Figure 4. Rapamycin protects from oxidative stress through increased MnSOD expression.

(A) Analysis of reactive oxygen species (ROS) levels by Dihydroethidium hydroethidine (DHE) staining 24 hours after radiation in NOK and HN12 cells pretreated or not (control) with rapamycin (upper panel) and FACS analysis of ROS levels by DHE staining in NOK after 10 or 40 days of continuous rapamycin treatment (lower panel). (B) Western blot of senescence (p16) and DNA damage (γH2AX) marker levels after 24 hs of H₂O₂ treatment in NOK pretreated or not (control) with rapamycin. (C) Western blot of DNA damage (γH2AX) marker levels after 24 hs of H₂O₂ treatment in the HNSCC HN12 cells pretreated or not (control) with rapamycin. (D) Western blot analysis of protein levels of ROS scavenging enzymes (MnSOD, Cu-ZnSOD and catalase) in NOK and HN12 cells after 72 hs of rapamycin treatment. mTOR inhibition by rapamycin is shown by the levels of pS6. (E) Quantification of MnSOD protein expression levels by western blot in NOK and HN12 cells after 72 hs of rapamycin treatment. (F) Western blot analysis of p16 and γH2AX levels in control (Con) or rapamycin (Rap) treated human primary NOK transfected with control siRNA (siCon) or siRNA targeted against MnSOD (siMnSOD). (G) Western blot analysis of MnSOD expression and colony forming efficiency from clonogenic assays of human primary NOK transfected with control siRNA (siCon) or siRNA targeted against MnSOD (siMnSOD). Cells were pretreated or not (control) with rapamycin for 72 hs after siRNA transfection, irradiated with 0 or 3 grays (Gy) and 24 hs later subjected to clonogenic assays. See also Figure S3.