Cyan adp 9 c analyser

5 × 10⁵ cells were prepared for each condition. They were trypsinized, then washed and incubated for 15 min, 30 min or 1 h with growth medium supplemented with 10% FBS at 37 °C. Following this step, the cells were washed with PBS/FBS 2% and fixed with PFA 2% for 20 min at 4 °C. They were then washed with PBS and incubated with cold FBS for 15 min at 4 °C before addition for 1 h at 4 °C of the anti-E-cadherin primary antibody recognizing the ectodomain of E-cadherin (#3195 S, Cell Signalling) diluted in PBS/FBS 2%. The cells were washed twice and the secondary antibody was added for 30 min at room temperature (RT) in the dark. They were then washed 3 times and re-suspended in 500 μl PBS for analysis on a CyAn ADP 9 C analyser (Beckman Coulter). At least 10⁴ cells were analysed per condition (n = 3).

Cells were plated in 6-well plates (TPP), and incubated for 24 h, before treatment with palbociclib, ribociclib or the corresponding vehicle. After 94 h of treatment, 10 μM EdU was added to the cell culture media for 2 h. The cells were then collected and washed twice with PBS. Click-it reactions were performed using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Kit (ThermoFischer Scientific) according to the manufacturer's recommendations. The cells were then counterstained with propidium iodide for 30 min. The cell cycle profile was generated using a CyAn ADP 9C analyser (Beckman Coulter). The analysis was performed with the Flowjo software (LLC). Cell cycle was studied on technical duplicates, in three independent experiments.