Fig 1: Cell cycle analyses of senescent cells subjected to Rapamycin, or combined Rapamycin and Spautin-1, treatment: (A) AGS and MCF-7 cells subjected to the indicated pharmacological treatments (in all cases, 96 h of treatment) were suspended in 1×PBS, fixed in ethanol, incubated with RNAse A, and stained with Propidium Iodide. Cells were then analyzed by flow cytometry in order to determine the relative proportion of cells in different phases of the cell cycle. (B) AGS and MCF-7 cells were exposed to the indicated drugs, or drug combinations, for 96 h. The number of viable cells was quantified with LUNA II counter. Error bars represent the mean ± SEM of three independent experiments. **** p < 0.0001, ** p < 0.01, * p < 0.05, based on two-way ANOVA with Tukey’s post-test. (C) AGS and MCF-7 cells were exposed to the indicated drug, or combination of drugs, for 96 h. Cells were then subjected to senescence-associated b-galactosidase assay (SA-β-Gal) and visualized with bright field microscopy at 40x magnification. Error bars represent the mean ± SEM of three independent experiments. **** p < 0.0001, ** p < 0.01, * p < 0.05, ns = no significant difference compared to DMSO based on two-way ANOVA. (D) Results of fluorometry-based β-galactosidase activity assays (ENZ-KIT129-0120, Enzo Life Sciences) following 96 h exposure of AGS and MCF-7 cells to the indicated drugs, or combination of drugs, are shown. Fluorescence values were corrected by the respective amounts of total protein. The results of at least three independent experiments were compiled. Error bars represent the mean ± SEM of three independent experiments, ** p < 0.0021, * p < 0.0332, ns p < 0.1234 (no significance) in comparison to DMSO treatment; based on two-way ANOVA with Geisser–Greenhouse correction and multiple comparisons with Tukey’s post-test.