Fig. 1: Screening of an in-house library of natural compounds to identify senolytic substances.

A HCT116 cells were seeded in 96-well plates the day before they were exposed to 10 Gy gamma-irradiation. Seven days later, after the senescent phenotype was fully formed (see B), the cells were exposed to the natural compounds in duplicates in concentrations ranging from 30 µM to 100 nM. Proliferating non-irradiated cells were seeded the day before treatment with the natural compounds. 24 h after treatment, cell death was assessed by staining the surviving cells using a crystal violet-based cytotoxicity assay. Each substance was tested in at least three independent experiments. B Proliferating or senescent (day 6 after γIR) HCT116 cells treated as described in (A), but in a 6-well plate, were stained for senescence-associated beta-galactosidase activity. Positive cells display a strong blue color. All microscopic pictures were taken with the same ×10 magnification (scale bar 100 µM) and are representative for at least three independent experiments. The specified values are the mean percentage of counted blue cells ± SD from these at least three independent experiments (at least 200 to 1000 counted cells per condition and experiment). C All compounds were classified based on their cytotoxic effect on proliferating and/or senescent cells. For each class, one exemplary substance is shown. All data shown are the mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. D Distribution of the 178 tested compounds into four different groups (cytotoxic/non-cytotoxic towards proliferating/senescent cells). Two substances, Kahalalide F and wortmannin, exerted senolytic properties towards HCT116 cells.