Wash buffer

PC3 and LNCaP cells were cultured on Poly L-Lysine coated coverslips in RPMI with 10% FBS, which was replaced 24 h before the experiment with RPMI containing 2% FBS. For the PI3K/FAK inhibition, cells were incubated with either Calphostin C (100 nM) or Wortmannin (10 μM) 2 h before addition of CXCL16. The cells were fixed with 4% paraformaldehyde in PBS for 10 minutes after 5 minutes CXCL16 treatment. After fixation cells were permeabilized for 5 minutes with permeabilization buffer (Cytoskeleton Inc. Denver). After washing with wash buffer (Cytoskeleton Inc. Denver), cells were incubated for 40 minutes with 100nM Rhodamine Phallodin and 20μl Alexa Fluor 488 conjugated Mouse anti-Ezrin (pY353) (BD Biosciences). After three washings the coverslip was mounted on slide in presence of aqueous permanent mounting medium (DAKO). Images were captured using Olympus microscope with 60X oil immersion objective.

In order to analyze changes in actin cytoskeleton structure in BEAS-2B cells treated with CuO NP, cells were cultured on coverslips and exposed to 0.01 µg/cm2 CuO NP for 24 h at 37°C. Cells were then washed for 30 sec at 4°C in wash buffer (Cytoskeleton Inc.), fixed for 10 min in 4% paraformaldehyde, washed three times for 30 sec at 4°C in wash buffer. Cells were permeabilized for 5 min with permeabilization buffer, and washed for 30 sec at 4°C in wash buffer, and stained for 30 min with Rhodamine Phalloidin by using the F-actin Visualization Biochem Kit (Cytoskeleton Inc.) following manufacturer's instructions. The cells were then covered with mounting medium containing DAPI (Vectashield). The images were collected by using Confocal Laser Scanning Microscopy (Zeiss Axiovert 200 M Inverted Research Microscope) and processed by using LSM Image Browser (4.2.0.121). Images from at least four replicates were collected for each condition.