Fig. 5.
DJ-1 paralogs regulate basal redox status of the cell.(A, B) Measurements of overall cellular ROS levels. An equivalent amount of cells from WT, Δhsp31, Δhsp34, and Δ31Δ34 strains were harvested and treated with H2-DCFDA and subsequently analyzed using flow cytometry. Median fluorescence values were obtained from FACS analysis plotted and represented as a bar graph (A). Similarly, H2-DCFDA treated strains were analyzed for cellular ROS levels by microscopic analysis, left panels (DIC image), middle panels (DCFDA fluorescence), and right panels (merge) (B). (C, D) Measurement of cytosolic superoxide levels. The yeast strains (WT, Δhsp31, Δhsp34, and Δ31Δ34) were grown till mid-log phase and treated with 10 μM oxidation-sensitive dye, DHE, and subjected to flow cytometry (C) and microscopic analysis (D). Scale bar (10 μm). All the panels imaged at identical exposure conditions. (E, F) Effect of ROS scavengers on mitochondrial mass. Yeast strains were grown overnight in YPD (Δ31Δ34 grown in YPD in the presence of either GSH or NAC) media and treated with 10 μM NAO-cardiolipin stain (E), and MitoTracker Deep-Red (F) followed by flow cytometry analysis. Error bars represent the standard deviation in the median values from 3 biological replicates. Asterisks indicate the p-value, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)