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. Author manuscript; available in PMC: 2016 Apr 1.
Published in final edited form as: Nat Cell Biol. 2015 Sep 7;17(10):1259–1269. doi: 10.1038/ncb3230

Figure 3. Peroxisomal ROS activates ATM to repress mTORC1 and induce autophagy.

Figure 3

(a) Representative images of FAO cells treated with vehicle (DMSO) or 0.25 mM clofibrate for 1 h, superoxide production was detected using dihydroethidium (DHE) staining. Scale bar, 30 μm. (b) Representative data of DCFDA assay from two independent experiments depicting the levels of ROS in FAO cells treated with clofibrate at the indicated doses for 1 h. Tert-butyl hydroperoxide (TBHP) 50 μM was used as a positive control for the assay. (c) ATM activation was monitored at the peroxisome in response to clofibrate at 3 h (1 mM) as indicated by western blot analysis of whole cell extract (WCE) and peroxisomal fractions (P) obtained from FAO cells. (d and e) FAO cells treated with 1 mM clofibrate for the indicated time points, activation of ATM-AMPK-TSC2 signaling and suppression of mTORC1 was monitored by western analysis for p-ATM (S1981), ATM, pS6K (T389), S6K, pS6 (S235/236), S6, p4E-BP1 (T37/46), 4E-BP1, pAMPK (T172), AMPK, pACC (S79), ACC, pULK1 (S757, mTORC1 site), pULK1 (S317, AMPK site), and ULK1. (f) Western analysis of HEK293 cells transfected with control or ATM siRNA and treated with 0.5 mM clofibrate for 6 h using anti-pS6K (T389), S6K, pS6 (S235/236), S6, p4E-BP1 (T37/46) and 4E-BP1 antibodies. (g) FAO cells treated with clofibrate (1mM) for indicated times. Induction of autophagy was monitored by western analysis of p62 and LC3-II. Uncropped images of western blots are shown in Supplementary Fig. S9, Source data for Fig. 3b can be found in Supplementary Table 1.