Fig. 4.

Mitophagy-dependent spermidine production promotes cytoprotective NO during heat stress. (A) Relative ROS in wild-type (WT), atg32Δ, and WT plus H₂O₂ yeast grown at 30°C and 37°C in YPGE medium as determined by ROS-Glo luminescence assay (Promega). P-values determined using a unpaired two-tailed Student's t-test (n=4) (B,C) Fluorescence microscopy images (B) and quantification (C) of relative NO levels in yeast grown in SGE medium and subjected to 2.5 h of heat stress (37°C) with or without spermidine (0.5 mM) in the presence of DAF-FM-DA. (C) P-values determined using a unpaired two-tailed t-test. Violin plot representing all measurements from three bioreplicates. n=200 cells/condition/experiment (n=600 cells/condition). Data are mean±s.e.m. (D) Percentage of cell death in cells grown in YPGE medium with or without 1 mM NO-scavenger CPTIO and/or 0.5 mM spermidine. Cell death measured using Sytox Blue and fluorescence microscopy. ∼200 cells per condition (n=3). P-values determined using a unpaired two-tailed Student's t-test. (E) Schematic outlining the process by which mitophagy facilitates heat stress tolerance by promoting the biosynthesis of SAM and spermidine necessary for cytoprotective NO production during heat stress. Scale bar: 10 µm. A.U., arbitrary units; N.S., not significant. The center line denotes the median value (50th percentile), while the box contains the 25th to 75th percentiles of the dataset. The black whiskers with a dot at the end of a line mark the 5th and 95th percentiles, and values beyond these upper and lower bounds are considered outliers, marked with black dots.