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. 2018 Jul 15;29(14):1718–1731. doi: 10.1091/mbc.E18-01-0015

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FIGURE 7: — Suppressor mutations relieve osmotic swelling and cell lysis of fab1∆ yeast. (A) Cell growth assay of the indicated yeast strains grown on rich agar medium (YPD) in the absence or presence of 1 M sorbitol at 26°C or 37°C for 3 d. (B) Quantitation of cell volume measurements of the indicated strains. Each dot represents a single cell, with the horizontal line representing the mean of all measurements (n > 100 per strain). Unpaired t tests with Welch’s correction were used to determine statistical significance; ***, P < 0.001. Unless otherwise indicated, all other mutant strains were not statistically different compared with the wild-type control. (C) Cartoon depiction of osmotic lysis assay. The total (T), pellet (P), and supernatant (S) were analyzed by Western blot, probing for the cytosolic protein Pgk1. Appearance of Pgk1 in the supernatant indicates cell lysis during spheroplast conversion. (D) Osmotic lysis assay as described in C of wild-type, fab1∆, or ppz1∆ppz2∆ yeast in the presence of 1 or 2 M sorbitol in the buffer. The inability to block the cell lysis of ppz1∆ppz2∆ cells also confirms that increasing osmotic support does not inhibit the conversion of cells into spheroplasts. Shown are representative blots of three independent experiments. (E) Quantitation of Pgk1 release of the experiment performed in D. Pgk1 release is determined as a percentage of Pgk1 detected in the supernatant compared with the total Pgk1 detected in the supernatant and pellet. Bars represent the mean and errors bars are the SD of three independent experiments. Unpaired t tests with Welch’s correction were used to determine statistical significance; n.s., not significant; *, P < 0.05. (F) Osmotic lysis assay as described in C and D of the indicated yeast strains. Shown are representative blots of two independent experiments.