Figure 3
The Ca2+/Mn2+ ATPase Pmr1p mediates αSyn cytotoxicity. (a and b) Quantification via fluorescence reader (a) and representative micrographs (b) of ROS production by assessing the ROS-driven conversion of dihydroethidium to ethidium (DHE→Eth) upon expression of αSyn for 24 h in WT yeast cells and indicated deletion mutants. Mean±S.E.M., n=8; ***P<0.001 and **P<0.01. (c and d) Flow cytometric quantification (c) and representative micrographs (d) of externalization of phosphatidylserine (AnnV+) and loss of membrane integrity (PI+) by Annexin V/PI co-staining of WT cells and indicated deletion mutants expressing αSyn for 48 h. Mean±S.E.M., n=6; ***P<0.001. (e) Western blot analysis of αSyn expression in WT cells and indicated deletion mutants. Blots were probed with antibodies against FLAG-epitope to detect FLAG-tagged αSyn and against glyceraldehyd-3-phosphate dehydrogenase (GAPDH) as loading control. (f) Survival determined by clonogenicity of WT and Δpmr1 yeast cells expressing αSyn or harbouring the vector control after 24 h and 48 h of expression on galactose media and plating on YEPD agar plates. Mean±S.E.M., n=10; ***P<0.001. (g) Quantification of ROS accumulation (DHE→Eth) in yeast cells in which the promoter region of PMR1 has been replaced by a doxycycline-repressible promoter (TetO-PMR1). Doxycycline (Doxy) was added in indicated concentrations and αSyn was expressed for 24 h or 48 h. Mean±S.E.M., n=8; ***P<0.001. (h) Q-PCR-based quantification of PMR1 mRNA levels in yeast cells described in (g) after treatment with 10 μg/ml Doxycycline (Doxy) and αSyn expression for 12 h. Data have been normalized to mRNA levels of actin. Means±S.E.M., n=3. Asterisks indicate significance between untreated and Doxy-treated cells, ***P<0.001
