Alexa fluor 488 phalloidin

The cells were cultured in the same way as for the other immunofluorescence stains. Then, the cells were washed three times with TBS before being incubated with protease-free donkey serum blocking and permeabilization buffer (5% in TBS with 0.1% Triton X-100) for 20 min at RT. Subsequently, cover slips were incubated with the primary antibody (SV40 T antigen mouse anti-human, Merck-Millipore, Darmstadt, Germany, 1:50) overnight at 4 °C in a humidifier chamber. Samples were rinsed three times with TBS prior to incubation with donkey anti-mouse cyanine (Cy)3 secondary antibody (Invitrogen, Waltham, MA, USA) combined with Alexa Fluor 488 phalloidin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) (diluted 1:200 and 1:100 in TBS with 0.1% Triton ×100 and 5% donkey serum) to visualize cytoskeletal F-actin organization for 1 h at RT in a humidifier chamber. During this incubation step, cell nuclei were counterstained using 4′,6-diamidino-2′-phenylindol (DAPI, Roche, Mannheim, Germany). Labelled cells were washed three times with TBS before being covered with Fluoromount mounting medium (Southern Biotech, Biozol Diagnostica, Eching, Germany). Photos were taken using confocal laser scanning microscopy (TCS SPEII; Leica, Wetzlar, Germany). The utilized antibodies are indicated in Table 2.

MDA-MB-231 Cells were incubated with Rhob Si@rMNP (0.022 mg/mL, 0.035 mg/mL, 0.17 and 0.35 mg/mL) for 24 h. After incubation, cells were washed and fixed with 4% paraformaldehyde (Boster Bio, Pleasaton, CA, USA) 1:1000 dilution of Alexa Fluor 488^® phalloidin (Santa Cruz) and 1:1000 dilution of DAPI (Sigma Aldritch). Cells were washed twice in 500 µL of PBS and mounted on to microscope slides by inverting onto Thermo Shandon Immuno Mount (ThermoFisher Scientific, Waltham, MA, USA) and the edges sealed with nail varnish. Imaging of cells was performed using a 60× objective on a Nikon TE200 Inverted Fluorescence and Phase Contrast Microscope (Nikon, Minato City, Tokyo, Japan). Images were taken using separate channels and merged using ImageJ software.