Figure 2.

SASP production is MTOR dependent. (a) HCA2 cells were infected with lentiviruses expressing GFP shRNA (control) or one of three different shRNAs targeting raptor. Cells were then irradiated; 7 days later, conditioned media were collected and analysed by ELISA for IL6. (b) HCA2 cells were infected with lentiviruses expressing GFP shRNA (control) or one of three different shRNAs targeting MTOR. Cells were then irradiated; 7 days later, conditioned media were collected and analysed by ELISA for IL6. (c) IL8 was quantified by ELISA in conditioned media from PSC27 cells in which S6K was depleted by siRNA or 4EBP1 overexpressed by transfection with a 4EBP1-encoding plasmid. Cells treated with rapamycin (Rapa) are shown for comparison. (d) After inducing senescence by ionizing radiation, protein extracts from PSC27 cells transfected with S6K siRNA or trans-4EBP1, or treated with rapamycin were analysed by western blotting for the indicated SASP factors. Unprocessed original scans of blots are shown in Supplementary Fig. 9. (e) Extracts from non-senescent (NS) or senescent (ionizing radiation; Sen (IR)) HCA2 cells, treated or not with rapamycin, were assayed for the indicated proteins by western blotting. β-actin served as the loading control. Unprocessed original scans of blots are shown in Supplementary Fig. 9. For a–c, shown is one representative of two independent experiments, each with triplicate samples. For d and e shown is one representative of two independent biological replicates; each replicate required multiple blots, which were probed at the same time. For raw data, see Supplementary Table 4.