FIGURE 1: Human caspase 3 compensates for ScMCA1 activity in yeast. (A) Schematic representation of yeast strain with hCasp3-GFP expression in the endogenous ScMCA1 gene locus. (B) Colorimetry assay was performed to detect Caspase 3 activity in BY4741 and hCasp3-GFP expressing strain subjected to H₂O₂ induced stress. Values are mean ± SEM of three independent experiments with p-value < 0.05. (C) Fluorescence imaging showing hCasp3-GFP expression in yeast strain subjected to 5mM hydrogen peroxide for 1.5hr and 21.5hr. (D) Spot assay was performed to assess viability of three strains BY4741, ΔScMCA1 and hCasp3-GFP subjected to heat stress (30°C & 42°C). The arrow above the panel indicates a 5-fold serial dilution of plated cells starting from left to right (1:1, 1:5, 1:25, 1:125). The colonies were counted across the three yeast strains as indicated in the graph. The data represents three independent experiments and are shown as mean ± SEM with p-value < 0.05. (E) FM4-64FX staining (red) of vacuoles was performed in BY4741, ΔScMCA1 and hCasp3-GFP yeast strain. The red fluorescence was detected using microscopy and the images were acquired using 63X objective under oil immersion. The graph represents vacuole count across three yeast strains performed in triplicates and represented as mean ± SEM with p-value < 0.05.