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. 2021 Mar 3;17(3):e1009407. doi: 10.1371/journal.pgen.1009407

Fig 2. Dbp4 enhances αSyn-mediated growth impairment in yeast.

Fig 2

(A) Growth assay of yeast cells expressing GAL1-driven αSyn-GFP from two genomic copies, either alone (αSyn) or co-expressed with DBP4 (αSyn+DBP4 oe). The isogenic background strain W303 was transformed with empty vectors (Control) or with 2μ plasmid overexpressing DBP4 (DBP4 oe). (B) Relative mRNA expression level of DBP4 in Tet-DBP4 and W303 strains determined by qRT-PCR in presence or absence of αSyn expression. Addition of doxycycline (Dox) efficiently downregulates the expression of Tet-DBP4. Expression of αSyn upregulates the mRNA levels of Tet-DBP4 upon downregulation of the Tet-promoter or of natively expressed DBP4 in wild type W303. Overexpression of DBP4 increases considerably the mRNA levels. Significance of differences was calculated with t-test (*p < 0.05; ***p < 0.001; ****p < 0.0001, n = 4). (C) Relative abundance of αSyn mRNA from three gene copies in Tet-DBP4 and from 2μ vector in W303 strains, determined by qRT-PCR. Significance of differences was calculated with t-test (n.s.; n = 4). (D) Western blot analysis of αSyn protein levels of cells from (C) with GAPDH antibody used as loading control. (E) Densitometric analysis of the immunodetection of αSyn relative to GAPDH loading control. Significance of differences was calculated with t-test (n.s.; n = 3). (F) Western blot analysis of Dbp4-GFP protein levels of cells expressing DBP4-GFP from native promoter with GAPDH antibody used as a loading control. (G) Densitometric analysis of the immunodetection of Dbp4-GFP relative to GAPDH loading control. Significance of differences was calculated with t-test (**p < 0.01; n = 3). (H) Fluorescence microscopy of yeast cells expressing αSyn-GFP from three gene copies in Tet-DBP4 strain in the presence (+) or absence (-) of doxycycline (left panels); from a 2μ vector in W303 strain (middle panels) or from three gene copies of αSyn-GFP in W303 strain (right panels) 6 h after induction of expression in galactose-containing medium. DBP4 was overexpressed from 2μ plasmid. EV = empty vector. Scale bar = 5 μm. (I) Quantification of the percentage of cells displaying αSyn-GFP inclusions in strains from H. Significance of differences was calculated with t-test (**p < 0.01, n = 4). (J) Promoter shut-off of cells from (H). Cells with inclusions were counted up to 6 h after GAL1-promoter shut-off and normalized to time point zero. Significance of differences was calculated with One-way Anova test (n.s.; n = 3).