Figure 2. PKA-dependent mitochondrial ROS production following Ypk1 inhibition.

(A) Ypk1-WT (PLY1083), Ypk1-AS (PLY1098), Ypk1-AS rho⁰ (PLY1528), Ypk1-AS tpk3Δ (PLY1529), and Ypk1-AS tpk3Δ rho⁰ (PLY1530) were grown in SCD-Ura media, then treated for 1 hr with 0.5 μM 2,3-DMB-PPI or with 30 μM myxothiazol (myxo) for 70 min (including a 10 min pre-treatment) where noted. ROS was determined and quantified as in Figure 1A. (B) Ypk1-WT, Ypk1-AS, and Ypk1-AS tpk3Δ were grown in SCD-Ura media, then treated for 1 hr with 0.5 μM 2,3-DMB-PPI or with 50 μM CCCP. All strains were incubated with 20 μM 3,3′-dihexyloxacarbocyanine iodine (DiOC₆) for the last 5 min prior to imaging by fluorescence microscopy. Quantification represents pixel intensities from several mitochondrial tubules from at least 5 different cells. (C) Ypk1-WT, Ypk1-AS, and Ypk1-AS tpk3Δ cells transformed with pPL585 (Cki1) were grown in SCD-Ura/-Leu media, then treated for 1 hr with 0.5 μM 2,3-DMB-PPI or 30 min with 200 nM rapamycin (Rap). Protein extracts were prepared and resolved by SDS/PAGE and immunoblotted with α-Myc antibody. Quantification describes the ratio of Cki-P/Cki1 relative to Ypk1- WT for three separate experiments. (D) PDE2 transcript levels were compared between Ypk1-AS and Ypk1-WT that had been treated with 0.5 μM 2,3-DMB-PPI for the indicated times (data from (Niles et al., 2012)). (E) Ypk1-WT, Ypk1-AS, and Ypk1-AS and Ypk1-AS tpk3Δ cells both transformed with pPL582 (PDE2) were grown in SCD-Ura/-Leu media, then treated for 1 hr with 0.5 μM 2,3-DMB-PPI. ROS was determined and quantified as in Figure 1A.