Supplementary Figure 2 : Peroxisomal activation of ATM signaling to AMPK-TSC2 to repress mTORC1 and induce autophagy.

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

Supplementary Figure 2

(a) FAO cells treated with clofibrate (0.25 mM) for 48 h activated PPARα to increase the mRNA level of EHHADH by RT–PCR analysis. The data shown is representative of two independent experiments. (b) FAO cells treated with clofibrate (0.25 mM) for 48 h activated PPARα to increase the ACAA1 mRNA levels as demonstrated by RT–PCR analysis. (mean ± s.d., n = 3 independent experiments, Student’s t test). P < 0.05, P < 0.01. (c) Western analysis of FAO cells treated with clofibrate at 0.25 mM for indicated times with ACAA1 and EHHADH antibodies. (d) MCF7 cells stably transfected with GFP–LC3 were treated with clofibrate (0.25 mM) for indicated times, and activation of ATM-AMPK-TSC2 signaling and induction of autophagy monitored by western analysis using pATM (S1981), ATM, pAMPK (T172), AMPK, pACC (S79), ACC, pS6K (T389), S6K, pS6 (S235/236), S6, p62 and LC3. (e) Representative images of GFP–LC3 puncta from MCF7 cells stably expressing GFP–LC3 and treated with clofibrate (0.25 mM) for 6 h. Scale bar, 10 μm. (f) HepG2 cells treated with 1 mM clofibrate for indicated times, were monitored for ATM-AMPK-TSC2 signaling, suppression of mTORC1, and induction of autophagy by western analysis using pATM (S1981), ATM, pAMPK (T172), AMPK, pACC (S79), ACC, pS6K (T389), S6K, pS6 (S235/236), S6, p4E-BP1 (T37/46), 4E-BP1, pULK1 (S757), pULK1 (S317), ULK1, p62 and LC3. (g) TSC2+/+ and TSC2−/− MEF cells were treated with clofibrate for 6 h at the indicated doses, and suppression of mTORC1 monitored by western analysis using pS6 (235/236), and S6. (h) Western analysis of FAO cells pre-incubated in the presence or absence of Bafilomycin A1 (Baf A1, 200 nM) for 1 h before treatment with 1 mM clofibrate for 3 h using anti-p62 and LC3-II antibodies. Uncropped images of western blots are shown in Supplementary Fig. 9, statistic source data for Supplementary Fig. 2a, b can be found in Supplementary Table 1.