Phalloidin alexa fluor 488

To visualize actin filaments an immunofluorescence analysis was performed. Cells were cultivated 15 days onto 2D-matrices after, then they were washed in PBS, fixed in 4% paraformaldehyde in PBS for 15 min at 4 °C and permeabilized with 0,5% Triton-X100 in PBS for 10 min at room temperature. After blocking with 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature cells were incubated with Phalloidin Alexa Fluor 488 (Immunological Sciences, Rome, Italy) 1:40 for 20 min at room temperature. Cells were ultimately washed in PBS and incubated with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, Thermo Fisher Scientific, Massachusset, USA) to visualize the nuclei and analyzed with Leica DM microscope (Leica Microsystem, Milan, Italy).

To visualize cell morphology and actin filaments, immunofluorescence experiments were performed. A total of 30 × 10³ cells per well were seeded in eight-well-ibidi plates and cultured for 24 and 72 h in the presence of 30 µg/mL of UA and with PLLA-UA20X, PGA-20X, and PLLA-PGA-UA20X nanoparticles containing 30 µg/mL of UA. At the end of the treatments, the cells were washed in PBS, fixed in 100% ethanol for 15 min, at room temperature, and permeabilized with 0.5% Triton-X 100 in PBS, for 10 min, at room temperature. After blocking with 3% bovine serum albumin (BSA) in PBS for 30 min, at room temperature, the cells were incubated with Phalloidin Alexa Fluor 488 (Immunological Sciences, Rome, Italy) 1:40, for 20 min, at room temperature. The cells were ultimately washed in PBS and incubated with DAPI (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) to visualize the nuclei. The images were captured with the optical microscope Leica DM IL LED, using a AF6000 modular Microscope (Leica Microsystem, Milan, Italy).