EMBO/soda-nel huggingface dataset:
DNA activates the Nse2/Mms21 SUMO E3 ligase in the Smc5/6 complex (2018)
Figure 3

Figure 3. A positive-patch region on the surface of Smc5 ARM domain interacts with DNA.

(A) Western blot of the SUMO conjugation reaction in the presence of different oligonucleotides using the minimal Smc5/ARM-Nse2 construct. Reactions were run at 30°C and stopped after 60 minutes by adding SDS-loading buffer. 25b (μM), 34b (μM) and 50b (μM), stands for a 25, 34 and 50 bases oligonucleotides, respectively, and the indicated concentration is in μM units. 5kb (nM), stands for the virion ϕx174 and the indicated concentration is in nM units (N-S1, cNse4-SUMO1; N-2S1, cNse4-2SUMO1; and pS1, poly-SUMO1) (*Overexposed chemiluminescent signal).

(B) Left, Ubc9-thioester formation in the presence of E1, E2 enzymes, Alexa488-SUMO1 and ATP. Right, single turnover reaction of the SUMO conjugation reaction in the presence or absence of ssDNA (50b) using Arm/Smc5-Nse2 as E3. Samples were run in the presence (below) or absence of β-mercaptoethanol (above).

(C) Ribbon representation of the complex between the ARM domain of Smc5 (yellow and orange) and Nse2 (pink) (PDB 3HTK) (Duan et al, 2009). Lysine residues forming the positive-charged patch in the surface of the coiled-coil Smc5 ARM are labeled and shown in stick representation (blue). Zinc atom in the Nse2 RING domain is depicted as a yellow sphere.

(D) Bar diagram representation of the SUMO conjugation rates of activity assays of Arm/Smc5-Nse2 KE mutants in the presence (orange bars) or absence (red bars) of ssDNA (virion ϕx174), relative to wild-type (set to 1). Reactions rates were performed at least in three different independent experiments. Data values are mean ± s.e.m. and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. *P<0.05, **P<0.01, ***P<0.001.

(E) DNA binding properties of wild-type, K337E/K344E/K764E and K743E/K745E Arm/Smc5-Nse2 mutants, were determined by electrophoretic mobility shift assays (EMSA) saturation experiments. Protein complexes were incubated for 30 minutes at 30°C before loading the agarose gel electrophoresis. Numbers above gel indicate the molar ratio (x103) of protein over ssDNA (virion ϕx174) in each lane.
DNA activates the Nse2/Mms21 SUMO E3 ligase in the Smc5/6 complex (2018)
Figure 4

Figure 4. Binding of DNA induces distinct conformational changes in Arm/Smc5-Nse2 wild-type and mutants.

(A) Left, circular dichroism (CD) analysis of purified wild-type Arm/Smc5-Nse2. Black line, wild-type Arm/Smc5-Nse2 without bound ligand (no 5kb ssDNA); dashed black line, denatured protein; blue line, ligand/Smc5-Nse2; numbers in graphs (dotted black lines), molar ratio of ligand/Smc5-Nse2. Right, conformational changes induced by 5kb circular ssDNA quantified by ligand titration until signal change in mean residue ellipticity (MRE) at 222 nm achieved saturation. Blue squares Arm/Smc5-Nse2 (wild-type); grey triangles Arm/Smc5-Nse2 (K333E/K344E); green circles Arm/Smc5-Nse2 (K764E); pink triangles Arm/Smc5-Nse2 (K333E/K344E/K764E); red diamonds Arm/Smc5-Nse2 (K743E/K745E).

(B) Left, tryptophan intrinsic fluorescence of wild-type Arm/Smc5-Nse2. Black line in each panel, native Arm/Smc5-Nse2 without bound DNA; blue line, 5kb circular ssDNA/Smc5-Nse2; pink line, 50b linear ssDNA/Smc5-Nse2; and green line, 25b linear ssDNA/Smc5-Nse2. Right, conformational changes of wild-type Arm/Smc5-Nse2 induced by different types of DNA quantified by changes in center of mass (CM, red shift) of fluorescence spectra upon titration of the ligand until signal change achieved saturation. Blue circles, 5kb circular ssDNA/Smc5-Nse2; pink diamonds and 50b linear ssDNA/Smc5-Nse2. Inset, titration curves of 5kb circular ssDNA/Smc5-Nse2 reactions containing 0 or 100mM NaCl (solid and hollow symbols). Below, table of the dissociation constants of the curves.

(C) Degree of conformational changes in CM induced by 60b linear ssDNA. Blue bar, Arm/Smc5-Nse2 (wild-type); grey bar, Arm/Smc5-Nse2 (K333E/K344E); green bar, Arm/Smc5-Nse2 (K764E); pink bar, Arm/Smc5-Nse2 (K333E/K344E/K764E); red bar, Arm/Smc5-Nse2 (K743E/K745E). Reactions were performed at least in three different independent experiments. Data values are mean ± s.d. and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. ****P < 0.0001.
DNA activates the Nse2/Mms21 SUMO E3 ligase in the Smc5/6 complex (2018)
Figure 5

Figure 5. A positively charged patch in Smc5 is required for DNA repair in vivo. (A) Ribbon representation of the complex between the ARM domain of Smc5 (yellow and orange) and Nse2 (red) (PDB 3HTK) (Duan et al, 2009), showing positions of mutated lysine residues covered with black asterisks (3KE: K743E/K745E/K764E; 4KE: K337E/K344E/K354E/K355E; and 7KE: K337E/K344E/K354E/K355E/K743E/K745E/K764E).

(B) Growth test analysis of wild-type, nse2-CH and the indicated smc5-KE mutants; 10-fold serial dilutions of the liquid cultures were spotted in YPD and pictures taken after 48 hours.

(C) Same as in (B) but using the nse2-CH, smc5-3KE, smc5-K743,745E, smc5-K743,745R, smc5-7KE and smc5-7KR mutants.
DNA activates the Nse2/Mms21 SUMO E3 ligase in the Smc5/6 complex (2018)
Figure 6

Figure 6. A DNA sensor in Smc5 participates in sumoylation in vivo.

(A) Protein extracts from exponentially growing wild-type, smc5-3KE, smc5-K743,745E, smc5-4KE and smc5-K743,745R cells were prepared under denaturing conditions; 6xHis-Flag tagged SUMO (HF-SUMO) was pulled down from protein extracts to purify sumoylated species and analyzed by western blot. A strain with no HF-tag was used as control; arrow points to unmodified form of the protein, vertical bar to sumoylated forms.

(B) Left, same as in (A), but using wild-type and two independent clones of smc5-7KE; right, same as in (A), but using wild-type, smc5-7KE and smc5-7KR mutant cells.

(C) Quantification of Smc5 sumoylated species in pull downs from the indicated smc5-KE mutants, relative to wild-type controls from at least three independent experiments. Boxes, 25-75% data range; whiskers, total data range; black bar, median; grey cross, mean. ****P < 0.0001; ***P<0.001; **P < 0.01; *P<0.05. 1-way ANOVA, n = number of samples analyzed.

(D) Co-immunoprecipitation analysis of the Smc5-Nse2 interactions. NSE2-6HA cells were transformed with centromeric plasmids expressing the indicated SMC5 alleles, and protein extracts from exponentially growing cells subjected to anti-HA immunoprecipitation. A strain with no HA tag was used as control.

(E) Same as in (D), but using SMC6-6HA cells.

(F) Quantification of Smc5-6HA signal from immunofluorescence on chromosome spreads prepared from exponentially growing cultures of the indicated genotypes. The mean value on wild-type spreads was arbitrarily set to 1; each dot represents one nucleus; red line, median. 1-way ANOVA, n=number of nuclei analyzed. ****P < 0.0001.
BRCA1/BARD1 mediate apoptotic resistance but not longevity upon mitochondrial stress in C. elegans (2018)
Figure 1

Figure 1 - Pro-longevity mitochondrial stress reduces germ cells proliferation and apoptosis in basal conditions.

A, B Quantification of mitotic (A) and meiotic (B) cells in the distal germline in wild-type animals fed bacteria transformed with either empty vector (con) or vector-expressing dsRNA against frh-1 (frh-1) or isp-1 (isp-1).

C-E Quantification of dissected distal germline from wild-type animal stained either anti-phosphohistone H3 (anti-PH3) (C), anti-CDK-1 (anti-CDK-1) (D) or 5-ethynyl-2′-deoxyuridine (EdU -green) (E) relative to the total number of mitotic cells of wild-type animal.

F Average number of apoptotic corpses in the meiotic compartment relative to the total number of meiotic cells in wild-type worms fed as in (A).

Data information: Data are presented as mean ± SEM, (A, B, F) or as mean ± SD (C-E). (A) N=3 at least 5 worms per replicate and condition, (B) N=3 at least 10 worms for condition (C-E) N=2 at least 5 worms per replicate and condition, (F) N=5 at least 10 worms per replicate and condition. * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey's multiple comparisons test).
BRCA1/BARD1 mediate apoptotic resistance but not longevity upon mitochondrial stress in C. elegans (2018)
Figure 2

Figure 2 - Mitochondrial stress protects against DNA-damage induced apoptosis.

A-D Quantification of apoptotic corpse in the meiotic compartment of CED-1::GFP (A, B, D) or wild-type strains (C) relative to the total number of meiotic cells, fed bacteria transformed with either empty-vector (con) or vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated with gamma radiation 125 Gy (A), UVC 400J/m2 (B), different doses of UVB (C) and 100µM cisplatin.

Data information: Data are presented as mean ± SEM. For each panel at least N=3 and 10 worms for replicate and condition. * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ## <0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey's multiple comparisons test.
BRCA1/BARD1 mediate apoptotic resistance but not longevity upon mitochondrial stress in C. elegans (2018)
Figure 3

Figure 3 - Mitochondrial stress does not impair the DNA-damage checkpoint.

A, B Time course quantification of apoptotic corpses relative to the total number of meitic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 (A) or isp-1 (B), and left untreated or exposed to 1200 J/m2 UVB. Apoptotic cells were counted 12, 24 and 36 hours after exposure.

C-F Real Time PCR gene expression analysis of egl-1 (C-D) and ced-13 (E-F) genes, performed in wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated with gamma radiation (C, E) or with UVB (D, F).

Data information: Data are presented as mean ± SEM, N=3 (A, B) N=2 (C, E), N=4 (D, F). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey's multiple comparisons test.
BRCA1/BARD1 mediate apoptotic resistance but not longevity upon mitochondrial stress in C. elegans (2018)
Figure 4

Figure 4 - Mitochondrial stressed animals overcome DNA-damage checkpoint.

A, B Representative fluorescence pictures and quantification (below the fluorescence pictures) of dissected adult gonads relative to the number of mitotic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 and stained with DAPI (blue) and either anti-PARP-1 (red) (A) or anti-RPA-1 (green) and anti-RAD-51 (red) antibodies and treated with gamma radiation (125 Gy) (B) (untreated animals displayed no staining of RPA-1 and RAD-51, not shown). Scale bar: 20µm (A), 15µm (B)

C, D Quantification of mitotic cells in wild-type animals fed as (A) and left untreated or treated with gamma radiation 125 Gy (D) or with 1200 J/m2 UVB (E).

Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and condition.
BRCA1/BARD1 mediate apoptotic resistance but not longevity upon mitochondrial stress in C. elegans (2018)
Figure 5

Figure 5 - Moderate mitochondrial stress promotes DNA-damage protective responses

A, B Quantification to the number of mitotic cells of dissected gonads from wild-type animals stained with either anti-phosphohistone H3 (PH3) (A), anti-CDK-1 (CDK-1) (B) transformed with either empty vector (con) or vector-expressing dsRNA against frh-1 (frh-1) or isp-1 (isp-1) and treated with gamma radiation 125 Gy. Quantification is expressed as fold changes of positive stained cells treated over the positive untreated cells.

C-H Fertility (C-E) and fecundity (F-H) quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, and left untreated or treated with gamma radiation (C, F), UVB (D, G) or Hydroxyurea (E, H) at the indicated doses.

Data information: Data are presented as mean ± SD (A, B) or as mean ± SEM (C-H). N=2 (A, B), N=3 (C-H) * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. One-way ANOVA Tukey's multiple comparisons test (A, B), 2-way ANOVA Tukey's multiple comparisons test (C-H).
BRCA1/BARD1 mediate apoptotic resistance but not longevity upon mitochondrial stress in C. elegans (2018)
Figure 6

Figure 6 - BRC-1/BRD-1-dependent and -independent germline responses to mitochondrial-stress.

A Quantification of BRD-1 protein and representative western blot from wild-type worms fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1.

B Quantification of mitotic nuclei in single gonad arm of wild-type (WT), brc-1(tm1145) and brd-1(gk297) strains fed as in (A).

C, D Quantification of apoptotic corpse in wild-type (WT), brc-1(tm1145) and brd-1(gk297) strains fed as in (A) and left untreated (C) or treated with ionizing radiation 125 Gy (IR) (D). Bar graphs are mean ± SEM. Data information: Data are presented as mean ± SD N=2 (A,B) or mean ± SEM N=4 (C,D), 8-10 worms per replicate and condition, * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; ### P<0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey's multiple comparisons test (C-D).
BRCA1/BARD1 mediate apoptotic resistance but not longevity upon mitochondrial stress in C. elegans (2018)
Figure 7

Figure 7 - BRC-1 and BRD-1 do not mediate mitochondrial-stress extension of lifespan.

A-E Kaplan-Meier survival curves of wild-type (WT) and, ced-9(n1653ts) (A), brc-1(tm1145) (B), brd-1(gk297) (C), glp-1(e2141) (D), glp4(bn2) (E), strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1.

F, G Kaplan-Meier survival curves of wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against isp-1 (F) or isp-1 (G) and left untreated or treated with 600, 1200 or 2400 J/m2 UVB. Comparison between curves was done using the log-rank test and the summary of the statistic is reported in Table 1.
BRCA1/BARD1 mediate apoptotic resistance but not longevity upon mitochondrial stress in C. elegans (2018)
Figure 8

Figure 8 - Mitochondrial stress mediates germline apoptosis and lifespan through different cell non-autonomous mechanisms.

A Kaplan-Meier survival curves of wild-type (WT) and, rrf-1(pk1417), ppw-1(pk2505) strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against isp-1. Comparison between curves were done using the log-rank test and the summary of the statistic is reported in Table 1.

B Quantification of apoptotic corpses in wild-type (WT) and, rrf-1(pk1417), ppw-1(pk2505) strains fed as (A) and left untreated or treated with 600 J/m2 UVB.

Data information: Data are presented as mean ± SEM, N=3, 9-10 worms per replicate and condition ** P < 0.01 vs con; ## P < 0.01 vs WT; ooo P < 0.00 and oooo P < 0.0001 vs untreated . Not significant if nothing is specified. 2-way ANOVA Tukey's multiple comparisons test.

C In the wild-type animals under normal conditions a signal from the germline limits lifespan.

D Systemic mitochondrial stress concurrently triggers a pro-longevity signal from the soma (independent from the germline) and a cell-autonomous germline signal partially inhibiting the limiting signal from the gonad, ultimately resulting in significant lifespan extension in the wild-type and further extending lifespan in germline less strains. Moreover, it induces a soma-to-germline signal that protects against IR-induced apoptosis in a brc-1/brd-1 dependent manner. In a germline competent strain, somatic isp-1 RNAi would only trigger the pro-longevity signal in the soma, which in the presence of the lifespan limiting effects from the germline turns into detrimental effects on lifespan. When isp-1 is instead only silenced in the germline, the anti-apoptotic effect is lost, while the absence of the pro-longevity signal from the soma and the partial suppression of the limiting signal in the germline would result in a lifespan extending effects, yet reduced compared to systemic MRC stress.

Expanded View Figure Legends
A tuberous sclerosis complex signalling node at the peroxisome regulates mTORC1 and autophagy in response to ROS (2013)
figf1

(a) Representative images of rat liver (FAO) cells immunostained with TSC2, TSC1 or Rheb (green) and PMP70 (peroxisome marker) or LAMP1 (lysosome marker) (red) antibodies. Scale bars, 10 μm. (b) Pearson’s correlation coefficient for TSC1 or TSC2 co-localization with PMP70 or LAMP1 calculated using Imaris software. Quantification was performed on 8–12 cells from each of the 4 independent experiments, giving rise to a total of 40 cells. All error bars represent s.e.m.,
***P0.001. (c) Representative images using HeLa cells transfected with Myc–TSC1 and Flag–TSC2 wild type (WT) and stained with anti-Flag (green) and anti-Myc (red) antibodies. Scale bar, 10 μm. (d) Representative images of HepG2 cells transfected with control or TSC2 siRNA immunostained for TSC2 (green) and PMP70 (red). Individual boundaries are shown to identify cells with TSC2 knockdown (white) versus cells that retain TSC2 (yellow). Scale bars, 10 μm. (e) Corresponding immunoblots for HepG2 cells transfected with control or TSC2 siRNA showing the extent of knockdown (average over population of cells). (f) HepG2 cells from d were analysed for Pearson’s correlation coefficient of TSC2 co-localization with PMP70 using Imaris software. Quantification was performed on 8–12 cells from each of the 4 independent experiments, giving rise to a total of 40 cells. All error bars represent s.e.m.,
***P0.001. Uncropped images of western blots are shown in Supplementary Fig. S6. Source data for the statistical analysis are shown in Supplementary Table S1.
A tuberous sclerosis complex signalling node at the peroxisome regulates mTORC1 and autophagy in response to ROS (2013)
figf2

(a,b) Subcellular fractionation of FAO (a) or HepG2 (b) cells demonstrated the localization of TSC2, TSC1, Rheb, TBC1D7 (FAO, a) and AKT in various subcellular compartments. Catalase, PMP70 and lamin A/C were used as subcellular markers for the peroxisome (P) and nuclear (N) fractions, respectively. EEA1, LAMP1 and VDAC were used as markers for endosomes, lysosomes and mitochondria, respectively. WCE, whole-cell extracts; M, membrane; C, cytosol. (c) Subcellular fractionation of TSC2+/+ and TSC2−/− mouse embryonic fibroblasts (MEFs) demonstrating the localization of TSC2, TSC1, Rheb and AKT. PMP70, catalase (peroxisome fraction, P), LDH (cytosolic fraction, C), β-integrin (membrane fraction, M) and lamin A/C (nuclear fraction, N) were used as subcellular markers. Uncropped images of western blots are shown in Supplementary Fig. S6.
A tuberous sclerosis complex signalling node at the peroxisome regulates mTORC1 and autophagy in response to ROS (2013)
figf3

(a) Western analysis of whole-cell extracts (WCE), membrane (M), cytosolic (C) and peroxisome (P) fractions from parental and MCF-7 cells stably expressing constitutively active myristoylated AKT (myr-AKT) immunoblotted for TSC2, LDH, catalase and PMP70. Light and dark represent short and long autoradiographic exposures, respectively. (b) Western analysis of whole-cell extracts, membrane, cytosolic and peroxisome fractions from MCF-7 cells stimulated with insulin (200 nM) for 30 min after 1 h of serum starvation and immunoblotted for TSC2, LDH, catalase and PMP70. Light and dark represent short and long autoradiographic exposures, respectively. (c) Representative western analysis of HEK293 whole-cell extracts, cytosolic and peroxisome fractions, immunoblotted for phospho-TSC2 (S939; inactive), TSC2 and phospho-AKT (S473 and T308; activated). LDH and catalase were used as markers for the cytosolic and peroxisome fractions, respectively. (d) Proteinase K protection assays performed in the presence or absence of Triton X-100 to disrupt peroxisomes on equal masses of peroxisome fractions (HEK293 cells) collected at the indicated time points. The lysates were immunoblotted for TSC2, TSC1, Rheb, catalase and PMP70. Uncropped images of western blots are shown in Supplementary Fig. S6.
A tuberous sclerosis complex signalling node at the peroxisome regulates mTORC1 and autophagy in response to ROS (2013)
figf4

(a) Representative merged images using GFP–LC3 MCF-7 cells expressing Flag–TSC1, Flag–TSC2 or both Flag–TSC1 and Flag–TSC2 showing GFP–LC3 (green) puncta. Scale bars, 10 μm. (b) Quantification of GFP–LC3 puncta was performed and the results are represented as the average puncta fluorescence per cell (±s.e.m.,
n = 3 independent experiments) from 100 cells per experiment as shown in a.
***P0.001; NS, not significant. (c) Representative immunocytochemistry images using MCF-7 cells transfected with an mRFP–GFP–LC3 construct. Cells treated with 0.4 mM H2O2 were analysed at 0 h (Control), 1 h, 3 h and 6 h. Scale bars, 10 μm. (d) Quantification of autophagosomes (AP, GFP–LC3) and autolysosomes (AL, RFP–LC3) per cell in different conditions as shown in Fig. 4c. The results are represented as the average puncta fluorescence per cell (±s.e.m.,
n = 3 independent experiments) from 100 cells per experiment.
**P0.01;***P0.001. (e) Western analysis of FAO cells treated with 50 μM Wy-14643 (WY) or vehicle (DMSO) for PPAR-alpha-inducible proteins (EHHADH and ACAA1), mTORC1 signalling proteins ((pS6 (S235/236), S6, pS6K (T389) and S6K)) and autophagy markers (LC3 and p62). (f) Representative images of FAO cells treated with vehicle (DMSO) or 50 μM Wy-14643 (WY) for 1 h, with superoxide production detected using dihydroethidium (DHE). Scale bars, 30 μm. (g) Representative images of LC3 puncta (green) in FAO cells following treatment with 50 μM Wy-14643 (WY) or vehicle (DMSO) for the indicated time periods. Scale bars, 10 μm. (h) Quantification of LC3 puncta per cell in response to Wy-14643 (WY) or vehicle (DMSO). The results are represented as the average LC3 puncta fluorescence per cell (±s.e.m.,
n = 3 independent experiments) from 100 cells per experiment as shown in Fig. 4a.
*P0.05;
**P0.01. (i) Transmission electron microscopy of FAO cells treated with vehicle (DMSO) or 50 μM Wy-14643 (WY) for 24, 48 and 72 h. Peroxisomes and autophagosomes are indicated with red and yellow arrows, respectively. Scale bars, 500 nm. Uncropped images of western blots are shown in Supplementary Fig. S6. Source data for the statistical analysis are shown in Supplementary Table S1.
A tuberous sclerosis complex signalling node at the peroxisome regulates mTORC1 and autophagy in response to ROS (2013)
figf5

(a) Representative images of Zellweger cells (GM13267) showing endogenous TSC2, TSC1 and Rheb (green) co-localization with PMP70 (red). Scale bars, 15 μm. (b) Western analysis of human fibroblasts obtained from a Zellweger (GM13267) or a corresponding control patient (with Ehlers–Danlos syndrome (GM15871)) treated with the indicated doses of H2O2 for 1 h. mTORC1 signalling was assessed by western analysis for pS6K (T389), S6K, pS6 (S235/236), S6, p4E-BP1 (T37/46), 4E-BP1, pATM (S1981), ATM, pAMPK (T172), AMPK, p62 and LC3. (c) Western analysis of human fibroblast (GM13427) cells pre-incubated with 3 mM
N-acetyl cysteine (NAC; ROS scavenger) for 1 h before being treated with 0.4 mM H2O2 for 1 h using anti-pS6K (T389), S6K, p62 and LC3 antibodies. (d) Representative western analysis using cell extracts from human fibroblasts obtained from a Zellweger patient (GM13267) or control fibroblasts (GM15871) treated with amino-acid-free media for 60 min, and stimulated with a mixture of amino acids for 10 min. mTOR signalling was monitored using anti-pS6K (T389), S6K, pS6 (S235/236), S6, p4E-BP1(T37/46) and 4E-BP1 antibodies. Uncropped images of western blots are shown in Supplementary Fig. S6.