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. 2020 Oct 19;9:e58155. doi: 10.7554/eLife.58155

Figure 1. Genetic evidence of a role for Ser57 phosphorylation in the cellular response to stress.

Ubiquitin variants were shuffled into the SUB280 yeast background (as described in the Materials and Methods) to generate strains that express exclusively wildtype, S57A, or S57D ubiquitin. (A) Yeast strains expressing the indicated ubiquitin variants (wildtype, S57A or S57D) were plated in serial dilutions onto the indicated synthetic dextrose medium (SDM) agar plates. (B) Serial dilutions of yeast cells were plated onto YPD agar plates and incubated at 26°C, 37°C, or 39°C for 18 hr and shifted back to 26°C for recovery. (C) Fluorescence microscopy and (D) quantification of cells stained with propidium iodide (PI) after incubation at 65°C for 5 min. Dead cells are stained with PI. Results are means (n = 3) of % PI-positive cells ± SD (error bars). Double asterisk (**) indicates p value < 0.005 using a Student’s t-test. (E) With a starting OD600 of 0.1, cells were grown in liquid culture (-URA synthetic medium) at 39°C and OD600 was measured at different time points. Data was collected from four biological replicate experiments (n=4) and the mean OD600 ± SD (error bars) was calculated for each time point. Double asterisk (**) indicates p value < 0.005 using a Student’s t-test. For all statistical analysis associated with Figure 1, p values are reported in the Figure 1—source data 1.

Figure 1—source data 1. Quantification and statistical analysis for viability stain measurements (Figure 1D) and cell growth measurements (Figure 1E and Figure 1—figure supplement 1).

Figure 1.

Figure 1—figure supplement 1. Yeast cells expressing S57A ubiquitin are sensitive to thermal stress.

Figure 1—figure supplement 1.

Cells in liquid culture (-URA synthetic medium) were grown at 38°C with a starting OD600 of 0.1 and growth was measured after 24 hr of incubation. Values are means of OD600 from three replicates (n = 3) ± SD (error bars). Double asterisk (**) indicates p value < 0.005 using a Student’s t-test. For all statistical analysis associated with this figure supplement, p values are reported in the Figure 1—source data 1.