Cell culture chamber slides

Fibronectin matrix assembly was analyzed based on the immuno-fluorescence staining of insoluble fibronectin fibrils as described previously [6 (link), 7 (link)]. Briefly, NIH 3T3 fibroblasts, stably transfected with pBabe empty vector, CA-Akt1 or DN-Akt1 were co-transfected with Ad-DN-Mek1 Ad-CA-Mek1, particles, Retro-CA-Akt1 co-transfected with Ad-DN-Mek1 and Retro-DN-Akt1 co-transfected with Ad-CA-Mek1 particles, were plated on cell culture chamber slides (Fisher scientific, Pittsburgh, PA) at 90–100 % of confluence and cultured for the next 8 h in serum free medium. Next, cells were fixed with 2% paraformaldehyde in 1 × PBS for 30 min followed by blocking with 5 % BSA for 1 h at room temperature. The fixed and blocked cells were incubated with primary antibody against fibronectin (dilution 1:1000) overnight at 4°C, followed by washing with 1 × PBS (3 × for 15 min each). Next, Alexa Fluor 488-labeled secondary antibodies were added and incubated room temperature for 1 h (dilution 1:1000). Slides were washed and mounted with Vectashield (Vector Laboratories). The images were taken using Zeiss fluorescent microscope (Zeiss Axiovert100M, Carl Zeiss, Germany).

Phalloidin immunofluorescence staining of the cells was performed as described previously (Goc et al., 2013 (link)). Briefly, PC3 or DU145 cells were plated on cell culture chamber slides (Fisher Scientific, Pittsburgh, PA) at low density were subjected to treatment with TGFβ1 (5 ng/ml) every day. After 3 days, cells were washed 2 times with 1X PBS and fixed with 1% paraformaldehyde in PBS. Cells were permeabilized using 0.1% Triton X-100 in PBS. The nonspecific staining was blocked with 2 % BSA for 1 h at room temperature. Slides were incubated with Alexa Fluor 555-labeled phalloidin for 20 min at room temperature and washed 4 times with 1X PBS. The slides were mounted with Vectashield (Vector Laboratories), and the images were taken by an inverted fluorescence microscope (Zeiss Axiovert100M, Carl Zeiss, Germany).

We subjected human T24 metastatic bladder cancer cells for immunocytochemistry analysis to determine the endogenous expression and localization of three Akt isoforms Akt1, Akt2 and Akt3. T24 cells were plated on cell culture chamber slides (Fisher scientific, Pittsburgh, PA) followed by fixation with 1 % paraformaldehyde in 1 X PBS. Cells were permeabilized with 0.1% triton X-100 in PBS and blocked with 2% BSA for 1 h, and incubated with 1:1000 primary antibodies over-night in 4 °C. Alexa Fluor 488 labeled secondary antibodies were applied for 1 h at room temperature washed and mounted with Vectashield (Vector Laboratories, Burlingame, CA). Images were taken by confocal fluorescent microscope (Zeiss Axiovert100M, Zeiss, Germany) and analyzed using NIH Image J software (Al-Husein et al., 2013 (link)).