Figure 7 : Induction of pexophagy by ROS.

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

Figure 7

(a) HepG2 cells treated with either H2O2 (0.4 mM) for the indicated times or CCCP (50 μM, 6 h). Western analysis of the peroxisome proteins PEX1 and PEX14 (SDHA and VDAC as mitochondrial markers, p62 and LC3-II as autophagy markers). (b) Quantification of PEX1 and PEX14 intensity normalized to GAPDH from a (mean ± s.d., n = 3 independent experiments, Student’s t-test). P < 0.05 and P < 0.001; NS, not significant. (c) Representative images using HepG2 cells transfected with an mRFP–EGFP–SKL construct and treated with H2O2 for 6 h. Scale bars, 10 μm. High-magnification images of the outlined areas are indicated to the right (scale bars, 2.5 μm). (d) Pearson’s correlation coefficient for co-localization between mRFP–SKL and EGFP–SKL was calculated from c. n = 4 independent experiment; 10 cells were analysed in each experiment. All error bars represent s.d., (Student’s t-test) P < 0.001. (e) HEK293 cells transfected with Myc–PEX5-WT or Myc–PEX5-K209R or Myc–PEX5-Ser141 for 24 h following prior siRNA knockdown of PEX5 for 48 h, treated with H2O2 (0.4 mM) for 6 h. Western analysis was performed using anti-PEX1, PEX14, phosphor-ATM (Ser 1981), ATM, Myc and GAPDH antibodies. Corresponding immunoblots for HEK293 cells transfected with control or PEX5 siRNA showing levels of PEX5. (f) Quantification of PEX1 and PEX14 intensity normalized to GAPDH from e. (Mean ± s.d., n = 4 independent experiments, Student’s t-test.) P < 0.001; NS, not significant. (g) WT (GM08399) and AT (GM05849) fibroblasts were treated with H2O2 (0.4 mM) for 6 h and immunoblotted with PEX1, PEX14, phospho-ATM, ATM and GAPDH antibodies. (h) Representative images using WT (GM08399) and AT (GM05849) fibroblasts transfected with an mRFP–EGFP–SKL construct and treated with H2O2 for 6 h. Scale bars, 15 μm. (i) Pearson’s correlation coefficient for co-localization between mRFP–SKL and EGFP–SKL was calculated from h. Quantification was performed from Supplementary Fig. 6a on n = 3 independent experiments; 10 cells were analysed in each experiment. All error bars represent s.d., (Student’s t-test) P < 0.01; NS, not significant. Unprocessed original scans of blots are shown in Supplementary Fig. 9. Statistic source data for b,d,f and i can be found in Supplementary Table 1.