*2.8. Adhesion Strength*

The cells were seeded at a density of 100 000 cells/mL/sample in 24 well tissue culture plates and cultured for either 4 or 24 h at 37 ◦C with 5% CO2. At those time points, samples were transferred to new tissue culture plates, and half of the plates were subjected to shaking at 500 rpm for 5 min using a tissue culture plate shaker (Thermomixer Comfort, Eppendorf, Hamburg, Germany). The reference samples (unshaken) were incubated at 37 ◦C. The number of cells remaining adhered to the surface was measured by replacing the growth medium with 0.1% crystal violet solution in 20% ethanol for 30 min. Then cells were washed with tap water. Before the measurement of absorption, dye was solubilised with 0,1% acetic acid solution in 50% ethanol. Optical density proportional to cell number was measured using a Varioskan Flash microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 570 nm and comparing it to analogously dyed, unshaken cell monolayers. A total of 3 independent experiments were performed with 3 repeats per material within each experiment.

#### *2.9. Signalling Protein Expression and Phosphorylation*

The cells were seeded on the samples at a density of 100,000 cells/mL/sample and cultured for either 4 or 24 h. At these timepoints, the samples were transferred to new tissue culture plates and gently washed with PBS. The PBS was then replaced with a lysis bu ffer consisting of 8 M urea, 2 M thiourea and 50 mM DTT. Cells were collected from 5 samples for each timepoint by pipetting 5–10 times. The lysates were then centrifuged for 10 min at 20,000 × G at RT. The supernatants of each vial were then transferred to new vials and frozen at −20 ◦C until further use. A total of 3 independent experiments was carried out.

Protein concentrations were normalised by running an SDS-PAGE gel, staining with Coomassie brilliant blue, taking images using a transilluminator (UVP, Upland, CA, USA) using analysing by ImageJ software. After diluting the highest concentration samples using lysis bu ffer, the concentration-equalised samples were subjected to gel electrophoresis again at 200 V for 45 min using a BioRad (Hercules, CA, USA) electrophoresis apparatus. The proteins were then transferred to a PVDF membrane (Carl Roth, Karlsruhe, Germany) using a Biometra Fastblot transfer device (Biometra GmbH, Göttingen, Germany) at 25 V and 300 mA. The membranes were blocked using 1% BSA (Sigma-Aldrich, St. Louis, MO, USA) in TBS with 0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA).

The membranes were subsequently treated with primary antibodies against p-Akt (Ser473 and Thr308, Cell Signalling technology, Danvers, MA, USA), Akt (Molecular Probes, Eugene, OR, USA), FAK (BD Biosciences, San Jose, CA, USA) and fluorescently-labelled antiα-tubulin (Sigma-Aldrich, St. Louis, MO, USA) overnight according to manufacturer's instructions at 4 ◦C.

Next day, the membranes were washed three times using wash bu ffer and incubated with secondary antibodies. FAK and Akt were treated with HRP-conjugated goa<sup>t</sup> anti-mouse (Invitrogen, Carlsbad, CA, USA) and HRP-conjugated anti-rabbit (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions for 1 h. Finally, the membranes were washed again three times using wash bu ffer. FAK and Akt were detected using by treating with ECL reagent, which upon catalysis by HRP yields chemiluminescence that was detected using a transilluminator (UVP, Upland, CA, USA).

P-Akt (Ser473) and p-Akt (Thr308) were treated with secondary goat-anti-rabbit antibodies conjugated to a fluorescent infrared dye (IRDye 800CW, LI-COR, Lincoln, NE, USA) and detected using an infrared imaging system (Odyssey, LI-COR, Lincoln, NE, USA).

Membrane image analysis was performed using ImageJ software (National Institutes of Health, USA).
