Figure 1

14-3-3β protects against various triggers of cell death in yeast. (a) Freshly saturated cultures of yeast cells harboring control empty plasmid (Vector), as well as plasmids expressing the Bax suppressors dUTPase and 14-3-3β/α (14-3-3) were serially diluted and aliquots were spotted onto YNB galactose-containing nutrient agar with no additions (Control) or with 1.5 mM cadmium sulfate (+Cadmium). The plates were incubated at 30°C for 3 days. (b) Freshly saturated cultures of yeast cells harboring control empty plasmid (Vector) and the plasmid expressing and 14-3-3β/α (14-3-3) were diluted and grown in galactose nutrient media for 4 h. Cells were then grown for 6 h without (black bars) or with 1.5 mM cadmium sulfate (gray bars). Viability was determined by microscopic examination of cells stained with the vital dye trypan blue. The data are shown as the percentage (%) of cells that does not stain with trypan blue and are thus viable (trypan blue negative). The data represent the mean of four independent experiments (±S.E.M.). *, indicates a significant difference between the viabilities of cadmium-treated Vector cells versus cadmium-treated 14-3-3-expressing cells (Student’s t-test P<0.001). (c–e) Human 14-3-3β/α prevents cycloheximide-mediated apoptosis. Freshly saturated cultures of yeast cells with empty plasmid (Vector) and the 14-3-3β/α-expressing plasmid (14-3-3) were diluted and grown in galactose nutrient media for 4 h prior to the addition of cycloheximide. (c) Viability of the cells was determined after a 24 h treatment with the indicated concentrations of cycloheximide using the clonogenicity assay. The data represent the mean of four independent experiments (±S.E.M.). * and ** indicates significant difference between the viabilities of control vector cells versus 14-3-3-expressing cells (*P<0.05 and **P<0.01). (d) Fluorescence was monitored in DHE-treated cells using a fluorescent cell sorter. The results are reported as the mean (±S.D.) of two experiments that were performed in triplicate. (e) Cells were simultaneously challenged with fluorescently labeled annexin V and propidium iodide, and the proportion of cells labeled with either or with both were determined by cell sorter as described.²⁷ The data represent the mean of four independent experiments (±S.E.M.). * and ** indicates significant differences between the control vector cells versus 14-3-3-expressing cells (*P<0.05 and **P<0.01). (f) Rapamycin-mediated cell death is inhibited by human 14-3-3β/α. Freshly saturated cultures of yeast cells with empty plasmid (circles) and the 14-3-3β/α-expressing plasmid (squares) were diluted and grown in galactose nutrient media for 4 h. Rapamycin was added (500 nM) and viability was monitored daily using the vital dye trypan blue over an 8-day period. The data are shown as the percentage (%) of cells that does not stain with trypan blue (trypan blue negative). The results are reported as the mean (±S.D.) of two experiments that were performed in triplicate