Figure 6.

Rapamycin does not reverse cellular senescence. (a) Senescent (ionizing radiation; Sen (IR)) HCA2 cells were treated with DMSO or rapamycin (Rapa) for 6 days, and the percentage of cells expressing SA-β-gal was determined by light microscopy (right panel) and counting (left panel). (b) Cells were treated with rapamycin or DMSO for 28 days following ionizing radiation exposure, washed, and incubated in drug-free media for 14 days, at which point cell numbers were determined. (c) HCA2 cells, non-senescent (NS) or made senescent by ionizing radiation exposure and treated with DMSO or rapamycin for 6 days, were pulsed with BrdU for 24 h and the fraction that incorporated BrdU was determined by fluorescence microscopy. (d) Clonogenic assays comparing the effects of chronic treatment with rapamycin or PP242 (500 nM) of non-senescent (NS) and senescent (ionizing radiation; Sen (IR)) HCA2 cells. Cells were plated and drugs were added at the indicated times before or after ionizing radiation exposure and cells were cultured for 10–14 days (one representative experiment shown). (e) HCA2 cells were infected with a control lentivirus (L3P) or lentivirus carrying oncogenic RAS. Cell were grown in drug for three days (acute) and released or continuously treated (chronic) for 10–14 days after which clonogenic staining was performed. (f) HCA2 cells were co-infected with RAS and lentiviruses carrying shRNAs to deplete the indicated proteins. Transcripts for IL6 were quantified by qPCR. (g,h) HCA2 cells were infected with lentiviruses carrying shRNAs to deplete the indicated proteins. Clonogenic assays were performed on the infected cells induced to senesce by oncogenic RAS (g) or ionizing radiation (h) (one representative experiment is shown). (i) HCA2 cells were infected with a lentivirus carrying shRNAs against GFP (control) or raptor, induced or not (NS) to senesce by 10 Gy ionizing radiation (IR) or 250 nM doxorubicin (Doxo) for 24 h and treated with DMSO or rapamycin. Cells were cultured for 10–14 days and clonogenic staining was performed using crystal violet (one representative experiment is shown). (j) Cells were treated as in i. Seven days after senescence induction, conditioned medium was collected and analysed for IL6 by ELISA. For a–c, f and j shown is one representative of two independent experiments, each with triplicate cell culture samples. For d, e, g–i, shown is one representative clonogenic assay experiment replicated once. For raw data, see Supplementary Table 4.