Skip to main content
. 2015 Sep 4;290(43):25876–25890. doi: 10.1074/jbc.M115.678508

FIGURE 6.

FIGURE 6.

Analysis of mitochondrial iron, ROS levels and mitochondrial function in COXPD19 related ISD11R68A mutant. A and B, representation of iron levels in yeast mitochondria expressing WT Isd11 or WT ISD11 or ISD11R68A, measured by iron-specific colorimetric assay (A) and AAS (B). C and D, mitochondrial superoxide levels in WT and ISD11R68A yeast strains were estimated using MitoSOX Red dye by flow cytometry and represented in the overlaying histograms (C) and mean fluorescence intensity obtained by flow cytometry was quantitated (D). WT Isd11 cells treated with 1 mm rotenone was utilized as positive control. E, yeast strains were treated with MitoSOX Red dye (red fluorescence) and subjected to microscopic analysis using DeltaVision Fluorescence Microscope (×100 objective). Left column, bright field; middle column, MitoSOX red fluorescence; and right column, merged. F, overall cellular peroxide levels were measured by staining using H2DCFDA dye (green fluorescence), followed by fluorescence microscopy analysis using Delta Vision Fluorescence Microscope (×100 objective). Left, bright field; middle, H2DCFDA fluorescence, and right columns, merged. G and H, mitochondrial mass in yeast strains expressing WT Isd11 or WT ISD11 or ISD11R68A was assessed using NAO dye. The fluorescence was measured through flow cytometry and represented as histogram (G) or mean fluorescence intensity (MFI) obtained in flow cytomety was quantitated and represented as bar diagram (H). I, JC-1 staining was used for the estimation of mitochondrial membrane potential in yeasts strains. An emission scan from the 500 to 620 nm wavelength was performed in JC-1-stained yeast cells and the multimer (590 nm) to monomer (530 nm) ratio was quantified and represented. Mitochondria treated with valinomycin were used as a positive control. Data represented as mean ± S.E. The values were obtained from three independent experiments (n = 3). p value of <0.05 was defined as significant, and asterisks are used to denote significance, where: *, p < 0.05; **, p < 0.01; ***, p < 0.001. Scale bar, 10 μm. A.U., arbitrary unit.