Epigenetic drug screening with natural compound library identifies toyocamycin as the most promising hit. (A) Schematic description of YB5 epigenetic reporter system in untreated condition (left), showing GFP expression upon drug-induced active chromatin (middle), or after drug-induced DNA demethylation and consequent chromatin activation (right). (B) Heatmap showing percentages of GFP-expressing cells after natural products (NP) screening in YB5 cells with the 5 different schedules (n = 1). NP10 means treatment with natural products at 10 µM for 72 h. NP50 means treatment with natural compound library at 50 µM for 24 h. NP TSA means treatment with natural products at 10 µM for 72 h followed by 24 h treatment with 200 nM trichostatin A. DAC+NP means 72 h treatment with decitabine (DAC) in simultaneous combination with 10 µM NP. DAC → NP means 72 h treatment with decitabine at 50 nM followed by NP at 50 µM. Each horizontal line represents a compound. Toyocamycin is highlighted with a star on the heatmap (*). (C) Flow cytometry graphical representations of YB5 cells for GFP fluorescence (x-axis) counterstained with propidium iodide (P.I., y-axis) for dead cell staining in untreated and treated cells (drugs and doses are indicated on the graphs, 10,000 cells were acquired). (D) Percentage of GFP-expressing cells in YB5 cells after vorinostat (SAHA), DAC, and toyocamycin treatments (at doses indicated on the graph, n = 3). (E) Percentage of GFP-expressing cells in HCT116 cells after depsipeptide and toyocamycin treatments (at doses indicated on the graph, n = 3).