Ouabain inhibits the FA/BRCA pathway.

<p>(A) The data from chemical library screening and chemical structure of ouabain. U2OS cells were pre-treated with 5 µM of each compound for 1 h prior to incubation in 200 ng/ml MMC for 24 h. Representative data from plate containing ouabain compound are shown. Each dot in the scattergram represents FANCD2 foci area per cell after treatment with each compound tested. (B) Ouabain inhibits MMC-induced FANCD2 foci formation. U2OS cells were pretreated with the indicated concentration of ouabain or curcumin for 1 h and then treated with 200 ng/ml MMC for 24 h. After incubation, the cells were fixed and processed for FANCD2 immunofluorescence and the FANCD2 foci were analyzed with an IN Cell Analyzer. Representative graphs and images from three independent experiments are shown. Values represent the means ± SEM (Student’s <i>t</i>-test, ***, <i>P</i> < 0.001; *, <i>P</i> < 0.05). (C) U2OS cells treated as described in (B) were analyzed with an IN Cell Analyzer. % foci total area/ cell represents relative % to MMC-only treated samples. IC₅₀ was calculated using GraphPad Prism version 5.03. Graphs and values represent the means ± SEM from three independent experiments. (D) Ouabain inhibits MMC-induced FANCD2 monoubiquitination. Protein extracts fromU2OS cells treated as described in (B) were analyzed by Western blotting using antibodies against FANCD2 and β-actin. Values shown represent the ratio of FANCD2 (L)/FANCD2 (S). (E) Ouabain inhibits FANCD2 monoubiquitination after cisplatin treatment. Western blotting was performed with protein extracts from U2OS cells treated with 10 µM cisplatin.