Facs attune

As part of mitochondrial ROS measurement, adherent cells were incubated at 37 °C for 30 min with DMSO-dissolved stock MitoSOX (Invitrogen) added to culture media at a final concentration of 5 μM. Cells were then trypsinised and prepared as single-cell suspension in U-bottom 96-well plates as described. A total of 1:1 PBS:AccuMax solution was removed following centrifugation and resuspended in PBS. Cell suspension was analysed via FACS Attune (Thermo Fisher) BL2 laser.
MitoTracker Deep Red vs MitoTracker Green plots were used to determine populations of cells with dysfunctional mitochondria (deep red − , green +) that have lost their membrane potential [138 (link)]. Adherent cells were incubated at 37 °C for 45 min with DMSO dissolved MitoTracker Deep Red (Invitrogen) and MitoTracker Green (Invitrogen) added to culture media at final concentrations of 30 nM and 20 nM, respectively. The resulting cell pellet was resuspended in 1-mL PBS and analysed via FACS Attune (Thermo Fisher) BL1 (Green) and YL2 (Deep Red) laser after cells were gated using FSC-A vs SSC-A plots.

To analyse cell cycle profile, cells were pulse‐labelled with 10 μM EdU for 30 min, harvested by trypsinisation and fixed in 70% ethanol. For analysis of chromatin bound proteins, cells were pulse‐labelled with 10 μM EdU for 30 min where indicated, harvested by trypsinisation, washed in PBS, pre‐extracted in pre‐extraction buffer (25 mM HEPES pH 7.4, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl₂, 0.3 M sucrose, 0.5% Triton X‐100) 5 min on ice, centrifuged and fixed with 4% PFA 15 min at room temperature (RT). Fixed cell samples were permeabilised with 0.5% Triton X‐100 for 15 min and blocked in 1% BSA. Click reaction was performed in buffer containing 0.1 M Tris–HCl pH 8.5, 0.1 M sodium ascorbate, 2 mM CuSO₄, 10 μM Alexa 647‐azide 30 min at room temperature. Cells were washed in PBS and incubated with primary antibodies for 2 h at RT before incubation with secondary antibodies 1 h at RT. Cells were resuspended in DAPI 5 μg/ml in PBS, and data were acquired using FACS Attune (Life Technologies) and analysed using FlowJo (TreeStar).

U‐2‐OS cells were seeded at 20,000 cells per well to 12‐well plate 1 day before transfection with siRNA. Cells were treated 1 day after siRNA transfection with indicated concentrations of Chk1i and analysed 3 days later. HeLa and CCNF K/O cells were seeded at 20,000 cells per well to 12‐well plate 1 day before treatment with treatment with 1 μM Chk1i. Cells were counted, viability was determined using PI staining at 0.2 μg/ml 24, 48 and 72 h by FACS Attune (Life Technologies), and data were analysed using FlowJo software (TreeStar).