Extended Data Fig. 7. Inhibition of S6K1 and S6K2 in IMR90 ER:RAS fibroblasts undergoing RAS-induced senescence.
a. Schematic depicting LY2584702 inhibiting both S6K1 and S6K2. b. IMR90 ER:RAS cells were treated with or without 4OHT in the presence of DMSO, LY2584702 (0.2-2 μM) or Torin1 (25 nM). Quantification of immunofluorescence staining for phosphorylated ribosomal protein S6S240/S244. n = 3 biological replicates of a single experiment. c-e. Quantification (c-d) and representative immunofluorescence images (e) for phosphorylated ribosomal protein S6S240/S244 and phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) following 7 days of treatment with or without 4OHT in the presence of DMSO, LY2584702 (2 μM) or Torin1 (25 nM). n = 3 independent experiments. f. IMR90 ER:RAS cells were treated with or without 4OHT in the presence of DMSO or LY2584702 (2 μM). Cell proliferation was assessed by colony formation assay (crystal violet staining) following 13 days in culture. g-h. IMR90 ER:RAS cells were treated with or without 4OHT in the presence of DMSO, LY2584702 or Torin1. Quantification of IF staining for BrdU incorporation (g) or senescence-associated beta-galactosidase (SA-β-Gal, h) following 7 days in culture. n = 3 independent experiments. i-j. IMR90 ER: RAS cells were treated with or without 4OHT in the presence of DMSO, LY2584702 or Torin1. Relative mRNA expression for IL1A (i) and IL1B (j) was assessed by RT-qPCR following 6 days of 4OHT treatment. mRNA expression was normalized to the Rps14 housekeeping gene. n = 3 independent experiments. Data are expressed as mean ± SEM. Statistical significance was calculated using one-way analysis of variance with Dunnett’s multiple comparison test. Scale bar, 100 μm.