A375-SM cells or HFFs were spread for 120 min at 37 °C, 5% (v/v) CO2 on glass-bottomed dishes (10 mm glass diameter, 35 mm plate diameter; MatTek Co., Ashland, MA, USA) coated with 10 μg ml−1 FN. Where applicable, spread cells were incubated with the CDK1-specific inhibitor RO-3306 (10 μM; Millipore), CGP74514A (5 μM, Millipore), Roscovitone (20 μM, Cell Signaling) or DMSO for 60 min at 37 °C, 8% (v/v) CO2. Cells were fixed in 4% (w/v) paraformaldehyde for 10 min, washed twice with PBS and permeabilized using 0.5% (w/v) Triton-X-100 in PBS for 5 min. Cells were then washed twice with PBS and glass-bottomed dishes were blocked using 0.1 M glycine in PBS (60 min, RT). Cells were incubated with primary antibodies (30 min, RT), and then washed with PBS and incubated for 30 min with the appropriate secondary antibodies and, where applicable, Alexa 594-conjugated Phalloidin (Invitrogen). Finally, glass-bottomed dishes were washed with PBS a further three times before imaging.
Images were acquired on an inverted confocal microscope (TCS SP5 Acousto-Optical Beam Splitter; Leica, Wetzlar, Germany) using a × 63 objective (HCX Plan Apochromat, NA 1.25) and LCS software (Leica). Alternatively, images were acquired on a spinning-disk confocal inverted microscope (Marianas; 3i, Denver, CO, USA) using a × 63 objective (Plan Apochromat, NA 1.4) and SlideBook 5.0 software (3i).
Images were acquired on an inverted confocal microscope (TCS SP5 Acousto-Optical Beam Splitter; Leica, Wetzlar, Germany) using a × 63 objective (HCX Plan Apochromat, NA 1.25) and LCS software (Leica). Alternatively, images were acquired on a spinning-disk confocal inverted microscope (Marianas; 3i, Denver, CO, USA) using a × 63 objective (Plan Apochromat, NA 1.4) and SlideBook 5.0 software (3i).