Fig 1: Growth of yeast strains containing two and three copies of the human a-syn gene, and analysis of cell death, MMP loss, ROS activity, and nuclear DNA fragmentation. (A–F) Growth of yeast cells harboring one copy, two copies, and three copies of the human a-syn gene on minimal medium solid agar plates that contained either glucose (SD) or galactose (SG). Panels (A–F), upper half (4 clones): strains containing one copy of the a-syn gene or two and three copies of the a-syn gene. Panels (A–F), lower half (4 clones): strains containing an empty plasmid or empty plasmids, without any a-syn gene, under the control of the GAL1 promoter. The solid agar plates were incubated at 30 °C for 96 h. (G) Western blot analyses of cells expressing one to three copies of HA-tagged a-syn protein. On lanes 1, 2, and 3 were loaded total protein (10 µg) obtained after lysis of cells that express one copy, two copies, and three copies of a-syn. The upper panel was probed with an antibody that recognizes the HA epitope (Proteintech, 51064-2-AP) and the lower panel with a ß-actin antibody (Proteintech, 60008-1-Ig); levels of ß-actin were used as a loading control, ß-actin being a housekeeping gene. (H) Growth of yeast strains that contain one copy, two copies, and three copies of the a-syn gene in liquid minimal medium yeast cell culture that contained galactose as a carbon source. OD600 represents the optical density of cells at 600 nm. The OD600 values at each time point represent the average of three independent experiments. (I) Analysis of cell death—percentage of yeast cells undergoing death after culturing strains expressing one copy, two copies, and three copies of a-syn for 20 h in galactose. Cells were stained with Phloxine B for detection of dead cells.33 The data represent mean ± SD of five independent fields of observation. Around 200 cells were counted in each field; (J) comparing the MMP of cells expressing one copy, two copies, and three copies of a-syn with cells containing empty plasmids as controls. Cells were stained with the JC-10 dye to detect MMP loss.34 The data represent mean ± SD of three independent experiments. (K) Total ROS activity of yeast strains containing one copy, two copies, and three copies of a-syn compared with strains than contain empty plasmids. Cells were stained with dihydroethdium to detect ROS.35b The data represent mean ± SD of three independent experiments. (L) Comparative levels of nuclear DNA fragmentation in yeast strains which express one copy, two copies, and three copies of a-syn, using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, compared with strains that contain empty plasmids.35b The data represent mean ± SD of three independent experiments. For all four sets of analyses (I–L), cells were cultured for 20 h in galactose, for induction of a-syn protein expression, before treatment of cells, as required by the assay [statistical analysis was performed in (I–L) for comparison between the 1C, 2C, and 3C a-syn with the respective control group, ***p < 0.01].