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Figure 2 | BMC Complementary and Alternative Medicine

Figure 2

From: Muscadine grape skin extract reverts snail-mediated epithelial mesenchymal transition via superoxide species in human prostate cancer cells

Figure 2

LNCaP prostate cancer cells stably transfected with Snail display increased superoxide in vitro . (A) Snail expression was tested by Western blot analysis in various LNCaP cell clones stably expressing Snail or empty vector (Neo5), as well as parental LNCaP cells. (B) 20,000 LNCaP Neo control or Snail-transfected clones were plated in 6-well plates overnight. 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 prepared from Neo- or Snail-transfected ARCaP cells 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 LNCaP-Neo5 or LNCaP-Snail high 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.

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