Figure 1

Snail is overexpressed in ARCaP prostate cancer cell line is associated with increased superoxide in vitro . (A) ARCaPE cells transfected stably with empty vector (ARCaP-NeoL) or Snail cDNA (ARCaP-Snail med, -Snail high) were utilized to analyze Snail levels by Western blot analysis. Quantification of the Western blot results by densitometry with normalization to actin levels was done using the Quantity One quantification software (BioRad). (B) 20,000 ARCaP Neo control or Snail-transfected clones were plated in 6-well plates overnight. The next day the cells were serum starved for 3 h, then treated with 50 μM PMA plus or minus 500 U/ml SOD for live cells. Subsequently, the media was replaced with HEPES/PBS buffer containing 25 μM of Hydro-Cy3 for 1 h followed by OD measurement at 530/590 nm. (C) Whole cell lysate was prepared from Neo- or Snail-transfected ARCaP cells treated with 1 or 50 μM PMA plus or minus 500 U/ml SOD for 1 h. 100 μl was mixed with HEPES/PBS buffer and 25 μM of Hydro-Cy3 for 1 h followed by OD measurement at 530/590 nm. Results were normalized to protein concentrations that were measured I whole cell lysate using BCA assay. (D) 5000 ARCaP-NeoL or ARCaP-Snail med cells were plated in duplicate, overnight in 16-well chamber slides. The following day, cells were incubated with 5 μM MitoSOX reagent in the dark at 37°C for 10 minutes. The cells were then washed three times with warm HBS/CA/Mg buffer. Cells stained with MitoSOX (red) were counter-stained with DAPI (blue) to view the nucleus and images taken with a fluorescence microscope. The results are representative of three independent experiments. Magnification ×40. Data are reported as mean + SD (*p < 0.05; **p < 0.005, compared to PMA).