Incubating chamber

500 μM Syto16 (Invitrogen, Italy) was used to stain living-cell nuclei. Syto 16 was administered 24 h after cell seeding (30 min incubation time at room temperature in culture medium without serum). Four independent time-lapse experiments were performed in epifluorescence by using a 20× air Nikon objective, N.A. 0.45, PlanFluor and an Eclipse Ti inverted microscope (Nikon, Japan) equipped with an incubating chamber (Okolab, Italy), a CCD ORCA R2 (Hamamatzu, Japan), a mercury arc lamp, and 3 blocks of filters (UV-2E/C, B-1°, G-2E/C, Nikon, Japan). Images were acquired for 15 hours, sampling every 15 minutes. Movies were analyzed with the ImageJ manual tracking plugin to extract the coordinates of single cells as a time function. Data were analyzed by a custom-made application written in Matlab. The following parameters were measured: cell displacement (R) (distance from the origin after 15 hours), total path covered in 15 hours (S), migration step (dS) and average speed (V) (calculated for intervals of 15 minutes). Directionality and speed of each step was measured and classified in two populations: parallel (dS_∥, V_∥) and perpendicular (dS_⊥, V_⊥) steps (Fig. 6c–g). dS was considered parallel if the angle between the step and the pattern was less than 15°, while it is considered perpendicular if this angle was between 75° and 90°.