Extended Data Figure 6. Sertraline selectively induces apoptosis in XL413-induced senescent cells through suppression of mTOR signalling.
a, Schematic outline of the GPCR compound screen. Huh7 cells were treated with 10 μM XL413 for 5 days prior to seeding in 96-well plates. All compounds were tested at 4 different concentrations for 6 days and cell viability was measured using CellTiter-Blue assay. b, c, Graph depicting the effects of compounds on cell viability. Each point represents a single compound, with % activity calculated by dividing the cell viability score in the presence of 5 μM of that compound by the mean viability of the negative control. Blue dots indicate compounds inducing cell death in both control and XL413-induced senescent cells. Sertraline (red dot) induced selective cell death in XL413-induced senescent cells. Representative images of the effects of different compounds on XL413-treated and untreated cells were shown. d, Control cells and XL413-induced senescent cells were sequentially cultured with increasing concentrations of sertraline for 48 h and apoptotic cells were visualized by caspase-3/7 apoptosis assay. e, Control and XL413-treated cells were sequentially exposed to 10 μM sertraline and growth curves were measured by Incucyte live cell assay (Graphs represent mean ± s.d. from three technical replicates). f, Control and XL413-treated cells were sequentially treated with vehicle or 10 μM sertraline for 96 h in colony formation assay. g, Control and XL413-treated Huh7 and Hep3B cells were treated with sertraline (10 μM) for 24 h and western blot analyses of the indicated mTOR signalling pathway proteins were performed. h, Hep3B and Huh7 cells were treated with 10 μM XL413 for 10 days prior to sequential sertraline treatment (10 μM, 24 h) and RNA sequencing was performed. GSEA indicates that the gene set related to downregulation of mTOR signalling was negatively enriched in liver cancer cells sequentially treated with XL413 and sertraline (See methods for more detailed information). i-j, TP53 mutant liver cancer cell lines (SNU449 and PLC/PRF/5) and lung cancer cell lines (NCI-H358 and PC9) were treated with 10 μM XL413 or vehicle for 5-7 days and sequentially exposed to increasing concentrations of AZD8055. Apoptotic cells were visualized by caspase-3/7 apoptosis assay 96 h post-AZD8055 treatment. k, TP53 mutant Hep3B and Huh7 cells were treated with 10 μM XL413 or vehicle for 7-10 days. Control cells and XL413-induced senescent cells were plated and exposed to increasing concentrations of the mTORC1/2 inhibitor AZD2014 prior to caspase-3/7 apoptosis assay at 96 h. l, TP53 wild-type liver cancer cell lines (SK-Hep1 and Huh6) were treated with 10 μM XL413 or vehicle for 5-7 days prior to exposure to increasing concentrations of AZD8055. Apoptotic cells were visualized by caspase-3/7 apoptosis assay 96 h post-AZD8055 treatment. m, Control cells and XL413-induced senescent cells were treated with AZD2014 for 48 h. Western blot analysis was performed with the indicated antibodies (left panel) and the levels of p-S6RP and p-4EBP1 was normalized to total-S6RP and total-4EBP1, respectively (right panel), showing that AZD2014 treatment leads to strong mTOR signalling inhibition in XL413-induced senescent cells. For gel source images, see Supplementary Figure 1. Data in a-f are representative of three independent biological experiments. Data in h-m are representative of two independent biological experiments.