Columbus software

On day 14 of differentiation, cells were treated with indicated compounds for 6 h, medium was removed and the cells were washed twice with PBS + /+ (containing Mg²⁺ and Ca²⁺, Sigma Life Sciences). By adding 4% PFA with 0,1% Triton-X100, cells were fixed and permeabilized. After three washing steps with PBS + 0,1% Triton-X100, cells were incubated for 1 h at room temperature for blocking with 5% normal goat serum (Thermo Fisher Scientific). Cells were again washed three times with PBS + 0,1% Triton-X100 and incubated with Hoechst 33342 (Sigma, 1:10,000) for 20 min at room temperature. Imaging was performed using an Operetta high-content system and images were analyzed with the Columbus software (PerkinElmer).
For live/dead discrimination, cells were live-stained with 5 µg/ml Propidium iodide (Invitrogen) after 6 h of compound treatment and imaged with an Operetta high-content system. Image analysis was done with the Columbus software (PerkinElmer).

For SA-β-Galactosidase assay, cells were washed with PBS 1X, fixed for 5 min in 2% formaldehyde/0.2% glutaraldehyde, rinsed twice in PBS 1X, and incubated at 37 °C overnight in SA-β-Galactosidase staining solutions, freshly prepared (40 mM citric acid/Na phosphate buffer, 5 mM K3[Fe(CN)6], 5 mM K4[Fe(CN)6] 3H2O, 150 mM sodium chloride, 2 mM magnesium chloride, 1 mg/mL X-gal in distilled water). For crystal violet assay, cells were washed with PBS 1×, fixed for 15 min in 3.7% formaldehyde and stained with crystal violet. For EdU Assay, Click-iT™ EdU Alexa Fluor™ 488 Imaging Kit was used according to manufacturer’s recommendations (ThermoFisher Scientific). For JC1, JC1-Mitochondrial Membrane Potential Assay Kit (ab113850, Abcam) was used. JC1 monomers and aggregates were both excited at 488 nm. Detection of fluorescence for JC1 monomers and aggregates were performed respectively at 530 and 590 nm. Ratio F(aggregate)/F(monomer) was subsequently evaluated using Columbus™ software (PerkinElmer) at the single-cell level. Cell Meter™ Mitochondrial Hydroxyl Radical Detection Kit (ATT Bioquest) allowed detection of mitochondrial ROS according to manufacturer’s recommendations. Excitation was monitored at 488 nm, and fluorescence emission was measured at 530 ± 30 nm using Columbus™ software (PerkinElmer) at the single-cell level.

Cells were pre-extracted for 1 min in ice cold PBS 0.2% Triton-X100, fixed with 4% formaldehyde for 20 min. Coverslips were blocked in 1% BSA for 1 h and incubated in primary antibodies overnight at 4 °C, RPA32 (RPA2) (Ms, MABE285, 1:500) and Phospho-Histone H2A.X (γH2AX) (Ser139) (Rb, 20E3, Cell Signalling Technology, 1:400). Coverslips were incubated in secondary antibodies for 1 h at RT; anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 647 1:2000 (Life Technologies). Coverslips were then incubated in Hoechst (Invitrogen) 1:10,000 for 5 min and mounted with Fluoroshield (Sigma). Images were obtained by confocal microscopy with a Leica TCS SP5 or SPE2 ×63 objective lens using LASAF. Images were processed in Fiji.
For EdU incorporation evaluated by microscopy cells were reverse transfected and seeded at density 6000 cells/well in 96-well plate (PerkinElmer, CellCarrier). Twenty-four hours after transfection, cells were incubated with 10 µM EdU for 30 min, then were fixed with 4% formaldehyde for 20 min, permeabilised with 0.5% TritonX in PBS for 5 min and EdU was detected by ClickIT reaction (Life Technologies). Cells were stained with 2 µg/ml DAPI solution in PBS for 30 min. Images were acquired by Opera Phenix HCS System with Harmony (PerkinElmer) and analysed in Columbus software (PerkinElmer) and custom-made RStudio script available upon request.