Fig. 5: Fld1-∆LR restores the defect of fld1∆ ldb16∆ mutants similar to hSeipin.
A Lack of Fld1 or Ldb16 or both Fld1 and Ldb16 results in an indistinguishable LD morphology defect. Cells of the indicated genotypes were grown in YPD media, stained with BODIPY and imaged in a fluorescent microscope. Representative image shown from three independent experiments. Scale bars: 5 µm. B Fld1-∆LR rescues LD phenotype of fld1∆ ldb16∆ double mutant. Yeast fld1∆ ldb16∆ double mutants expressing either Fld1 alone or Fld1-∆LR as an mCherry fusion were grown in SC media, stained with LD dye BODIPY and subjected to fluorescence microscopy. White arrowheads denote colocalization between LDs with either Fld1 or Fld1-∆LR. Representative image shown from three independent experiments. Scale bars: 5 µm. C, D Quantification of LD number (C) and size distribution (D) in cells of the indicated genotypes. Data represent mean ± s.d. and were analysed with two-way ANOVA and Tukey’s multiple comparisons. n = 100 cells. E Human Seipin rescues the lack of Fld1 and Ldb16 in yeast. Yeast fld1∆ and fld1∆ ldb16∆ cells expressing GFP-hSeipin on a plasmid were grown in selective media, stained with LD dye, MDH and visualized by fluorescence microscopy. White arrowheads denote colocalization between hSeipin marked puncta and LDs. Representative image shown from three independent experiments. Scale bar: 5 µm. F Fld1-∆LR compliments terbinafine growth sensitivity of fld1∆ ldb16∆ cells like hSeipin. Indicated yeast strains were cultivated in either YPD or SC media, serially diluted, and spotted on YPD plates containing either DMSO or terbinafine at a final concentration of 100 μg/mL. Representative image shown from three independent experiments. G Western blot analysis. Western blot showing expression of Fld1-mCherry or Fld1-∆LR-mCherry or Fld1-∆LR-mCherryP31G. PGK1 serves as a loading control. H Fld1-∆LR-mCherryP31G gets mislocalized. Yeast fld1∆ and fld1∆ lbd16∆ cells expressing Fld1-∆LR-mCherryP31G were grown in selective media, stained with BODIPY and imaged. Representative image shown from three independent experiments. Scale bar: 5 µm. I, J Quantification of LD number (I) and size distribution (J) in cells of the indicated genotypes. Data represent mean ± s.d. and were analysed with two-way ANOVA and Tukey’s multiple comparisons. n = 100 cells. K Terbinafine growth assay. Yeast strains of the indicated genotypes were grown in either YPD or SC media, serially diluted, and spotted on YPD plates containing either DMSO or terbinafine at a final concentration of 100 μg/mL. Representative image from three independent experiments is shown. Source data are provided as a Source Data file.
