Extended Data Fig. 6: Sertraline selectively induces apoptosis in XL413-induced senescent cells through the suppression of mTOR signalling.

a, Schematic of the GPCR-compound screen. Huh7 cells were treated with 10 μM XL413 for 5 days before seeding in 96-well plates. All compounds were tested at four concentrations for six 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 per cent 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 that induce 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 compounds on XL413-treated and untreated cells are 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 and caspase-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 assays. 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 proteins of the mTOR signalling pathway were performed. h, Hep3B and Huh7 cells were treated with 10 μM XL413 for 10 days before sequential treatment with sertraline (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 that were sequentially treated with XL413 and sertraline (Methods). i, j, Liver cancer cells with TP53 mutations (SNU449 and PLC/PRF/5) and lung-cancer cell lines with TP53 mutations (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 and caspase-7 apoptosis assay 96 h after treatment with AZD8055. k, Liver cancer cells with TP53 mutations (Hep3B and Huh7) 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 and mTORC2 inhibitor AZD2014. Apoptotic cells were visualized by caspase-3 and caspase-7 apoptosis assay 96 h after treatment with AZD2014. l, Liver cancer cell lines with wild-type TP53 (SK-Hep1 and Huh6) were treated with 10 μM XL413 or vehicle for 5–7 days before exposure to increasing concentrations of AZD8055. Apoptotic cells were visualized by caspase-3 and caspase-7 apoptosis assay 96 h after treatment with AZD8055. 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) and the levels of phosphorylated S6RP and phosphorylated 4EBP1 were normalized to the total levels of S6RP and 4EBP1, respectively (right); this shows that treatment with AZD2014 leads to strong inhibition of mTOR signalling in XL413-induced senescent cells. For gel source images, see Supplementary Fig. 1. Data in a–f are representative of three independent biological experiments. Data in h–m are representative of two independent biological experiments.