Figure 1.
Abrogation of respiration suppresses apoptosis and ROS production in a maximal proliferating solid cell population.
(A) Isolated colonies were grown from single cells, manipulated onto agar plates. Clonogenic assay of cells removed from colonies of the wild-type strain, rho0 (no mitochondrial DNA) and two single gene-deletion strains (mgm1 and oxa1), grown on SCGlu. After 3 days 500 cells of whole colonies, after 5 days 500 cells of the central colony-region were analysed (mean±SEM, n = 2). Statistical significance of p<0.006 compares colony forming units (cfu) of mutant strains to wild-type strain at respective time-points. (B) Cells from the whole colony (3 days) as well as from the central region (5 days) were stained for reactive oxygen species (ROS) with dihydroethidium and analysed by FACSAria flow cytometry (mean±SEM, n = 2; **p<0.01, ***p<0.001 compared to deletion strains at respective time-points). (C) Approximately 50 (2 days) and 10 (3 days) colonies grown on SCGlu plates were washed off and clonogenic assays were performed with the collected cells (mean±SEM, n = 5; **p<0.01, ***p<0.001). (D) Approximately 50 colonies grown on SCGlu plates for 2 days were stained for phosphatidylserine exposition (AnnV) and DNA breakage (TUNEL) and analyzed by FACSAria flow cytometry (mean±SEM, n = 3; **p<0.01, ***p<0.001). (E) Fluorescence microscopy from central and outer region of 5 days old wild-type colony cells, harbouring plasmid dsRED-MLS.
Figure 2.
An oxa1 deletion does not influence the strains growth rate.
(A) Growth curves of an oxa1 deletion strain and the corresponding wild-type strain. Experiment was performed in triplicate. The y-axis is scaled logarithmically. (B) Size of isolated colonies of the indicated strains grown for 3 and 5 days on SCGlu plates, respectively. Diameters were evaluated by processing the photos with Metamorph Imaging (mean±SEM, n = 2).
Figure 3.
Forced enhancement of respiration triggers cell death, suppresses growth, and elevates ROS production.
(A) Clonogenic assay of cultures pre-grown in liquid SCGlu. 500 cells were plated on SCGlu, SCGal, and SCGly agar plates, respectively (mean±SEM, n = 3; ***p<0.001). (B) Clonogenic assay of cultures pre-grown in liquid SCGly. 500 cells were plated on SCGly and SCGlu media plates, respectively (mean±SEM, n = 2; **p<0.01). (C) Cells from the whole colony (3 days) as well as from the central region (5 days) were stained for reactive oxygen species (ROS) with dihydroethidium (DHE). Values of relative fluorescent units (RFU) were normalized to wild-type strain values (mean±SEM, n = 2; **p<0.01, ***p<0.001). (D) Size of isolated wild-type colonies grown on SCGlu and SCGal media plates were monitored by taking photos at indicated time-points. Diameters were evaluated by processing the photos with Metamorph Imaging (white bar represents 1 cm) (mean±SEM, n = 3; *p<0.05, **p<0.01).
Figure 4.
Administration of GSH diminishes ROS levels and enhances survival within wild-type colonies, as well as during seeding on highly respiratory media.
(A) Approximately 100 colonies were grown for 36 hours on SCGlu plates with or without 5 mM GSH, washed off and clonogenic assays were performed with the collected cells (mean±SEM, n = 3; **p<0.01). Additionally equal amounts of cells were stained for reactive oxygen species (ROS) with dihydroethidium (DHE) and quantified using a fluorescence microplate reader (Genios-Pro). Values of relative fluorescent units (RFU) are displayed (mean±SEM, n = 3; **p<0.01) (B). (C) Clonogenic assay of cultures pre-grown in liquid SCGlu. 500 cells were plated on SCGlu, SCGal, or SCGly agar plates, respectively, with or without 1 mM GSH (mean±SEM, n = 2; ***p<0.001).