Figure 7.

Sustained effect of rapamycin on the SASP. (a) HCA2 cells were treated with rapamycin (Rapa) for 1 day immediately after ionizing radiation exposure, and conditioned medium was collected 6 days later and analysed for IL6 secretion by ELISA; shown is one representative of two independent experiments, each with triplicate cell culture samples. (b) The amount of rapamycin was measured in non-senescent (NS) and senescent (ionizing radiation; Sen (IR)) HCA2 cells 1, 3, 7, 15 and 31 days after acute treatment (single 1-day dose immediately after ionizing radiation exposure). Rapamycin was measured by high-performance liquid chromatography at the Biological Psychiatry Laboratories Services of the University of Texas Health Sciences Center San Antonio. Six samples were collected and pooled in groups of two for each condition. (c) Western blot analysis of phospho-S6 and S6 using proteins extracted 1, 3, 7 and 15 days after acute treatment (as in a) of HCA2 cells with rapamycin (one representative or two representative experiments is shown). Unprocessed original scans of blots are shown in Supplementary Fig. 9. (d) IL6 secretion by X-irradiated (IR) senescent HCA2 cells at day 7, 13, 19 and 25 after rapamycin treatment, relative to non-senescent (NS) secretion; shown is one representative of two independent experiments, each with triplicate cell culture samples. (e) SA-β-gal activity in non-senescent and senescent cells treated with DMSO and rapamycin was determined by light microscopy 7 or 25 days after treatment (one representative of two representative independent experiments is shown). For raw data, see Supplementary Table 4.