Figure 4.

Rapamycin suppresses IL1A signalling. (a) HCA2 cells were infected with lentiviruses expressing shRNAs against GFP (control) or raptor. Senescent (ionizing radiation; Sen (IR)) cells, treated with rapamycin (Rapa) or DMSO for 10 days after ionizing radiation exposure, were analysed by flow cytometry for cell-surface IL1A using a FITC-tagged antibody. The fluorescence signal was divided by the forward scatter signals to account for cell size variations; 10,000 flow cytometry events were recorded. Shown is the result of one of two independent experiments. (b) HCA2 cells infected with lentiviruses expressing GFP shRNA or IL1A shRNA were irradiated and treated with DMSO (D) or rapamycin (R); 7 days later conditioned media were collected and analysed by ELISA for IL6. (c) Proteins were extracted from DMSO- and rapamycin-treated senescent cells and analysed by western blotting for IRAK1, IκBα, phospho-S6 and S6 at the indicated intervals after ionizing radiation exposure. Recombinant (r) IL1A protein was added to one senescent (ionizing radiation) sample treated with rapamycin (right lane). Unprocessed original scans of blots are shown in Supplementary Fig. 9. Shown is one of two independent biological replicates; each replicate required multiple blots, which were probed at the same time. (d) Non-senescent (NS) and senescent (ionizing radiation; Sen (IR)) HCA2 cells were treated with DMSO or rapamycin for 6 d, after which rIL1A in serum-free medium was added for 24 h. Conditioned media were collected and analysed by ELISA for IL6. For b and d, shown is one representative of two independent experiments, each with triplicate samples. For raw data, see Supplementary Table 4.