DOI:.38/ncb2822 a MTC02 FAO cells EEA1 b +/+ MEFs /DAPI -/- MEFs /DAPI -/- MEFs //DAPI c HEK 293 cells WCE N M C P AKT TBC1D7 Lamin A/C EEA1 VDAC d HeLa cells WCE N M C P AKT Lamin A/C EEA1 VDAC Figure S1 Localization at the peroxisome. (a) Representative images of FAO cell showing endogenous (green) and MTC02 (mitochondria marker) or EEA1 (endosome marker) (red). (Scale bar - μm). (b) Representative images of +/+ and -/- MEFs showing endogenous (green) and (red). (Scale bar - μm). (c and d) Subcellular fractionation of HEK 293 (c) or HeLa (d) cells to separate nuclear (N), cytosolic (C), membrane (M), and peroxisome (P) fractions. Immuno-blot analyses were performed using antibodies to,,, TBC1D7 (HEK 293, Fig. ), AKT, catalase, and lamin A/C. The degree of enrichment for peroxisomes in fractionated lysates was evaluated by assessing markers for endosomes (EEA1), lysosomes () and mitochondria (VDAC). WCE whole cell extracts. Uncropped images of western blots are shown in Supplementary Fig.. WWW.NATURE.COM/NATURECELLBIOLOGY 1 13 Macmillan Publishers Limited. All rights reserved.
Ratio of /Actin Ratio of /Actin Ratio of /Actin SUPPLEMENTARY INFORMATION a GFP-LC3 MCF7 cells H 2 O 2 + + + + + + + Time (hr) 0 1 2 4 6 8 16 24 24 p p Actin b 6.0 * * * * * 4.0 *** * 2.0 NS 0.0 H 2 O 2 _ + + + + + + + _ Time (hr) 0 1 2 4 6 8 16 24 24 c light dark H 2 O 2 - + - + Baf A1 - - + + GFP-LC3 MCF7 cells d 2 ** ** NS NS 1.5 * ** 1 5 0.5 0 0 H 2 O 2 - + - + H 2 O 2 - + - + Baf A1 - - + + Baf A1 - - + + Actin e Atg5 +/+ MEFs Atg5 -/- MEFs H 2 O 2 - + - + Atg5 Figure S2 Induction of autophagy in response to ROS. (a) Western analyses of MCF-7 stably expressing GFP-LC3 cells treated with 0.4 mm H 2 O 2 for the indicated time with markers for autophagy ( and LC3), mtorc1 signaling (p (T389),, p (S235/236) and ). (b) Quantitation of the ratio of /Actin from Fig. S. (±s.e.m., n = 3 independent experiments). *p < 0.05, *** p < 0.001, NS, not significant. (c) Western analysis of GFP-LC3 MCF7 cells pre-incubated in the presence or absence nm Bafilomycin A1 (BafA1) for 1hr before treatement with 0.4 mm H 2 O 2 for 7hr using anti- and LC3 antibodies. (d) Quantitation of the ratio of /Actin and /Actin from Fig. S2c. (±s.e.m., n = 3 independent experiments). *p < 0.05, ** p < 0.01, NS, not significant. (e) Western analysis of Atg5 +/+ MEFs and Atg5 -/- MEFs treated with 0.4 mm H 2 O 2 for 24hr using anti- and Atg5 antibodies. Uncropped images of western blots are shown in Supplementary Fig.. Source data of statistical analysis are shown in Supplementary Table S1. 2 WWW.NATURE.COM/NATURECELLBIOLOGY 13 Macmillan Publishers Limited. All rights reserved.
Ratio of /Actin Ratio of /Actin SUPPLEMENTARY INFORMATION a H 2 O 2 (mm) p p p4ebp1 GM13427 (control) GM13269 (ZW) 0.0.05.1.2.4 1 0.0.05.1.2.4 1 b GM871 (control) GM13267 (ZW) H 2 O 2 - + - + - + - + Baf A1 - - + + - - + + LC3-I LC3-II Actin 4EBP1 patm ATM pampk AMPK (light) (dark) LC3-I LC3-II c 5 control ZW control ZW 4 ** ** 2.5 2 * * 3 1.5 * NS NS 2 *** ** ** 1 NS 1 0.5 *** 0 H 2 O 2 - - + - + - + + - - + - + - + H 2 O 0 2 + - - + - + - + + - - + - + - Baf A1 + Baf A1 + d Amino acids p p p4ebp1 4EBP1 GM13427 GM13269 (Control) (ZW) - + - + Figure S3 mtorc1 signaling and autophagy in Zellweger cells with ROS and amino acids. (a) Western analysis of human fibroblasts obtained from Zellweger (GM13269) or corresponding control patient with Ehlers-Danlos syndrome (GM13427) treated with indicated doses of H 2 O 2 for 1hr. mtorc1 signaling monitored by western analysis for p (T389),, p (S235/236),, p4ebp1 (T/46), 4EBP1, patm (S1981), ATM, pampk (T172), AMPK, and LC3. (b) Western analysis of Zellweger cells (GM13267) or control fibroblasts (GM871) cells pre-incubated in the presence or absence of nm Bafilomycin A1 (BafA1) for 1hr before treatment with 0.4 mm H 2 O 2 for 1hr using anti- and LC3 antibodies. (c) Quantitation of the ratio of /Actin and /Actin from Fig. S3b (±s.e.m., n = 3 independent experiments). *p < 0.05, ** p < 0.01, *** p < 0.001, NS, not significant. (d) Representative western analysis using cell extracts from human fibroblasts obtained from a Zellweger patient (GM13269) or control fibroblasts (GM13427) treated with amino acid free media for 60 min, and stimulated with amino acid containing media for min. mtorc1 signaling was monitored using anti-p (T389),, p (S235/236),, p4ebp1(t/46) and 4EBP1. Uncropped images of western blots are shown in Supplementary Fig.. Source data of statistical analysis are shown in Supplementary Table S1. WWW.NATURE.COM/NATURECELLBIOLOGY 3 13 Macmillan Publishers Limited. All rights reserved.
Fold change of p/ SUPPLEMENTARY INFORMATION a c Calnexin WT RQ RQ HeLa cells VDAC RQ HeLa cells RG d HEK 293 cells RW IgG Myc- L1624P WT RQ RG RW G294E IP: Myc b Myc- Input RQ HeLa cells RG e - HEK 293 cells Mock WT RQ 1.5 1 ** * RW p 0.5 mtor Myc- 0 Mock WT RQ mtor Figure S4 Localization of PEX5 binding mutants. (a) Representative images using HeLa cells transfected with Myc- and wild type (WT) and or the PxBS mutants (RQ, RG, RW) stained with (red) and (peroxisome marker, green). (Scale bar - μm). (b) Representative images using HeLa cells transfected with Myc- and mutants (RQ, RG and RW) stained with (green) and (marker for lysosome, red). As a control, the cells were stained by anti-mtor (green) and anti- (red). (Scale bar - μm). (c) Representative images using HeLa cells transfected with Myc- and mutant (RQ) stained with (red) and either calnexin (marker for endoplasmic reticulum, green) or VDAC (marker for mitochondria, green). (Scale bar - μm). (d) HEK 293 cells were transfected with Myc- and wild type (WT) or the mutants (RQ, RG, RW) or L1624P (GAP mutant) or G294E ( binding mutant). The lysates were immunoprecipitated using anti-myc and control IgG, and samples were analyzed using anti- and anti-myc antibodies. (e) Functional assays were performed using HEK 293 cells expressing -, myc-, and wild type (WT) or mutant (RQ). mtor signaling was monitored by measuring the ratio of p (T389) to level. Graph represents densitometric quantitation of the ratio of phospho- to total (±s.e.m., n = 3 independent experiments). *p < 0.05, ** p < 0.01. Uncropped images of western blots are shown in Supplementary Fig.. Source data of statistical analysis are shown in Supplementary Table S1. 4 WWW.NATURE.COM/NATURECELLBIOLOGY 13 Macmillan Publishers Limited. All rights reserved.
Figure S5 Model for TSC complex localization on peroxisomal membranes, and activation of the -- signaling node by peroxisomal ROS to repress mtorc1 and induce autophagy, or inactivation by AKT phosphorylation of, with subsequent binding of by 14-3-3 and sequestration in cytosol. WWW.NATURE.COM/NATURECELLBIOLOGY 5 13 Macmillan Publishers Limited. All rights reserved.
1e 2 1 1e 2 1 2 1 AKT TBC1D7 Lamin A/C EEA1 2 1 2 1 VDAC 2 1 2 1 Figure Original scans of Western blots. Black boxes indicate cropped region and are labeled for corresponding figure, panel and antibody 6 WWW.NATURE.COM/NATURECELLBIOLOGY 13 Macmillan Publishers Limited. All rights reserved.
AKT Lamin A/C EEA1 2 1 2 1 VDAC 2c 2 1 2c AKT 2c 2c 2c 2c 1 LDH β-integrin 2c 2c 2 1 Lamin A/C 2c Figure continued WWW.NATURE.COM/NATURECELLBIOLOGY 7 13 Macmillan Publishers Limited. All rights reserved.
3a (light) 2 1 3a (dark) 2 1 3a LDH 3a 3a 3b (light) 2 1 3b (dark) 2 1 LDH 3b 3b 3b Figure continued 8 WWW.NATURE.COM/NATURECELLBIOLOGY 13 Macmillan Publishers Limited. All rights reserved.
3c 3c p (S939) pakt (S473) 2 1 3c 3c 2 1 3c pakt (T308) LDH 3c 3d 2 1 3d 2 1 3d 3d 3d EHHADH 4e ACAA1 4e 4e p 4e Figure Con=nued Figure continued WWW.NATURE.COM/NATURECELLBIOLOGY 9 13 Macmillan Publishers Limited. All rights reserved.
p 4e 4e 4e 4e 4e p p p4ebp1 4EBP1 patm pampk (light) pampk (dark) 2 1 ATM AMPK 2 1 Figure Con=nued Figure continued WWW.NATURE.COM/NATURECELLBIOLOGY 13 Macmillan Publishers Limited. All rights reserved.
5c p 5c 5c 5c 5c 5d p 5d p4ebp1 5d p 5d 4EBP1 5d 5d 5d IP:PEX5 IP:PEX5 IP:PEX5 IP:PEX5 2 2 mtor 1 1 2 PEX5 1 IP:PEX5 IP:PEX5 (dark) (light) IP:PEX19 IP:PEX19 IP:PEX19 mtor 2 2 1 1 IP:PEX19 IP:PEX19 IP:PEX19 PEX1 2 1 PEX19 2 1 Figure Con=nued Figure continued WWW.NATURE.COM/NATURECELLBIOLOGY 11 13 Macmillan Publishers Limited. All rights reserved.
PEX5 IP: IP: IP: 2 6d 1 6d 6d Input 2 1 IP:PEX5 6d 2 1 IP:PEX5 PEX5 6d IP:PEX5 6d Input 2 1 6e 2 1 6e PEX5 6f 6f 2 1 6f Input 2 1 WCE M P 2 1 2 1 6g 2 1 β-integrin 6g 1-6h IP:PEX19 6h IP:PEX19 Input - 2 1 2 1 IP:PEX19 PEX19 6h 6i LDH 6g - 6i Lamin A/C 6i Figure Con=nued Figure continued 12 WWW.NATURE.COM/NATURECELLBIOLOGY 13 Macmillan Publishers Limited. All rights reserved.
PEX5 7a p (light) 7a p (dark) 7a 7a 7a p (light) 7a p (dark) 7a - 7b 7b 260 160 1 80 60 7b 30 - p 7b 80 60 260 160 1 7b 30 32 p 7b - 7c 30 260 160 1 7b 7b 4EBP1 p4ebp1(s65) 30-7d 2 1 7f 30 7c p 7d - 7f 260 160 1 HA- 7d 7f Figure continued WWW.NATURE.COM/NATURECELLBIOLOGY 13 13 Macmillan Publishers Limited. All rights reserved.
2 1 2 1 AKT TBC1D7 Lamin A/C EEA1 2 1 2 1 VDAC 2 1 2 1 AKT Lamin A/C EEA1 2 1 1 VDAC S S p S S Figure continued 14 WWW.NATURE.COM/NATURECELLBIOLOGY 13 Macmillan Publishers Limited. All rights reserved.
p S S Actin S S2c light S2c dark S2c Actin S2c Atg5 S2e S2e p p patm p4ebp1 4EBP1 ATM pampk 2 1 P62 (light) 2 1 AMPK Figure continued WWW.NATURE.COM/NATURECELLBIOLOGY 13 Macmillan Publishers Limited. All rights reserved.
P62 (dark) P62 S3b S3b S3b Actin p S3d S3d p S3d p4ebp1 4EBP1 S3d S3d S3d S4d S3d IP: Myc Myc- p S4e Input 80 60 2 1 IP: Myc S4d S4e IP: Myc S4d Input 2 1 2 S4d 1 - S4e Myc- S4e IP: Myc Myc- 2 1 2 1 Figure continued 16 WWW.NATURE.COM/NATURECELLBIOLOGY 13 Macmillan Publishers Limited. All rights reserved.
Supplementary Table 1 Statistics Source data WWW.NATURE.COM/NATURECELLBIOLOGY 17 13 Macmillan Publishers Limited. All rights reserved.