Localization of oxidated thiols in A375 melanoma cells.

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Localization of oxidated thiols in A375 melanoma cells. Localization of oxidated thiols in A375 melanoma cells. A, after incubation with 150 μmol/L CNP and 5 μmol/L doxorubicin (DOX) alone or in combination, cells were fixed for an immunochemical staining was performed by using the α-hapten antibody raised against the oxidation product of sulfenic acid and dimedone, which was added to the cells for the last 2 hours of incubation. In addition, nuclei were stained with DAPI. Presented is 1 of 3 independent experiments. B, Western blot analysis was carried out with cytoplasmic and nuclear cell extracts from melanoma cells that were treated with 0.5 μmol/L doxorubicin or 150 μmol/L CNP for 2 hours, as well as co-incubated. CNP caused thiol oxidation mainly in the cytosol, whereas doxorubicin showed strong formation of sulfenic acids in the nuclei as well. Three independent experiments were performed. α-Tubulin was used for the cytoplasmic extract and PARP for the nuclear extract as loading control. The figure represents 1 of 3 independent experiments that were analyzed by densitometry with ImageJ. The x-fold increase of the untreated controls is presented. Maren Sack et al. Mol Cancer Ther 2014;13:1740-1749 ©2014 by American Association for Cancer Research