Accuri c6

Ploidy was estimated with a modified version of the method of Popolo and colleagues [55 (link)]. Briefly, cells patched on YPD overnight were suspended in 1 mL sterile ice-cold water to OD600 ~1, centrifuged (5 minutes, 5,000 rpm), and fixed in 1 mL cold 70% ethanol for 24 hours at 4°C (to minimize cell aggregation during ethanol fixation, cells were first resuspended in 300 μL cold sterile water followed by dropwise addition of 700 μL cold absolute ethanol while vortexing). Fixed cells were next treated with 100 μL 1 mg/mL RNAse A for 90 minutes at 37°C after centrifugation to remove the ethanol. RNAse A–treated cells were centrifuged and the pellet stained with 100 μL 0.05 mg/mL propidium iodide at 4°C for 24 hours. For cell viability assays, cells were washed once with PBS by centrifuging at low speed (5 minutes, 5,000 rpm), stained directly with the abovementioned volume and concentration of propidium iodide in PBS. Stained samples were incubated for 30 minutes at room temperature before flow cytometry. Approximately 50 μL of stained cells was diluted to 500 μL with ice-cold water and run on a Becton Dickinson Accuri C6 flow cytometer during ploidy determination and on the CytoFLEX LX (Beckman Coulter) during viability assays on slow fluidics. Data were analyzed using Flowjo software (Flowjo, LLC).

ROS levels in SHSY-5Y cells were determined according to Reactive Oxygen Species Assay Kit (Beyotime, Nanjing, China). SH-SY5Y cells were seeded into 6-well plates at a density of 2.0×10⁵ cells per well and pre-treated with different formulations for 6 h. Cells were then exposed to 2 mM MPP⁺ for 2 h at 37°C and stained with 10 μM DCFH-DA for 20 min at 37°C. ROS expression was detected by flow cytometry (Accuri C6, Beckman, NJ) and analyzed using Beckman Coulter software.

Exponentially growing
cdc2
^asM17 cells (OD
₆₀₀ = 0.1–0.2; 1–2 × 10
⁶ cells/mL) were treated with 2µM 3-Br-PP1 (abcam, ab143756) for 3 h. A 50mL fraction 3-Br-PP1-treated culture was centrifuged (1000 × g, 5 min, 25°C) and the cell pellet washed with 50 mL of fresh YES medium pre-heated to 30°C. Washed cells were re-suspended in 50 mL of fresh pre-heated YES and incubated at 30°C. In 15-min intervals, 1mL aliquots of synchronous cell culture were centrifuged (1000 × g, 3 min, 25°C) and collected cells fixed in 1 mL of 70% ethanol. For each time point, 500 µL of fixed cells were centrifuged (1000 × g, 3 min, 25°C), the supernatant was discarded, and the cell pellet re-suspended in 500 µL of sodium citrate (50 mM, pH = 7) containing 1 mg/mL RNase A (NEB, T3018L). The resulting cell suspension was incubated for 3 h at 37°C, and then mixed with 500 µL of sodium citrate (50 mM, pH = 7) containing 2µM SYTOX Green (Invitrogen, S7020). Samples were analysed using an Accuri C6 flow cytometry system (Beckman Coulter). Data were analysed by BD CSampler software (version 1.0.264.21) and R (version 4.0.0) (
https://www.R-project.org;
R Core Team, 2020 ).