Research Paper Volume 14, Issue 16 pp 6381—6414

Figure 1. Scheme of the experimental setup and data analysis. (A) The summary of used senescence models, including their designation, the number of cells seeded, and time of senescence development. The seeding area was 0.322 cm² per well of 96 well-plate. (B) Summary of the experimental procedure. Twenty-four hours after cell seeding, the senescent insult was applied to develop a senescent phenotype for a period depicted in (A). Control proliferating or quiescent cells were seeded 24 hours before the drug exposure. After 24-hour of single or combined compound treatment, the cultures were stained with crystal violet to quantify the amount of surviving cells by absorbance reading (A = 595). The cell viability was expressed as the percentage of the untreated population. (C) The analytical workflow. After treatment, the viability of senescent cells expressed as a percentage of a mean (n = 3) relative to untreated cells, scaled from 0 (red) to 100 percent (green), was subsequently analyzed by the SynergyFinder to determine the area of synergy for senescent cells transformed back into the matrix representing compound concentration (orange region; note that only compound combinations are presented). In parallel, the matrix of selective senolytic effect was obtained by subtracting the viability of senescent cells from the control population. The green gradient, which indicates the region where senescent cell viability was lower than the control population, was marked blue to highlight the region with a selective senolytic effect (note that only compound combinations are presented). Next, the compound concentrations giving selective senolytic effect were statistically evaluated by a two-tailed Student's t-test (*, P > 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Finally, to obtain drug concentrations with synergistic selective senolytic effect, synergy and selective senolytic effect matrices were merged to get an effective window of compound concentrations (blue-orange fields). CMD – compound.