Drug screening to isolate pharmacological suppressors of lethality of an msp1^P300S strain grown at restrictive temperature in a galactose-based medium. (A) Experimental strategy: a yeast strain expressing a thermosensitive form of the Msp1p protein (msp1^P300S) was grown at the permissive temperature (25°C) and then spread on agar-based solid medium containing galactose. Then, filters were deposited onto the agar surface and individually loaded with single pharmacological compounds from repurposed drug libraries (3 µl at 10 mM or DMSO as a control onto the top left filter) and Petri plates were then incubated at the restrictive temperature (37°C) for 5-7 days and photographed. (B) The presence of a white halo around the filter indicates yeast growth. The presence of a dark halo indicates the absence of growth and therefore toxicity of the compound at high concentration (close to the filter). The names of the compounds and their chemical structures are indicated. The indicated quantities of drugs were added onto the filters. (C) Drops containing 800 cells expressing WT (msp1^WT) or thermosensitive (msp1^P300S) Msp1p protein were deposited on agar-based solid medium containing either galactose (gal) without (-) or with 6 μM vanoxerine, 30 μM hexestrol, 15 μM clomifene, 1 μM ketoconazole or 9 μM terconazole, or dextrose (dex) without (-) or with 1 μM vanoxerine, 10 μM hexestrol, 15 μM clomifene, 1 μM ketoconazole or 1 μM terconazole, as indicated. The plates were then incubated at 37°C for 3 days and photographed. C is representative of three independent experiments.

Effects of hexestrol and clomifene on mitochondrial morphology and maintenance of mtDNA. (A,B) Yeasts expressing the mitochondrial protein Arg11p fused to the fluorescent mCherry protein (Arg11p-mCherry), together with either WT (strain msp1^WT) or mutated (strain msp1^P300S) Msp1p protein, were cultured at 37°C for 18 h on dextrose liquid medium, without (-) or with 15 μM hexestrol (Hex) or 4 μM clomifene (Clo) as indicated, and then fixed and labeled with DAPI before being observed with a fluorescence microscope. Scale bar: 5 μm. High magnifications are shown in insets (×1.7). A and B are representative of five experiments. (C) The number of mitochondrial nucleoids was counted in the msp1^WT and msp1^P300S strains cultured at 37°C in dextrose liquid medium for 18 h without (-) or with 15 μM hexestrol (Hex) or 4 μM clomifene (Clo), as indicated. Data represent the mean±s.d. of two independent experiments with 50 cells per condition. They were statistically analyzed using Kruskal–Wallis Dunn's multiple comparison tests to compare, for each strain, the values obtained with drugs (Hex, Clo) with that obtained for the control (-) (****P<0.0001). (D) qPCR was performed on DNA extracted from the msp1^WT and msp1^P300S strains cultured at 37°C on dextrose liquid medium for 18 h without (-) or with 15 μM hexestrol (Hex) or 4 μM clomifene (Clo), as indicated. The results of three independent experiments performed in triplicate are expressed as a percentage relative to those obtained for the msp1^WT strain cultured at 37°C (Student's t-tests; **P<0.01; ***P<0.001).