Extended Data Fig. 6. Confirmation of S6K1/2 depletion or inhibition in IMR90 ER: RAS fibroblasts.
IMR90 fibroblasts were stably transduced with the pLNC-ER:RAS retroviral vector and treated with 4-hydroxytamoxifen (4OHT) for senescence induction. a-c. IMR-90 ER:RAS cells were reverse transfected with either Allstars (scrambled sequence - siControl) or the indicated siRNAs. Cells were treated with or without 4OHT on the following day to induce senescence. Relative mRNA expression for S6K1 (a), S6K2 (b) and Il6 (c) was assessed by RT-qPCR following 4 days of 4OHT treatment with the indicated siRNAs in IMR90 ER: RAS cells. S6K1 and S6K2: n = 3 for all conditions. IL6: n = 4 for siControl, siS6K1_2, siS6K1_4, siS6K2_4 and siS6K2_5 and n = 3 for siS6K1/2 and siC/EBPβ. mRNA expression was normalized to the Rps14 housekeeping gene. d-e. Quantification (d) and representative immunofluorescence images (e) for phosphorylated ribosomal protein S6S240/S244 staining of the indicated cells (n = 2). Data are expressed as mean ± SEM. Statistical significance was calculated using one-way analysis of variance with Tukey’s multiple comparison test. Scale bar, 100 μm.