Figure 2.

Fluorescence images of the PAA-OH substrates coated with a solution of fibronectin (25μg/mL) and Alexa488 conjugated Fibrinogen (2μg/mL), for two different stiffnesses (a,b: soft 0.32kPa, c: stiff, 50kPa) and two drying timing (for the soft hydrogel) after FN incubation. For each picture it is shown an example of cell (MCF10A) seeded on that substrate (bright field images d, e, f). FN fibers are visible in the rectangle of figure e. From the comparison between a,d and b,e pictures it is evident that softer hydrogels are more sensitive to the drying time and require shorter (5’) drying times to avoid formation of adhesive bundles or nets instead of a homogeneous protein coating on a relatively large area hydrogel.

Immunofluorescence images of YAP/TAZ subcellular localization of U2OS cells seeded on: soft PAA-OH (g), FN fibers conjugated to soft PAA-OH (h) and stiff PAA-OH (i). From the staining are visible: nuclei (in blue) and YAP/TAZ (in green).

1) quantifications of the nuclear/cytoplasmic (N/C) ratio of YAP/TAZ subcellular localization in U2OS seeded on soft PAA-OH (0.32 kPa), FN fibers conjugated to soft PAA-OH (0.32 kPa) and stiff PAA-OH (50 kPa). Number of cells for each lane in Figure 2l is: lane 1:36; lane 2:19; lane 3:48. Scale bar: 100 μm (a,b and c), 200 μm (d,e and f) and 50 μm (g,h and i).