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

From: ATM functions at the peroxisome to induce pexophagy in response to ROS

Figure 3

(a) Representative images of FAO cells treated with vehicle (dimethylsulphoxide) or 0.25 mM clofibrate for 1 h; superoxide production was detected using dihydroethidium (DHE) staining. Scale bars, 30 μm. (b) Representative data of the 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,e) FAO cells treated with 1 mM clofibrate for the indicated time points; activation of ATM–AMPK–TSC2 signalling and suppression of mTORC1 was monitored by western analysis for pATM (Ser 1981), ATM, pS6K (Thr 389), S6K, pS6 (Ser 235/236), S6, p4E-BP1 (Thr 37/46), 4E-BP1, pAMPK (Thr 172), AMPK, pACC (Ser 79), ACC, pULK1 (Ser 757, mTORC1 site), pULK1 (Ser 317, 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 (Thr 389), S6K, pS6 (Ser 235/236), S6, p4E-BP1 (Thr 37/46) and 4E-BP1 antibodies. (g) FAO cells treated with clofibrate (1 mM) for the indicated times. Induction of autophagy was monitored by western analysis of p62 and LC3-II. Unprocessed original scans of blots are shown in Supplementary Fig. 9. Source data for b can be found in Supplementary Table 1.