Fig. 3. Characterization of mitochondrial quality.

A) DiOC₆ was used to visualize mitochondrial membrane potential in wild-type cells. Images were acquired using a 470 nm LED at 100% power, 50 ms exposure time and standard GFP filters for DiOC₆ and 200 ms exposure time and metal-halide lamp with standard DsRed filters for Tom70-mCherry. Left panels: DiOC₆:Tom70-mCherry ratios overlaid on bright-field images. Color scale indicates ratio values; higher numbers and warmer colors indicate higher membrane potential. Middle panels: DiOC₆ images. Right panels: Tom70-mCherry images. Cell outlines were drawn from brightfield images. Scale bar = 1 μM. B) Notched dot box plot of the average DiOC₆:Tom70-mCherry ratio in wild-type cells grown in glucose, 10 mM FCCP, and glycerol. Cells were treated with FCCP for 15 minutes prior to addition of DiOC₆ or grown in YPG overnight and allowed to recover in SC with glucose for 3 hrs. n = 50 cells for each strain. Data is representative of 3 experiments. *** = p-value < 0.001 using non-parametric Kruskal-Wallis testing with pairwise Bonferroni correction. C) Mito-roGFP was used to visualize the redox environment of mitochondria in wild-type cells. Left panels: reduced:oxidized roGFP ratios overlaid on phase images. Images were acquired using a standard GFP filter with excitation filter removed, and LED excitation at 365 nm (25% power) and 100 ms exposure for the oxidized channel and 470 nm (100% power) and 100 ms exposure for the reduced channel. Color scale indicates ratio values; higher numbers and warmer colors indicate more reducing mitochondria. Middle panels: reduced roGFP. Right panels: oxidized roGFP. Cell outlines were drawn from bright-field images. Scale bar = 1 μM. D) Notched dot box plot of the average reduced:oxidized mito-roGFP1 ratio in wild-type cells untreated and treated with 5 mM H₂O₂ and 5 mM DTT. Cells were treated with H₂O₂ or DTT for 30 min prior to imaging. n = 50 cells for each strain. Data is representative of 3 experiments. *** = p-value < 0.001 using non-parametric Kruskal-Wallis testing with pairwise Bonferroni correction. E) DHE was used to visualize mitochondrial ROS levels in wild-type cells. Left panels: DHE:Cit1-GFP ratios overlaid on bright-field images. Cells were stained with DHE at 40 μM for 30 min and imaged using a standard rhodamine filter and 100 ms exposure for DHE and a standard GFP filter and 100 ms exposure for Cit1-GFP. Color scale indicates ratio values; higher numbers and warmer colors indicate lower ROS levels. Middle panels: DHE. Right panels: Cit1-GFP. Cell outlines were drawn from bright-field images. Scale bar = 1 μM. F) Notched dot box plot of the average DHE:Cit1-GFP ratio in wild-type cells untreated and treated with 2.5 mM paraquat (PQ) and 100 μM Tempol. Cells were treated with PQ or Tempol for 30 min prior to addition of DHE. n = 50 cells for each strain. Data is representative of 2 experiments. *** = p-value < 0.001 using non-parametric Kruskal-Wallis testing with pairwise Bonferroni correction.