Cell lines were cultured in 50 mm diameter glass-bottomed dishes (WillCo Wells, Amsterdam, The Netherlands) for use on the Bioscope Catalyst II AFM (Bruker, Coventry, UK). After the desired treatment regime, the culture media was replaced with 2 ml of pre-warmed DMEM/F-12 phenol red free media (Gibco, Thermo Fisher, UK) prior to live cell imaging. Cells were imaged over a maximum time of 90 mins at 37 °C. ScanAsyst Fluid cantilevers (Bruker, Coventry, UK) were used with a nominal spring constant of 0.7 N/m and a tip apex radius of 20–60 nm. The indentation force was kept below < 1 nN, scan rate was 0.5 Hz, resolution was 128 samples/line and scan area encompassed 50–150 µm of the cell monolayer. Processing of PFQNM images was undertaken with the Bruker Nanoscope software, following standard protocol [53, 54]. Data shown is based on a minimum of three biological repeats, analysed as parametric data using 2 tailed T test. Significance given as * p < 0.05, ** p < 0.001 and *** p < 0.0001.

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