Fig. 1: Design of SSK1 and validation of its ability to selectively kill senescent cells.

a Quantification of cell viability of non-senescent and replication-induced senescent new born dermal fibroblasts (NBFs) incubated with increasing concentrations of gemcitabine for 3 days (n = 3). b Molecular structure of the prodrug SSK1. c Metabolism of SSK1 into gemcitabine in replication-induced senescent NBFs and their non-senescent counterparts incubated with SSK1 (0.5 µM) for 3 days (n = 3). d Quantification of cell viability of senescent and non-senescent NBFs incubated with increasing concentrations of SSK1 for 3 days (n = 4). e Cell viability of GLB1 knockdown (shGLB1-1, -2 and -3) or shControl senescent cells treated with vehicle or SSK1 (0.5 µM) for 3 days (n = 4). For cell viability analysis in (a), (d) and (e), cell numbers were quantified using Hoechst 33342 staining and dead cells were excluded by propidium iodide (PI) staining. f Detection of phos-p38 MAPK in senescent cells or non-senescent cells incubated with SSK1 (0.5 µM) for 3 days by western blot. g Representative flow cytometric plots (left) and quantification (right) of the percentage of viable (Q4: PI⁻ annexin V⁻) and apoptotic (Q2 and Q3: PI⁺ annexin V⁺ and PI⁻ annexin V⁺) cells in vehicle- or SSK1-treated senescent cells by annexin V and PI staining after vehicle or SSK1 (0.5 µM) treatment for 3 days (n = 3). ‘n’ represents the number of biological replicates. Data are presented as means ± SEM. Unpaired two-tailed t-test for (c), (d), and (g), two-way ANOVA test for (e), *P < 0.05, ***P < 0.001, ****P < 0.0001.