells were plated at a cell density of 16105 cells in 3 cm dishes with inset cover slips. LNCaP and MCF-7 cells were incubated for at least for 48 hours, depending on cell adherence and morphology, before the beginning of experiments. All other cell lines were plated 24 hours before the beginning of experiments. At the start of each experiment samples were incubated with BTP plus any additional experimental conditions detailed below for one hour before viewing. Experiments that were carried out in the absence of serum or in the presence of R1881 or flutamide were incubated PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19639654 for twenty-four hours before the addition of BTP. Images from all microscopy experiments were processed using the FV10-ASW 3.1 Viewer. Cell phosphorescence for 10 cells per plate was quantified using ImageJ. Cell phosphorescence was calculated as corrected total cellular phosphorescence according to the following equation: CTCP~integrated pixel intensity{ This method takes into account the area of the cell when determining pixel intensity of a cell. Images have been falsecolored green for easier viewing. Detection of HIF-1a by Western Blotting For a positive control for the detection of HIF-1a, cells were treated with 200 mM CoCl2 for 6 hours before nuclear extract collection. For samples that were exposed to hypoxia, the cells were incubated at 0.2% oxygen for 6 hours before nuclear extract collection. Nuclear extracts were collected by first addition of 10 mM HEPES buffer plus 1 mM DTT and D-α-Tocopherol polyethylene glycol 1000 succinate site protease and phosphatase inhibitors at a ratio of 400 ml per 26106 cells. After the cells were allowe