Enhanced mitochondrial superoxide production by MM cells.

<p>A) Equivalent numbers of LP9 and HM cells were deprived of serum for 72 hr, then incubated in medium with either low serum (0.25% FBS) or high serum (10% FBS) and loaded with the fluorescent reporter hydro-Cy3. After 30 min relative fluorescence was measured in triplicate; data are expressed as relative fluorescence units (RFU) +/− the standard error of the mean. B) LP9 mesothelial cells (row b) and HM MM cells (row c) were incubated with nitroblue tetrazolium (NBT) and MitoTracker Deep Red and imaged by confocal microscopy. Colocalization of the NBT and MitoTracker Deep Red signals indicated that the majority of superoxide in LP9 and HM cells is derived from mitochondria. Row a represents images of LP9 cells without staining with NBT. C) H2373 and HM MM cells and LP9 controls were loaded with MitoSOX Red for 30 min and analyzed by flow cytometry for mitochondrial superoxide production. D) The indicated cell lines were transfected with an expression vector for mitochondrial roGFP, a genetically-encoded reporter that is responsive to mitochondrial redox status. The relative redox status of the different cell lines was examined by ratiometric live cell imaging as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039404#pone.0039404-Hanson1" target="_blank">[67]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039404#pone.0039404-Lohman1" target="_blank">[68]</a> Higher 400∶495 ratios are indicative of an increased oxidation state in cellular mitochondria. E) Total RNA was prepared from LP9, HM and H2373 cells and relative levels of expression of PRX3 and TR2 mRNA in comparison to HPRT were determined by qRT-PCR. Data are plotted as fold-increase in the MM cell lines as compared to LP9. F) Cell extracts were prepared from LP9, HM and H2373 cells and assayed for total cellular thioredoxin reductase (TR) activity. Data are expressed as arbitrary units (AU) per 25 µg cell extract.