Phalloidin tetramethylrhodamine b isothiocyanate tritc

A549 investigated cells were fixed in 3.8% paraformaldehyde solution containing 0.2% Triton X-100 (Sigma-Aldrich, USA) for 5 min at 37°C and subjected to immunostaining. To evaluate the effect of angiotensin-(1–7) and miRNA transfections in A549 cellular morphology, the actin filaments were stained in a 1% bovine serum albumin (BSA) solution containing 100 μg/ ml of phalloidin-tetramethylrhodamine B isothiocyanate (TRITC) (Sigma-Aldrich, USA) for 1 h. Next, the nuclei of the cells were counterstained in 3.33 ng/ ml 4’,6 diamino-2-phenylindole (DAPI, Sigma-Aldrich, USA) solution for 5 min. After extensive washes with saline phosphate buffer (PBS, 2.7 mM KCl, 1.5 mM KH₂PO₄, 137 mM NaCl, and 8 mM Na₂HPO₄, pH 7.4), the coverslips containing cells were subsequently mounted onto slides and subjected to microscopic immunofluorescence analysis. Images were obtained with an Olympus BX51 microscopy equipped with corresponding filter sets and a DP71 CCD camera (Tokyo, Japan). One hundred randomly selected cells were analyzed in each investigated group. For phase-contrast analyses required in the in vitro scratching assays (wound healing) and in the cellular invasion chamber assays, the same microscopy was used and images were monitored and quantified using the Image J software (http://rsb.info.nih.gov/ij/).

Human tumor cells were purchased from Banca Biologica and Cell Factory (IRCCS Azienda Ospedaliera Universitaria San Martino-IST Istituto Nazionale per la ricerca sul cancro, Genova, Italy). The human cancer cell line MCF-7 was cultured in high glucose DMEM (Euroclone) supplemented with 10% FBS (Euroclone), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in an atmosphere of 5% CO₂. The cells were maintained in an exponential growth phase during experiments.
Epidermal growth factor (EGF) was from Prospec. Primary antibodies were from Santa Cruz: β-Catenin (sc-1496), α-Tubulin (sc-23948), Cofilin (sc-33779), phospho-Cofilin (sc-21867-R); or Cell Signaling: β-Actin (#8457), c-Myc (#13987), fatty acid synthase (FASN, #3180), and Acetyl CoA Carboxylase (ACC, #3676). Secondary antibodies (HRP-conjugated) were from Santa Cruz Biotechnology (goat anti-mouse IgG-HRP, sc-2005; goat anti-rabbit IgG-HRP, sc-2004) or Cell Signaling (anti-rabbit IgG-HRP, #7074). Phalloidin-tetramethylrhodamine B isothiocyanate (TRITC) and Fluoroshield were from Sigma. All other reagents were from standard commercial sources and were of the highest grade available.

Cells were seeded on glass coverslips in 24 multiwell plates at a density of 18.000 cells/cm² and, once they reached the confluence, about 24 h post plating, they were switched to differentiation medium and were cultured for 3, 6, 9, and 12 days. For bright field, qualitative images were obtained directly from 24 multiwell plates using IM-3 OPTIKA inverted microscope equipped with a digital microscope camera (Leica DFC320, Wetzlar, Germany). For fluorescence microscopy, cells were fixed with 4% formalin in phosphate buffered saline (PBS) pH 7.4 for 1h at room temperature, rinsed with PBS, permeabilized with 0.5% Triton X-100 in PBS, and incubated with Phalloidin–Tetramethylrhodamine B isothiocyanate (TRITC), diluted 1:1000 (Sigma Aldrich, Milan, Italy) for 45 min at 37 °C. Cell nuclei were stained with 300 nM Diamidine-20-phenylindole dihydrochloride (DAPI, Sigma Aldrich, Milan, Italy) for 2 min. After rinsing, samples were processed in mounting medium (60% glycerol in PBS) and observed under a fluorescence microscope (DFC7000 T Leica, Wetzlar, Germany). Each treatment was performed in triplicate, and each experiment was repeated at least two times. Images were acquired (10 field/well) and analyzed to determine differentiation and fusion indexes. Representative images were taken for each condition and analyzed to evaluate myotube formation.