Figure 4.

PaCYPD mediates autophagy and participates in longevity-assurance under ROS stress. (A) Monitoring autophagy by western blot analysis using the cytosolic protein PaSOD1::GFP. 6 d old wild-type (WT) and ΔPaCypD strains expressing PaSod1::Gfp were cultured for 2 d in CM medium with or without 60 μM paraquat (PQ). “Free GFP” (indicated by arrow) and PaCYPD was detected by immunoblotting with anti-GFP and anti-PaCYPD in 100 μg total protein extracts. The positions of molecular mass markers are indicated on the right. (B) The GFP protein levels of three different isolates of each strain were normalized to the Coomassie-stained gels (loading control) and the protein amount present in the 6 d old wild type was set to one. Data represent average ± SEM (2-tailed student's t-test), not significant (n.s.), ^*P < 0.05, ^***P < 0.001. (C) Lifespan and (D) mean lifespan of at least 14 different isolates of the wild-type strain and at least 21 different isolates of the ΔPaCypD strains on M2 medium in the presence of 0, 20, 80, or 150 μM paraquat (PQ). Data represent average ± SEM (2-tailed Wilcoxon rank-sum test) Not significant (n.s.), ^**P < 0.01. At note: Survival curves of wild type and ΔPaCypD in presence of 150 μM PQ are largely overlapping and therefore difficult to distinguish.