Supplementary Figure 7 : Regulation of pexophagy by ROS is ATM-dependent.

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

Supplementary Figure 7

(a) Frame capture from time course imaging of pexophagy in live RPE cells stably expressing GFP–LC3 and DsRed-SKL following H2O2 (0.4 mM) treatment. Scale bar, 10 μm. (b) Western analysis of HepG2 cells pre-incubated in the presence or absence of Bafilomycin A1 (Baf A1, 200 nM) for 1 h before treatment with 0.4 mM H2O2 for 3 h using anti-PEX1, anti-PEX14, anti-p62 and LC3-II antibodies. (c) Quantification of PEX1 and PEX14 intensity normalized to GAPDH from Supplementary Fig. 7b (mean ± s.d., n = 3 independent experiments, Student’s t test). P < 0.05, P < 0.01, P < 0.001, NS = not significant. (d) Western analysis of whole cell extracts (WCE) and peroxisome fractions (P) of HepG2 cells treated with Baf A1 (200 nM) for 4 h immunoblotted using anti-ATM, anti-pPEX5(S141) and PEX5 antibodies. (e) Western analysis of HepG2 cells transfected with or without sip62 for 72 h and treated with 0.4 mM H2O2 for 3 h using anti-PEX1, anti-PEX14, anti-pATM, anti-ATM, anti-p62 and LC3-II antibodies. (f) Quantification of PEX1 and PEX14 intensity normalized to GAPDH from Supplementary Fig. 7e (mean ± s.d., n = 3 independent experiments, Student’s t test). P < 0.01, P < 0.001, NS = not significant. (g) Representative images of wild type (WT, GM08399) and ataxia telengiectasia (AT, GM05849) patient fibroblasts treated with H2O2 for 6 h, and immunostained with PEX14 antibody. Scale bar, 15 μm. (h) Quantification of PEX14 puncta per cell was performed from Supplementary Fig. 7g from n = 4 independent experiments, 100 cells were analyzed in each experiment. All error bars represent s.d., (Student’s t test) P < 0.001, NS = not significant. (i) Western analysis of AT (GM05849) fibroblasts transfected with Flag-ATM WT or Flag-ATM KD (kinase dead) mutant and treated with 0.4 mM H2O2 for 6 h using anti-PEX1, anti-PEX14, anti-pATM, anti-Flag and GAPDH antibodies. (j) Western analysis of HEK293 cells transfected with Pex2/10/12 siRNA for 72 h and treated with H2O2 (0.4 mM) for 6 h using anti-PEX1, anti-PEX14, anti-pATM, ATM and GAPDH antibodies. Uncropped images of western blots are shown in Supplementary Fig. 9. Statistic source data for Supplementary Fig. 7c, f, h can be found in Supplementary Table 1.