Reagents and antibodies
RPMI 1640 medium (1X with L-glutamine and without L-glutamine and phenol red medium) and penicillin-streptomycin were from Mediatech (Manassas, VA). Protease inhibitor cocktail was from Roche Molecular Biochemicals, Indianapolis, IN. Mouse monoclonal anti-human E-cadherin antibody and rat tail collagen were from BD Transduction Laboratories, Lexington, KY. Mouse monoclonal anti-human vimentin antibody and mouse monoclonal anti-human Stat3 was from Santa Cruz Biotechnology, Santa Cruz, CA. Rabbit monoclonal anti-phospho-Stat3 were from EMD Millipore (Billerica, MA). Phorbol 12-myristate 13-acetate (PMA), and mouse monoclonal anti-human actin antibody were from Sigma-Aldrich, Inc., St Louis, MO. Geneticin (G418) and superoxide dismutase (SOD) were from EMD Corp BioScience (Brookfield, WI). MSKE was kindly provided by Dr Tamaro Hudson, Howard University, Washington, DC, and the preparation of the extract has been previously described . Rat monoclonal anti-human Snail antibody and Horseradish perioxidase (HRP)-conjugated goat anti-rat antibody were from Cell Signaling Technology, Inc., Danvers, MA. HRP-conjugated sheep anti-mouse and the Enhanced chemiluminescence (ECL) detection reagent were purchased from Amersham Biosciences, Buckingham, England. Fetal bovine serum (FBS) and Charcoal/dextran treated FBS (DCC-FBS) were from Hyclone, South Logan, UT. Dihydroethidium bromide (DHE) and Mitosox staining kit were obtained from Invitrogen, Carlsbad, CA. Hydro-Cy3 dye was kindly provided by Dr Niren Murthy, Georgia Institute of Technology, Atlanta, GA. The Snail cDNA construct was kindly provided by Dr Mien-Chie Hung, University of Texas, Houston, TX.
Cell lines and culture
LNCaP human prostate cancer cells were obtained from ATCC (Manassas, VA). LNCaP cells stably transfected with constitutively active Snail cDNA has been described previously . Cells were grown in RPMI supplemented with 10% fetal bovine serum and 1X penicillin-streptomycin, at 37°C with 5% CO2 in a humidified incubator.
Stable transfection of Snail cDNA was performed in epithelial ARCaPE cells [23, 24] utilizing Lipofectamine 2000 (Invitrogen) as previously described  to generate ARCaP-Neo, ARCaP-Snail low, and ARCaP-Snail high clones.
Western blot analysis
Western blot was performed as described previously . The membranes were stripped using stripping buffer (Pierce Biotechnology, Inc., Rockford, IL) prior to re-probing with a different antibody. For treatments, 70% confluent cells were serum-starved in phenol red-free serum-free RPMI containing penicillin/streptomycin for 24 h prior to treatment with MSKE or SOD in phenol-free serum-free RPMI containing 5% FBS DCC-FBS for 3 days.
In vitro measurement of superoxide with DHE
For in vitro experiments, 70% confluent cells were washed with PBS followed by trypsin digestion. Cells were pelleted at 300 g for 2 min, the supernatant removed and the cells resuspended in 500 μL of HANKS with 5% FBS. 10 μM DHE (to detect superoxide) was added to cells, followed by incubation for 30 min while gently rocking in the dark. 20,000 cells were gated and analyzed by Fluorescence Activated Cell Sorting (FACS).
In vitro measurement of superoxide with HydroCy3
20,000 cells were plated in RPMI without antibiotics in a 6-well plate. The cells were then placed overnight in 37°C with 5% CO2 in a humidified incubator. The next day cells were serum starved in RPMI without L-glutamine and phenol red for three hours followed by replacement of media with 90 μL PBS/HEPES buffer plus 10 μL of 25 μM Hydro-Cy3 for 15 min at 37°C, and subsequent imaging with a fluorescence microscope. To measure superoxide in cell lysate, 100 μl whole cell lysates prepared from untreated or treated cells was mixed with 90 μL HEPES/PBS buffer and 10 μL of 25 μM of HydroCy3 for 1 h followed by OD measurement at 530/590 nm. Protein concentration was assayed with BCA reagent in whole cell lysates to be used to normalize OD readings.
5,000 cells were plated overnight in RPMI supplemented with 10% fetal bovine serum and 1X penicillin-streptomycin in 16-well chamber slides. The MitoSOX staining was performed as per manufacturer protocol. Briefly, 1 mL of 5 μM MitoSOX reagent was added to the cells, covered with foil and placed at 37°C with 5% CO2 in a humidified incubator for 10 minutes. The cells were then washed three times with warm HBS/CA/Mg buffer. Cells were counter-stained with DAPI to view the nucleus and images taken with a fluorescence microscope.
In vitro cell migration assay
We utilized Costar 24-well plates containing a polycarbonate filter insert with an 8-μ pore size, coated with collagen I. Following treatment with MSKE, N-acetylcysteine (NAC), or SOD for 3 days as described above, cells were trypsinized and 50,000 cells were plated in the upper chamber of the insert containing 0.1% fetal bovine serum (FBS) while the lower chamber contained 10% FBS. Five h later (for ARCaP) or 24 h later (for LNCaP), cells that had migrated to the bottom of the insert were fixed, stained, and counted to obtain the relative migration.
Statistical analysis was performed using ANOVA and Turkey’s Multiple Comparison Test as Post-Hoc test from Graph Pad Prizm3 software; *p < 0.05 was considered significant. Multiple experiments were performed in replicates of 3.