(A) Agar plates showing the growth of yeast cells when Bax gene is co-expressed with 1–3 copies of eGFP tagged α-synuclein gene. Bax(LEU2)::α-syn-eGFP(TRP1) (1); Bax(LEU2)::α-syn-eGFP(TRP1,HIS3) (2); Bax(LEU2)::α-syn-eGFP(TRP1,HIS3,URA3) (3). SD: glucose-containing minimal medium; SG: galactose-containing minimal medium. Plates were incubated at 30°C for 96 h. (B) Growth of cells co-expressing Bax and 1, 2 or 3 copies of the α-synuclein-eGFP fusion protein in galactose containing liquid SG minimal medium. The growth difference between one, and two copies with their own control was significant (P<0.05), while that of three copies was not significant (P>0.05). ANOVA test also revealed that there was no significant difference between strains co-expressing 1 copy and 2 copies of α-synuclein-eGFP with Bax (P>0.05), but there was a significant difference between 1-copy and 2-copy α-synuclein-eGFP co-expressing cells on the one hand and 3-copy co-expressing cells on the other (P<0.05). (C) The percentage cell death in the three different strains, co-expressing Bax and 1–3 copies of the α-synuclein-eGFP fusion protein, and their respective controls. This was determined by staining with Phloxine B. There was a significant difference between the yeast strains and their respective controls, and between strains expressing different copy numbers (P<0.05). (D) Representative microscopic images of Phloxine B stained dead cells showing an appreciable decrease in the death of cells expressing 2 copies of the α-synuclein-eGFP fusion protein. (E) Comparison of ROS produced in the three strains that co-express Bax and 1 to 3 copies of eGFP-tagged α-synuclein along with controls. There was a significant difference among the three yeast strains with their respective controls (P<0.05), ANOVA test revealed that there was a significant difference between one and two copies (P<0.05), and two and three copies (P<0.05), but there was no significant difference between one and three copies (P>0.05). (F) Comparison of the MMP of yeast strains co-expressing Bax and 1, 2 or 3 copies of α-synuclein-eGFP along with controls. There was a significant difference between the three yeast strains with their respective controls (P<0.05). ANOVA test revealed that there was no significant difference between 1 and 2 copies (P>0.05), and 1 and 3 copies (P>0.05), but there was a significant difference between two and three copies (P<0.05). (G) Measurement of NDF in the yeast strains that co-express Bax and 1, 2 or 3 copies of α-synuclein-eGFP along with controls via the TUNEL assay. There was a significant difference among the yeast strains both with their respective controls and between the copy numbers (P<0.05). (H) Shows a representative microscopic view of the TUNEL assay results of which were presented, in a bar-chart format. (I) Western blot of cell lysates co-expressing Bax and 1, 2 or 3 copies of the α-synuclein-eGFP fusion protein. 1-copy α-syn-eGFP (1); 2 copies α-syn-eGFP (2); 3-copies α-syn-eGFP (3), 1-copy control (4); 2-copies control (5); and 3-copies control (6). A total of 10 µg of total cellular protein was loaded in each lane. The HA-antibody was used to probe for expression of α-synuclein-eGFP, the c-myc antibody for Bax, and the β-actin antibody for β-actin, which was used as a loading control. (J) Monitoring of eGFP expression within cells, the strain expressing two copies did not show traces of aggregation.