( e ) Western of FAO treated with 50 μM Wy - 14643 ( WY ) or vehicle ( DMSO ) for PPAR - alpha - inducible ( EHHADH ACAA1 ) , mTORC1 signalling ( ( pS6 ( S235 / 236 ) , S6 , pS6K ( T389 ) S6K ) ) autophagy markers ( LC3 p62 ) . ( f ) Representative of FAO treated with vehicle ( DMSO ) or 50 μM Wy - 14643 ( WY ) for 1 h , with superoxide production detected using dihydroethidium ( DHE ) . Scale bars , 30 μm .

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(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 of LC3 ( green ) in FAO following treatment with 50 μM Wy - 14643 ( WY ) or vehicle ( DMSO ) for indicated time periods . Scale bars , 10 μm . ( h ) of LC3 per in response to Wy - 14643 ( WY ) or vehicle ( DMSO ) . results are represented as average LC3 per ( ± s . e . m . , n = 3 independent experiments ) from 100 per experiment as shown in Fig . 4a . * P0 . 05 ; * * P0 . 01 .

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(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 treated with vehicle ( DMSO ) or 50 μM Wy - 14643 ( WY ) for 24 , 48 72 h . Peroxisomes autophagosomes are indicated with red yellow arrows , respectively . Scale bars , 500 nm . Uncropped of western blots are shown in Supplementary Fig . S6 . Source data for statistical are shown in Supplementary Table S1 .

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(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 ) Representative of Zellweger ( GM13267 ) showing endogenous TSC2 , TSC1 Rheb ( green ) co - localization with PMP70 ( red ) . Scale bars , 15 μm .

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(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 of human fibroblasts obtained from a Zellweger ( GM13267 ) or a corresponding control ( with Ehlers - Danlos syndrome ( GM15871 ) ) treated with indicated doses of H2O2 for 1 h . mTORC1 signalling was assessed by western for pS6K ( T389 ) , S6K , pS6 ( S235 / 236 ) , S6 , p4E - BP1 ( T37 / 46 ) , 4E - BP1 , pATM ( S1981 ) , ATM , pAMPK ( T172 ) , AMPK , p62 LC3 .

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(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 of human fibroblast ( GM13427 ) 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 LC3 antibodies .

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(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 using extracts from human fibroblasts obtained from a Zellweger ( GM13267 ) or control fibroblasts ( GM15871 ) treated with - acid - free media for 60 min , stimulated with a mixture of for 10 min . mTOR signalling was monitored using anti - pS6K ( T389 ) , S6K , pS6 ( S235 / 236 ) , S6 , p4E - BP1 ( T37 / 46 ) 4E - BP1 antibodies . Uncropped of western blots are shown in Supplementary Fig . S6 .

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(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.


( a ) Immunoprecipitation was performed with anti - PEX5 ( left panel ) anti - PEX19 ( right panel ) in HEK293 followed by immunoblotting for endogenous TSC1 , TSC2 , mTOR ( negative control ) PMP70 , PEX1 ( PEX19 cargo ) or catalase ( PEX5 cargo ) .

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(a) Immunoprecipitation was performed with anti-PEX5 (left panel) and anti-PEX19 (right panel) in HEK293 cells and followed by immunoblotting for endogenous TSC1, TSC2, mTOR (negative control) and PMP70, and PEX1 (PEX19 cargo) or catalase (PEX5 cargo).


( c ) Representative of FAO transfected with DsRed - Del - ARL ( deleted ARL ; red ) GFP - PTS1 ( green ; top panel ) . FAO were also transfected with DsRed - ARL ( red ) stained for PMP70 or catalase ( green ) as indicated . Scale bars , 10 μm .

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(c) Representative images of FAO cells transfected with DsRed-Del-ARL (deleted ARL sequence; red) and GFP-PTS1 (green; top panel). FAO cells were also transfected with DsRed-ARL (red) and stained for PMP70 or catalase (green) as indicated. Scale bars, 10 μm.


( d ) HEK293 expressing Flag - TSC2 wild type ( WT ) or Flag - TSC2 mutants ( RQ , RW RG ) were co - immunoprecipitated using anti - Flag or anti - PEX5 , blotted for PEX5 Flag - TSC2 .

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(d) HEK293 cells expressing Flag-TSC2 wild type (WT) or Flag-TSC2 mutants (RQ, RW and RG) were co-immunoprecipitated using anti-Flag or anti-PEX5, and blotted for PEX5 and Flag-TSC2.


( e ) Representative experiment showing subcellular fractionation of HEK293 overexpressing Flag - TSC2 wild type Flag - TSC2 mutants ( RQ , RW RG ) . Lysates from peroxisome ( P ) or ( M ) fractions whole - extracts ( WCE ) were immunoblotted with Flag or PMP70 antibodies .

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(e) Representative experiment showing subcellular fractionation of HEK293 cells overexpressing Flag-TSC2 wild type and Flag-TSC2 mutants (RQ, RW and RG). Lysates from peroxisome (P) or membrane (M) fractions and whole-cell extracts (WCE) were immunoblotted with Flag or PMP70 antibodies.


( f ) Co - immunoprecipitation of HEK293 overexpressing Flag - TSC2 wild type , or Flag - TSC2 mutant ( RQ ) or Flag - TSC2 rescue mutant ( RQ - 9NT ) using anti - Flag antibody or control IgG immunoblotted with anti - Flag anti - PEX5 antibodies .

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(f) Co-immunoprecipitation of HEK293 cells overexpressing Flag-TSC2 wild type, or Flag-TSC2 mutant (RQ) or Flag-TSC2 rescue mutant (RQ-9NT) using anti-Flag antibody or control IgG and immunoblotted with anti-Flag and anti-PEX5 antibodies.


( g ) Subcellular fractionation of TSC2 − / − MEFs transfected with Flag - TSC2 wild type , or Flag - TSC2 mutants ( RQ or RQ - 9NT ) . β - integrin catalase were used as subcellular markers for peroxisome fractions , respectively . WCE , whole - extract . arrow indicates position of overexpressed Flag - TSC2 .

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(g) Subcellular fractionation of TSC2−/− MEFs transfected with Flag-TSC2 wild type, or Flag-TSC2 mutants (RQ or RQ-9NT). β-integrin and catalase were used as subcellular markers for membrane and peroxisome fractions, respectively. WCE, whole-cell extract. The arrow indicates the position of overexpressed Flag-TSC2.


( h ) HEK293 co - transfected with Flag - TSC1 Flag - TSC2 wild type ( WT ) or Flag - TSC2 G294E mutant ( TSC2 mutant that cannot bind TSC1 ) . Lysates were immunoprecipitated using anti - PEX19 blotted for PEX19 Flag .

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(h) HEK293 cells co-transfected with Flag-TSC1 and Flag-TSC2 wild type (WT) or Flag-TSC2 G294E mutant (TSC2 mutant that cannot bind TSC1). Lysates were immunoprecipitated using anti-PEX19 and blotted for PEX19 and Flag.


( i ) Representative blots from subcellular fractionation of HEK293 overexpressing wild type ( WT ) or mutant ( CaaX mutant ) Flag - Rheb . LDH , lamin A / C catalase were used as subcellular markers for ( Cp ) , peroxisome fractions , respectively . Uncropped of western blots are shown in Supplementary Fig . S6 .

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(i) Representative blots from subcellular fractionation of HEK293 cells overexpressing wild type (WT) or mutant (CaaX mutant) Flag-Rheb. LDH, lamin A/C and catalase were used as subcellular markers for the cytoplasmic (Cp), nuclear and peroxisome fractions, respectively. Uncropped images of western blots are shown in Supplementary Fig. S6.


( a ) HEK293 were transfected with either control shRNA or a PEX5 targeting shRNA , lysates were blotted using pS6K ( T389 ) , S6K , pS6 ( S235 / 236 ) S6 antibodies .

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(a) HEK293 cells were transfected with either control shRNA or a PEX5 targeting shRNA, and lysates were blotted using pS6K (T389), S6K, pS6 (S235/236) and S6 antibodies.


( b ) TSC2 functional was performed in HEK293 co - expressing myc - Rheb , Flag - TSC1 Flag - TSC2 wild type ( WT ) , or Flag - TSC2 mutants ( RG , RQ RW ) HA - S6K ( left panel ) or HA - 4E - BP1 ( right panel ) . transfected with empty vector ( EV ) with or without myc - Rheb were used as controls . Lysates were further analysed for phospho - S6K ( T389 ) , S6K , phospho - 4EB - P1 ( S65 ) , 4E - BP1 32P incorporation into S6 .

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(b) TSC2 functional assay was performed in HEK293 cells co-expressing myc-Rheb, Flag-TSC1 and Flag-TSC2 wild type (WT), or Flag-TSC2 mutants (RG, RQ and RW) and HA-S6K (left panel) or HA-4E-BP1 (right panel). Cells transfected with empty vector (EV) with or without myc-Rheb were used as controls. Lysates were further analysed for phospho-S6K (T389), S6K, phospho-4EB-P1 (S65), 4E-BP1 and 32P incorporation into S6.


( c ) Rheb GTPase activity assays were performed by co - immunoprecipitating TSC heterodimers from HEK293 expressing Flag - TSC1 TSC2 - TSC2 wild type , Flag - TSC2 mutants or Flag - TSC2 GAP mutant ( L1624P ) , for indicated times .

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(c) Rheb GTPase activity assays were performed by co-immunoprecipitating TSC heterodimers from HEK293 cells expressing Flag-TSC1 and TSC2-TSC2 wild type, Flag-TSC2 mutants or the Flag-TSC2 GAP mutant (L1624P), for the indicated times.


( d ) TSC2 functional was performed using TSC2 − / − MEFs co - transfected with Flag - TSC1 , HA - S6K Flag - TSC2 wild type or Flag - TSC2 mutants ( RQ or RQ - 9NT ) , with mock transfected as controls . Arrows denote positions of Flag - TSC1 Flag - TSC2 . ( e ) of of phospho - S6K to total HA - S6K from Fig . 7d . ( ± s . e . m . , n = 3 independent experiments ) . * P0 . 05 ; * * P0 . 01 .

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(d) TSC2 functional assay was performed using TSC2−/− MEFs co-transfected with Flag-TSC1, HA-S6K and Flag-TSC2 wild type or Flag-TSC2 mutants (RQ or RQ-9NT), with mock transfected cells as controls. Arrows denote the positions of Flag-TSC1 and Flag-TSC2. (e) Quantification of the ratio of phospho-S6K to total HA-S6K from Fig. 7d. (±s.e.m., n = 3 independent experiments). *P0.05;**P0.01.


( f ) In vivo guanine nucleotide loading of Rheb was measured using HEK293 overexpressing Flag - TSC1 Flag - TSC2 wild type , or Flag - TSC2 mutants ( RQ RQ - 9NT ) with or without Rheb as indicated . ( g ) Graph shows of of Rheb bound to GTP ( indicative of Rheb GTPase activity , ± s . e . m . , n = 4 independent experiments ) . * P0 . 05 ; * * * P0 . 001 .

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(f) In vivo guanine nucleotide loading of Rheb was measured using HEK293 cells overexpressing Flag-TSC1 and Flag-TSC2 wild type, or Flag-TSC2 mutants (RQ and RQ-9NT) with or without Rheb as indicated. (g) Graph shows quantification of the percentage of Rheb bound to GTP (indicative of Rheb GTPase activity, ±s.e.m., n = 4 independent experiments). *P0.05;***P0.001.


( h ) of axon number in hippocampal neurons co - transfected with GFP , Flag - TSC1 Flag - TSC2 WT or Flag - TSC2 mutants . was performed on 150 - 250 neurons from each of 3 independent experiment results are represented as polarity ( % ) . error bars represent s . e . m . * P0 . 05 ; * * P0 . 01 compared with GFP - only transfected control . Uncropped of western blots are shown in Supplementary Fig . S6 . Source data for statistical are shown in Supplementary Table S1 .

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(h) Quantification of axon number in hippocampal neurons co-transfected with GFP, Flag-TSC1 and Flag-TSC2 WT or Flag-TSC2 mutants. Quantification was performed on 150-250 neurons from each of the 3 independent experiment and the results are represented as polarity (%). All error bars represent s.e.m. *P0.05;**P0.01 compared with GFP-only transfected control. Uncropped images of western blots are shown in Supplementary Fig. S6. Source data for the statistical analysis are shown in Supplementary Table S1.


Phenotypic changes of HSCs by citral . Phase - contrast ( left ) , oil red O - stained ( middle ) , autofluorescence ( right ) are shown for HSCs after passage 1 with or without treatment of citral ( 100 μM ) for 24 h . Scale bar = 10 μm .

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Phenotypic changes of HSCs by citral. Phase-contrast images (left), oil red O-stained images (middle), and autofluorescence images (right) are shown for HSCs after passage 1 with or without the treatment of citral (100 μM) for 24 h. Scale bar = 10 μm.


Western blot of lysates from citral - treated HSCs in ( A ) . α - tubulin was used as a loading control . quantitative densitometric data are expressed as of untreated control represent means ( SD ) for three independent experiments . * * P - value = 0 . 002 , paired t - ( compared to control ) .

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Western blot analysis of cell lysates from the citral-treated HSCs in (A). α-tubulin was used as a loading control. The quantitative densitometric data are expressed as the percentage of untreated control and represent the means (SD) for three independent experiments. **P-value = 0.002, paired t-test (compared to control).


expression of ALDH1A1 ALDH1A2 during culture activation of HSCs . lysates were prepared from HSCs at 3 days after plating ( HSCs - d3 ) from HSCs after passage 1 ( P1 ) , 2 ( P2 ) , 3 ( P3 ) , analyzed by Western blotting .

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Protein expression of ALDH1A1 and ALDH1A2 during the culture activation of HSCs. Cell lysates were prepared from HSCs at 3 days after plating (HSCs-d3) and from HSCs after passage 1 (P1), 2 (P2), and 3 (P3), and analyzed by Western blotting.


Real - time PCR for expression of ALDH1A1 ALDH1A2 during culture activation of HSCs . PCR data are expressed as of HSCs - d3 . * * P - value , paired t - ( n = 3 ) ( compared to HSCs - d3 ) , ALDH1A1 / HSCs - P3 : 0 . 003 , ALDH1A2 / HSCs - P1 : 0 . 0018 , ALDH1A2 / HSCs - P3 : 0 . 001 .

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Real-time PCR analysis for the expression levels of ALDH1A1 and ALDH1A2 during the culture activation of HSCs. The PCR data are expressed as the percentage of HSCs-d3. **P-value, paired t-test (n = 3) (compared to HSCs-d3), ALDH1A1/HSCs-P3: 0.003, ALDH1A2/HSCs-P1: 0.0018, ALDH1A2/HSCs-P3: 0.001.


Effect of ALDH1A suppression on HSC activation . HSCs - P1 were transfected with non - targeting siRNA or siRNA specific to ALDH1A1 / or ALDH1A2 , after 48 h , whole - lysates were subjected to Western blotting . * * P = 0 . 0023 , paired t - ( n = 3 ) ( siRNA - ALDH1A1 / 2 compared to control ) .

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Effect of ALDH1A suppression on HSC activation. HSCs-P1 were transfected with non-targeting siRNA or siRNA specific to ALDH1A1 and/or ALDH1A2, and after 48 h, whole-cell lysates were subjected to Western blotting. **P = 0.0023, paired t-test (n = 3) (siRNA-ALDH1A1/2 compared to control).


Reverse - phase HPLC of whole - lysates from siRNA - ALDH1A - treated HSCs in ( E ) . Typical chromatograms of - trans retinoic acid 13 - cis retinoic acid . retention time is 14 min for 13 - cis - RA 23 min for - trans RA .

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Reverse-phase HPLC analysis of whole-cell lysates from the siRNA-ALDH1A-treated HSCs in (E). Typical chromatograms of all-trans retinoic acid and 13-cis retinoic acid. The retention time is 14 min for 13-cis-RA and 23 min for all-trans RA.


Phenotypic changes of HSCs by RAR antagonist . HSCs - d3 were incubated with AGN193109 ( 1 μM ) ± - trans RA ( 10 μM ) for 3 days subjected to Oil red O . Scale bar = 10 μm .

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Phenotypic changes of HSCs by RAR antagonist. HSCs-d3 were incubated with AGN193109 (1 μM) ± all-trans RA (10 μM) for 3 days and subjected to Oil red O staining. Scale bar = 10 μm.


Western blot of lysates from AGN193109 ± RA - treated HSCs in ( G ) . * * P = 0 . 003 , paired t - ( n = 3 ) ( AGN193109 compared to control ) .

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Western blot analysis of cell lysates from AGN193109 ± RA-treated HSCs in (G). **P = 0.003, paired t-test (n = 3) (AGN193109 compared to control).


Albumin R - III downregulate RA signaling . HSCs - P1 were either transfected with RARE Cignal reporter expression plasmid containing vehicle or albumin ( left panel ) or treated with R - III ( 25 - 250 nM ) after transfection with RARE Cignal reporter ( right panel ) . After 36 h , transfected were harvested for luciferase . Renilla luciferase activity was normalized to firefly luciferase activity . Ratios were normalized against transfected with vehicle plasmid . * * P - value , two - sample t - ( n = 9 ) ( compared to control ) , albumin : 0 . 0037 , R - III 125 nM : 0 . 0032 .

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Albumin and R-III downregulate RA signaling. HSCs-P1 were either transfected with RARE Cignal reporter and expression plasmid containing vehicle or albumin (left panel) or treated with R-III (25-250 nM) after transfection with RARE Cignal reporter (right panel). After 36 h, the transfected cells were harvested for luciferase assay. Renilla luciferase activity was normalized to firefly luciferase activity. Ratios were normalized against the cells transfected with vehicle plasmid. **P-value, two-sample t-test (n = 9) (compared to control), albumin: 0.0037, R-III 125 nM: 0.0032.


Phenotypic changes of HSCs by expression of albumin / or ALDH1A . HSCs - P1 were transfected with plasmids encoding albumin , ALDH1A1 , ALDH1A2 , either individually or in combination , incubated in presence of - trans retinoic acid ( 50 nM ) for 18 h . were analyzed by oil red O . Scale bar , 20 μm .

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Phenotypic changes of HSCs by the expression of albumin and/or ALDH1A. HSCs-P1 were transfected with plasmids encoding albumin, ALDH1A1, and ALDH1A2, either individually or in combination, and incubated in the presence of all-trans retinoic acid (50 nM) for 18 h. Cells were analyzed by oil red O staining. Scale bar, 20 μm.


Western blot of lysates from albumin - / or ALDH1A - transfected HSCs as in ( B ) . * * P = 0 . 002 , paired t - ( n = 3 ) ( albumin compared to control ) .

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Western blot analysis of cell lysates from albumin- and/or ALDH1A-transfected HSCs as in (B). **P = 0.002, paired t-test (n = 3) (albumin compared to control).


Reverse - phase HPLC of whole - lysates from albumin - / or ALDH1A - transfected HSCs as in ( B ) .

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Reverse-phase HPLC analysis of whole-cell lysates from albumin- and/or ALDH1A-transfected HSCs as in (B).


RAR agonist counteracts R - III action . HSCs - P1 were treated with His - tagged R - III ( final , 0 . 15 μM ) in presence or absence of RAR agonist ( AGN191183 , 1 μM ) for 18 h analyzed by Western blotting . * * P = 0 . 0024 , paired t - ( n = 3 ) ( R - III compared to control ) . separating lines demarcate bands that come from non - consecutive lanes of same gel .

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RAR agonist counteracts R-III action. HSCs-P1 were treated with His-tagged R-III (final concentration, 0.15 μM) in the presence or absence of RAR agonist (AGN191183, 1 μM) for 18 h and analyzed by Western blotting. **P = 0.0024, paired t-test (n = 3) (R-III compared to control). The separating lines demarcate bands that come from non-consecutive lanes of the same gel.


expression of STRA6 during culture activation of HSCs . lysates were prepared from HSCs at 3 days after plating ( HSCs - 3d ) , from HSCs after passage 1 ( P1 ) , 2 ( P2 ) , 3 ( P3 ) , analyzed by Western blotting . Lysates from NIH3T3 were used as a negative control . separating lines demarcate bands that come from non - consecutive lanes of same gel .

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Protein expression of STRA6 during the culture activation of HSCs. Cell lysates were prepared from HSCs at 3 days after plating (HSCs-3d), and from HSCs after passage 1 (P1), 2 (P2), and 3 (P3), and analyzed by Western blotting. Lysates from NIH3T3 cells were used as a negative control. The separating lines demarcate bands that come from non-consecutive lanes of the same gel.


Real - time PCR for expression level of STRA6 during culture activation of HSCs . PCR data are expressed as of HSCs - 3d . * * P - value , paired t - ( n = 3 ) ( compared to HSCs - d3 ) , HSCs - P2 : 0 . 002 , HSCs - P3 : 0 . 0022 .

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Real-time PCR analysis for the expression level of STRA6 during the culture activation of HSCs. The PCR data are expressed as the percentage of HSCs-3d. **P-value, paired t-test (n = 3) (compared to HSCs-d3), HSCs-P2: 0.002, HSCs-P3: 0.0022.


R - III uptake parallels STRA6 level . HSCs at different stages of activation were incubated with His - tagged R - III ( final , 0 . 15 μM ) for 30 min , washed , subjected to Western blotting for R - III uptake .

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R-III uptake parallels STRA6 level. HSCs at different stages of activation were incubated with His-tagged R-III (final concentration, 0.15 μM) for 30 min, washed, and subjected to Western blotting for R-III uptake.


STRA6 - mediated uptake of R - III . HSCs - P1 were transfected with non - targeting siRNA or one of two different STRA6 - specific siRNAs , after 48 h , were incubated with R - III for 30 min analyzed by Western blotting .

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STRA6-mediated uptake of R-III. HSCs-P1 were transfected with non-targeting siRNA or one of the two different STRA6-specific siRNAs, and after 48 h, cells were incubated with R-III for 30 min and analyzed by Western blotting.


Representative macroscopic pictures of livers from control CCl4 - , CCl4 / albumin - , CCl4 / RBP - , CCl4 / R - III - treated mice .

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Representative macroscopic pictures of livers from control and CCl4-, CCl4/albumin-, CCl4/RBP-, and CCl4/R-III-treated mice.


Liver sections were stained with H & amp ; amp ; E Sirius red , also subjected to immunohistochemistry for type I collagen . Scale bar , 200 μm .

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Liver sections were stained with H&amp;E and Sirius red, and also subjected to immunohistochemistry for type I collagen. Scale bar, 200 μm.


Hydroxyproline content in livers from control CCl4 - , CCl4 / albumin - , CCl4 / R - III - treated mice ( μg / g liver ) . * P = 0 . 037 , two - sample t - ( n = 10 ) ( CCl4 + R - III compared to CCl4 - treated mice ) .

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Hydroxyproline content in the livers from control and CCl4-, CCl4/albumin-, and CCl4/R-III-treated mice (μg/g liver). *P = 0.037, two-sample t-test (n = 10) (CCl4 + R-III compared to CCl4-treated mice).


Western blot on liver extracts from treated mice with antibody against type I collagen .

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Western blot analysis on liver extracts from the treated mice with antibody against type I collagen.


Immunohistochemical of α - SMA on representative liver sections from treated mice . Scale bar , 200 μm .

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Immunohistochemical analysis of α-SMA on representative liver sections from the treated mice. Scale bar, 200 μm.


Liver sections from control CCl4 - CCl4 / R - III - treated mice were immunohistochemically stained for TGF - β F4 / 80 . Scale bar , 50 μm .

Liver sections from control and CCl4- and CCl4/R-III-treated mice were immunohistochemically stained for TGF-β and F4/80. Scale bar, 50 μm.


Liver sections from treated mice were immunohistochemically stained for desmin , His - tag , α - SMA . His - positive signals largely overlapped with desmin / α - SMA . Scale bar , 50 μm .

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Liver sections from treated mice were immunohistochemically stained for desmin, His-tag, and α-SMA. His-positive signals largely overlapped with desmin/α-SMA staining. Scale bar, 50 μm.


Liver sections from control CCl4 - CCl4 / R - III - treated mice were stained with Sirius red . Scale bar , 200 μm .

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Liver sections from control and CCl4- and CCl4/R-III-treated mice were stained with Sirius red. Scale bar, 200 μm.


Hydroxyproline content in livers ( μg / g liver ) . * P = 0 . 034 , two - sample t - ( n = 10 ) ( CCl4 + R - III compared to CCl4 - treated mice ) .

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Hydroxyproline content in the livers (μg/g liver). *P = 0.034, two-sample t-test (n = 10) (CCl4 + R-III compared to CCl4-treated mice).


Liver sections from control , BDL , BDL / R - III - treated mice were stained with Sirius red . Scale bar , 200 μm . .

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Liver sections from control, BDL, and BDL/R-III-treated mice were stained with Sirius red. Scale bar, 200 μm..


Hydroxyproline content in livers ( μg / g liver ) . * P - value , two - sample t - ( n = 10 ) ( compared to BDL - treated mice ) ; BDL + R - III ( 10 μg ) : 0 . 031 , BDL + R - III ( 5 μg ) : 0 . 026 .

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Hydroxyproline content in the livers (μg/g liver). *P-value, two-sample t-test (n = 10) (compared to BDL-treated mice); BDL + R-III (10 μg): 0.031, BDL+R-III (5 μg): 0.026.


Epi - ID H3K79 methylation scores of deletion mutants of members of Rpd3L Rpd3S complexes , calculated as described in Appendix Supplementary Methods , where 0 means a wild - type H3K79me level ( log2 scale ) . gray dots indicate accessory subunits . UpTag DownTag are barcode reporters in a promoter terminator context , respectively . Data was obtained on Rpd3L / Rpd3S subunits except Sds3 .

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Epi-ID H3K79 methylation scores of the deletion mutants of members of the Rpd3L and Rpd3S complexes, calculated as described in Appendix Supplementary Methods, where 0 means a wild-type H3K79me level (log2 scale). The gray dots indicate accessory subunits. UpTag and DownTag are barcode reporters in a promoter and terminator context, respectively. Data was obtained on all Rpd3L/Rpd3S subunits except Sds3.


Mass spectrometry of H3K79 methylation in wild - type mutant strains . Mean data points of two biological replicates . p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) p < 0 . 001 ( * * * ) by two - way ANOVA , comparison to wild type .

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Mass spectrometry analysis of H3K79 methylation in wild-type and mutant strains. Mean and individual data points of two biological replicates. p<0.05 (*), p<0.01 (**) and p<0.001 (***) by two-way ANOVA, comparison to wild type.


Heatmaps of H3K79me1 , H3K79me3 , H2Bub H3 in wild - type , aligned on TSS . Genes were sorted on average H3K79me3 / H3K79me1 in first 500 bp .

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Heatmaps of H3K79me1, H3K79me3, H2Bub and H3 in wild-type cells, aligned on the TSS. Genes were sorted based on the average H3K79me3/H3K79me1 ratio in the first 500 bp.


Snapshot of depth - normalized ChIP - seq data tracks from wild - type rpd3Δ strains showing 6 kb surrounding DBP1 ORF , which is top gene in in panel ( F ) . tracks have same y axis ( 0 - 20 RPM ) . A snapshot of another top - regulated gene is shown in Fig EV1D .

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Snapshot of depth-normalized ChIP-seq data tracks from wild-type and rpd3Δ strains showing 6 kb surrounding the DBP1 ORF, which is the top gene in the heatmap in panel (F). All tracks have the same y axis (0-20 RPM). A snapshot of another top-regulated gene is shown in Fig EV1D.


of H3K79me3 / H3 change in rpd3Δ versus wild - type , aligned on TSS . Genes were sorted on average in first 500 bp .

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Heatmap of the H3K79me3/H3 change in rpd3Δ versus wild-type cells, aligned on the TSS. Genes were sorted based on the average ratio in the first 500 bp.


ChIP - seq - seq data for genes ranked on H3K79me3 / H3 in rpd3Δ / WT , smoothed using locally weighed regression . gray band around shows 95 % confidence interval . Vertical dashed lines separate 4 groups with distinct changes upon RPD3 deletion . ChIP - seq data of H3K79me1 , H3K79me3 H2Bub were generated in this study ( plotted is average coverage in reads per genomic content , RPGC ) , Rpd3 binding , H4ac WT gene expression data were from ( McKnight et al , 2015 ) , relative expression in rpd3Δ / WT from Kemmeren et al ( 2014 ) .

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ChIP-seq and RNA-seq data for genes ranked on H3K79me3/H3 in rpd3Δ/WT, smoothed using locally weighed regression. The gray band around the line shows the 95% confidence interval. Vertical dashed lines separate 4 groups with distinct changes upon RPD3 deletion. ChIP-seq data of H3K79me1, H3K79me3 and H2Bub were generated in this study (plotted is the average coverage in reads per genomic content, RPGC), Rpd3 binding, H4ac and WT gene expression data were from (McKnight et al, 2015), and the relative expression in rpd3Δ/WT from Kemmeren et al (2014).


H3K79me3 / H3 ChIP - qPCR efficiencies ( relative to a non - transcribed region , which was unaffected by RPD3 deletion ) in wild - type in rpd3Δ harboring empty or RPD3 - encoding CEN plasmids . H188A H150A - H151A mutations have previously been shown to abrogate catalytic activity ( Kadosh & Struhl , 1998 ) . Error bars indicate standard deviation of three biological replicates . p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) p < 0 . 001 ( * * * ) by two - way ANOVA , comparison to wild type .

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H3K79me3/H3 ChIP-qPCR efficiencies (relative to a non-transcribed region, which was unaffected by RPD3 deletion) in wild-type and in rpd3Δ cells harboring empty or RPD3-encoding CEN plasmids. The H188A and H150A-H151A mutations have previously been shown to abrogate catalytic activity (Kadosh & Struhl, 1998). Error bars indicate standard deviation of three biological replicates. p<0.05 (*), p<0.01 (**) and p<0.001 (***) by two-way ANOVA, comparison to wild type.


Gene set enrichment on genes ranked on H3K79me3 / H3 in rpd3Δ / WT ; genes have been ranked ranks of genes in subsets are indicated by vertical lines . Meiotic ( C ) Ume6 - bound ( D ) genes are enriched among genes at which Rpd3 represses H3K79 methylation , subtelomeric genes ( < 30 kb of telomere ) ( E ) are enriched among genes at which H3K79 methylation is decreased in rpd3Δ .

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Gene set enrichment analysis on genes ranked on H3K79me3/H3 in rpd3Δ/WT; all genes have been ranked and the ranks of the genes in the subsets are indicated by vertical lines. Meiotic (C) and Ume6-bound (D) genes are enriched among the genes at which Rpd3 represses H3K79 methylation, and subtelomeric genes (<30 kb of telomere) (E) are enriched among genes at which H3K79 methylation is decreased in rpd3Δ cells.


Mass spectrometry of H3K79 methylation in thymuses from 3 - week - old mice , either wild - type ( Cre - ) or with deleted Hdac1 or Dot1L alleles , as indicated . Mean data points of biological replicates ; H3K79me0 is predominant state , y axis is truncated at 70 % for readability . remaining H3K79 methylation after homozygous Dot1L deletion is probably due to presence of some in which Cre is not expressed ( yet ) . p < 0 . 01 ( * * ) p < 0 . 001 ( * * * ) by two - way ANOVA , comparison to wild type .

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Mass spectrometry analysis of H3K79 methylation in thymuses from 3-week-old mice, either wild-type (Cre-) or with deleted Hdac1 or Dot1L alleles, as indicated. Mean and individual data points of biological replicates; H3K79me0 is the predominant state, the y axis is truncated at 70% for readability. The remaining H3K79 methylation after homozygous Dot1L deletion is probably due to the presence of some cells in which Cre is not expressed (yet). p<0.01 (**) and p<0.001 (***) by two-way ANOVA, comparison to wild type.


Representative H & E immunohistochemical stainings on sequential sections of thymic lymphomas of indicated genotypes . A picture with lower magnification of independent samples is included in Fig EV3B .

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Representative H&E and immunohistochemical stainings on sequential sections of thymic lymphomas of the indicated genotypes. A picture with lower magnification of independent samples is included in Fig EV3B.


Kaplan - Meier curves of mice harboring thymocytes with indicated genotypes . An event was defined as or sacrifice of a mouse caused by a thymic lymphoma . Mice that died due to other causes or were still alive event - free at end of experiment were censored . Mice for which cause of could not be determined were removed from data . Wild - type mice were Cre - of mice that were used for other curves .

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Kaplan-Meier curves of mice harboring thymocytes with indicated genotypes. An event was defined as death or sacrifice of a mouse caused by a thymic lymphoma. Mice that died due to other causes or were still alive and event-free at the end of the experiment were censored. Mice for which the cause of death could not be determined were removed from the data. Wild-type mice were the Cre- littermates of the mice that were used for the other curves.


Immunoblots showing HDAC1 status H3K79me / H3K9ac in lysates of Hdac1 - proficient ( p53 - null ) Hdac1 - deficient thymic lymphoma lines . top four bottom two panels are from separate lysates of same lines .

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Immunoblots showing HDAC1 status and H3K79me/H3K9ac levels in nuclear lysates of Hdac1-proficient (p53-null) and Hdac1-deficient thymic lymphoma cell lines. The top four and bottom two panels are from separate lysates of the same cell lines.


Mass spectrometry of H3K79 methylation in lines from panel A . Mean data points of two independent lines ; H3K79me0 is predominant state , y axis is truncated at 80 % for readability . p < 0 . 01 ( * * ) p < 0 . 001 ( * * * ) by two - way ANOVA .

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Mass spectrometry analysis of H3K79 methylation in the cell lines from panel A. Mean and individual data points of two independent cell lines; H3K79me0 is the predominant state, the y axis is truncated at 80% for readability. p<0.01 (**) and p<0.001 (***) by two-way ANOVA.


Immunoblot showing H2BK120 ubiquitination in Hdac1 - proficient - deficient lines ( two independent lines each ) .

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Immunoblot showing H2BK120 ubiquitination levels in Hdac1-proficient and -deficient cell lines (two independent lines each).


Growth curves of Hdac1 - proficient - deficient lines that were left untreated or were infected with empty ( pLKO ) or shRNAs against Dot1L selected with puromycin . Growth curves were determined by a series of resazurin assays , which measure metabolic activity , starting four days post - infection . Error bars indicate range of two replicates from independent lines .

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Growth curves of Hdac1-proficient and -deficient cell lines that were left untreated or were infected with empty virus (pLKO) or shRNAs against Dot1L and selected with puromycin. Growth curves were determined by a series of resazurin assays, which measure metabolic activity, starting four days post-infection. Error bars indicate the range of two replicates from independent cell lines.


Inhibitor dose - response curves of two DOT1L inhibitors EPZ - 5676 ( Pinometostat ) SGC - 0946 in Hdac1 - proficient - deficient lines . viability was measured by a resazurin after three days of treatment measurements were normalized to DMSO - treated . Two independent lines are plotted separately ; error bars indicate range of two biological replicates .

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Inhibitor dose-response curves of the two DOT1L inhibitors EPZ-5676 (Pinometostat) and SGC-0946 in Hdac1-proficient and -deficient cell lines. Cell viability was measured by a resazurin assay after three days of treatment and measurements were normalized to DMSO-treated cells. Two independent cell lines are plotted separately; error bars indicate the range of two biological replicates.


Representative apoptosis FACS plots of lines treated with DMSO or DOT1L inhibitor SGC - 0946 for two days . Annexin V DAPI were performed on unpermeabilized to distinguish live ( Annexin V low ; DAPI low ) , apoptotic ( Annexin V high ; DAPI low ) dead ( Annexin V high ; DAPI high ) . of several independent FACS experiments , including experiment shown in panel ( A ) . Mean with data points of 2 - 4 replicates each of two independent lines per genotype . p < 0 . 001 ( * * * ) by two - way ANOVA , comparison to corresponding DMSO control .

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Representative apoptosis FACS plots of cell lines treated with DMSO or the DOT1L inhibitor SGC-0946 for two days. Annexin V staining and DAPI staining were performed on unpermeabilized cells to distinguish live (Annexin V low; DAPI low), apoptotic (Annexin V high; DAPI low) and dead (Annexin V high; DAPI high) cells. Quantification of several independent FACS experiments, including the experiment shown in panel (A). Mean with individual data points of 2-4 replicates each of two independent lines per genotype. p<0.001 (***) by two-way ANOVA, comparison to corresponding DMSO control.


DRiP labelling in GFP - NCL HeLa Kyoto that were either left untreated or treated with OP - puro ( 25 µM ) for 2 h . Where indicated were allowed to recover in drug - free medium ( control ) or in presence of MG132 ( 10 µM ) for 5 h . Scale bars : 5 µm . GFP - NCL HeLa Kyoto were treated as described in A , with exception that a lower of OP - puro was used ( 5 µM ) . distribution of DRiPs GFP - NCL was analyzed by STED super - resolution microscopy . Scale bars : 5 µm .

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DRiP labelling in GFP-NCL HeLa Kyoto cells that were either left untreated or treated with OP-puro (25 µM) for 2 h. Where indicated cells were allowed to recover in drug-free medium (control) or in presence of MG132 (10 µM) for 5 h. Scale bars: 5 µm. GFP-NCL HeLa Kyoto cells were treated as described in A, with the exception that a lower concentration of OP-puro was used (5 µM). The distribution of DRiPs and GFP-NCL was analyzed by STED super-resolution microscopy. Scale bars: 5 µm.


Isolation of nucleoli from HeLa Kyoto untreated or treated with puromycin ( 25 µM ) for 2 h . Puromycylated were detected by immunoblotting ( right panel ) . left panel shows purity of isolated nucleoli by western blotting , where fibrillarin was used as nucleolar marker , FUS as nucleoplasmic marker TUBA4A as marker .

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Isolation of nucleoli from HeLa Kyoto cells untreated or treated with puromycin (25 µM) for 2 h. Puromycylated proteins were detected by immunoblotting (right panel). The left panel shows the purity of isolated nucleoli by western blotting, where fibrillarin was used as nucleolar marker, FUS as nucleoplasmic marker and TUBA4A as cytoplasmic marker.


HeLa were left untreated or treated with heat shock ( HS ) at 42 ˚ C for 3 h , alone or with cycloheximide ( CHX ; 50 μg / ml ) , or Actinomycin D ( Act . D ; 3 µM ) followed by with dye Amylo - glo .

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HeLa cells were left untreated or treated with heat shock (HS) at 42˚C for 3 h, alone or with cycloheximide (CHX; 50 μg/ml), or Actinomycin D (Act. D; 3 µM) followed by staining with the dye Amylo-glo.


DRiP labelling in GFP - NCL HeLa Kyoto that were either left untreated or treated with HS at 42 ˚ C OP - puro ( 25 µM ) for 2 h . Where indicated , were cotreated with CHX ( 50 μg / ml ) .

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DRiP labelling in GFP-NCL HeLa Kyoto cells that were either left untreated or treated with HS at 42˚C and OP-puro (25 µM) for 2 h. Where indicated, cells were cotreated with CHX (50 μg/ml).


HSPA8 subcellular distribution recruitment inside nucleoli following exposure to HS at 42 ˚ C for 2 h , alone or combined with CHX , in GFP - NCL HeLa Kyoto .

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HSPA8 subcellular distribution and recruitment inside nucleoli following exposure to HS at 42˚C for 2 h, alone or combined with CHX, in GFP-NCL HeLa Kyoto cells.


DRiP labelling in HeLa treated with MG132 ( 10 µM ) alone or with CHX ( 50 μg / ml ) for 8 h ; where indicated , OP - puro ( 25 µM ) was added during last 45 min , prior to fixation .

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DRiP labelling in HeLa cells treated with MG132 (10 µM) alone or with CHX (50 μg/ml) for 8 h; where indicated, OP-puro (25 µM) was added during the last 45 min, prior to cell fixation.


Amylo - glo of HeLa left untreated or exposed to MG132 ( 10 µM ) alone or combined with CHX ( 50 μg / ml ) for 8 or 13 h .

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Amylo-glo staining of HeLa cells left untreated or exposed to MG132 (10 µM) alone or combined with CHX (50 μg/ml) for 8 or 13 h.


Amylo - glo of HeLa left untreated or subjected to transcriptional stress ( MG132 10 µM Act . D 4 µM ) for 6 hrs . Where indicated , translation was concomitantly inhibited with CHX ( 50 μg / ml ) . Amylo - Glo is also shown in HeLa treated with Act . D alone or with CHX for 6 hrs , as control .

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Amylo-glo staining of HeLa cells left untreated or subjected to transcriptional stress (MG132 10 µM and Act. D 4 µM) for 6 hrs. Where indicated, translation was concomitantly inhibited with CHX (50 μg/ml). Amylo-Glo staining is also shown in HeLa cells treated with Act.D alone or with CHX for 6 hrs, as control.


Amylo - glo of HeLa exposed to HS at 42 ˚ C for 3 h let to recover for 3 h in drug - free medium ( + rec . control ) or with VER ( 40 µM ; + rec . VER ) . Where indicated were treated with VER alone ( VER 3 h ) .

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Amylo-glo staining of HeLa cells exposed to HS at 42˚C for 3 h and let to recover for 3 h in drug-free medium (+ rec. control) or with VER (40 µM; + rec. VER). Where indicated cells were treated with VER alone (VER 3 h).


Amylo - glo of HeLa treated with MG132 ( 10 µM ) for 13 h let to recover for 5 h in drug - free medium ( + rec . control ) or with VER ( 40 µM ; + rec . VER ) . Where indicated were treated with VER alone ( VER 5 h ) .

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Amylo-glo staining of HeLa cells treated with MG132 (10 µM) for 13 h and let to recover for 5 h in drug-free medium (+ rec. control) or with VER (40 µM; + rec. VER). Where indicated cells were treated with VER alone (VER 5 h).


HeLa were transfected with vectors encoding for GFP , GFP - GR50 or RPL23a - GFP . 24 hrs post - transfection were fixed stained with Amylo - glo . of transfected with Amylo - Glo positive nucleoli is shown . Number of counted / condition : GFP , 412 ; RPL23a - GFP , 482 ; GFP - GR50 , 359 , in three independent experiments ; statistical significance via One - way ANOVA ; p < 10 - 7 , ± s . e . m .

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HeLa cells were transfected with vectors encoding for GFP, GFP-GR50 or RPL23a-GFP. 24 hrs post-transfection cells were fixed and stained with Amylo-glo. The percentage of transfected cells with Amylo-Glo positive nucleoli is shown. Number of cells counted/condition: GFP, 412; RPL23a-GFP, 482; GFP-GR50, 359, in three independent experiments; statistical significance via One-way ANOVA; p < 10-7, ± s.e.m.


DRiPs , polyUb ( FK1 ) PML labelling in HeLa treated with OP - puro ( 25 µM ) heat shock ( HS ) at 42 ˚ C for 2 h ; where indicated cycloheximide was also added ( CHX ; 50 μg / ml ) . Scale bars : 5 µm . Lower panel : STED super - resolution microscopy showing colocalization of DRiPs with PML - NBs in PML - GFP HeLa treated with a low of OP - puro ( 5 µM ) heat shock ( HS ) at 42 ˚ C for 2 h . PML - NBs were visualized using a PML specific antibody , followed by incubation with alexa fluor 647 . For convenience alexa fluor 647 signal is shown in green . Scale bars : 5 µm .

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DRiPs, polyUb proteins (FK1) and PML labelling in HeLa cells treated with OP-puro (25 µM) and heat shock (HS) at 42˚C for 2 h; where indicated cycloheximide was also added (CHX; 50 μg/ml). Scale bars: 5 µm. Lower panel: STED super-resolution microscopy showing colocalization of DRiPs with PML-NBs in PML-GFP HeLa cells treated with a low concentration of OP-puro (5 µM) and heat shock (HS) at 42˚C for 2 h. PML-NBs were visualized using a PML specific antibody, followed by incubation with alexa fluor 647. For convenience the alexa fluor 647 signal is shown in green. Scale bars: 5 µm.


PolyUb ( FK1 ) labelling in HeLa left untreated or treated with HS at 42 ˚ C for 2 h , alone or combined with CHX ( 50 μg / ml ) . Scale bars : 5 µm .

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PolyUb proteins (FK1) labelling in HeLa cells left untreated or treated with HS at 42˚C for 2 h, alone or combined with CHX (50 μg/ml). Scale bars: 5 µm.


STED super - resolution microscopy showing colocalization of endogenous polyUb ( FK1 ) PML - NBs in PML - GFP HeLa treated with HS at 42 ˚ C for 2 h . Scale bars : 5 µm .

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STED super-resolution microscopy showing colocalization of endogenous polyUb proteins (FK1) and PML-NBs in PML-GFP HeLa cells treated with HS at 42˚C for 2 h. Scale bars: 5 µm.


Quantitation of shown in A - C . Number of counted / condition : 712 - 1343 in three independent experiments ; statistical significance via One - way ANOVA ; p < 10 - 5 , ± s . e . m .

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Quantitation of cells shown in A-C. Number of cells counted/condition: 712 - 1343 in three independent experiments; statistical significance via One-way ANOVA; p < 10-5, ± s.e.m.


DRiPs , polyUb ( FK1 ) PML labelling in HeLa treated with MG132 ( 10 µM ) OP - puro ( 25 µM ) for 4 h ; where indicated , cycloheximide was also added ( CHX ; 50 μg / ml ) . Asterisks ( * ) indicate nucleoli . Scale bars : 10 µm . Lower panel : STED super - resolution microscopy showing colocalization of DRiPs with PML - NBs in PML - GFP HeLa treated with a low of OP - puro ( 5 µM ) MG132 ( 10 µM ) for 4 hrs . Scale bars : 5 µm . Quantitation of shown in E . Number of counted / condition : 298 - 1713 in three independent experiments ; statistical significance via One - way ANOVA ; p ≤ 0 . 001 , ± s . e . m .

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DRiPs, polyUb proteins (FK1) and PML labelling in HeLa cells treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h; where indicated, cycloheximide was also added (CHX; 50 μg/ml). Asterisks (*) indicate nucleoli. Scale bars: 10 µm. Lower panel: STED super-resolution microscopy showing colocalization of DRiPs with PML-NBs in PML-GFP HeLa cells treated with a low concentration of OP-puro (5 µM) and MG132 (10 µM) for 4 hrs. Scale bars: 5 µm. Quantitation of cells shown in E. Number of cells counted/condition: 298 - 1713 in three independent experiments; statistical significance via One-way ANOVA; p ≤ 0.001, ± s.e.m.


Intranuclear distribution of 20S proteasomes in HeLa left untreated or treated with MG132 ( 10 µM ) OP - puro ( 25 µM ) for 4 h . Asterisks ( * ) indicate nucleoli . Scale bars : 10 µm .

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Intranuclear distribution of 20S proteasomes in HeLa cells left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Asterisks (*) indicate nucleoli. Scale bars: 10 µm.


HeLa were lipofected with non - targeting siRNA control or a specific siRNA against PML . 72 h post - transfection , total were extracted expression of PML were analyzed by immunoblotting . TUBA4A was used as loading control .

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HeLa cells were lipofected with non-targeting siRNA control or a specific siRNA against PML. 72 h post-transfection, total proteins were extracted and expression levels of PML were analyzed by immunoblotting. TUBA4A was used as loading control.


HeLa were lipofected with non - targeting siRNA control or a specific siRNA against PML . 72 h post - transfection , were treated with MG132 ( 10 µM ) OP - puro ( 25 µM ) for 4 h . Left panel : automated quantitation of number of PML - NBs / nucleus is reported . Statistical significance via 2 ‐ Student ' s t ‐ . number analyzed : 334 - 364 ; p < 10 - 10 . Right panel : automated quantitation of number of DRiP foci / nucleus . number analyzed : 334 - 364 ; p < 10 - 10 . Automated DRiP segmentation is on azide signal .

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HeLa cells were lipofected with non-targeting siRNA control or a specific siRNA against PML. 72 h post-transfection, cells were treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Left panel: automated quantitation of the number of PML-NBs/nucleus is reported. Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 334 - 364; p < 10-10. Right panel: automated quantitation of the number of DRiP foci/nucleus. Cell number analyzed: 334 - 364; p < 10-10. Automated DRiP segmentation is based on azide signal.


DRiPs polyUb ( FK1 ) distribution in treated as described in ( A ) . Automated DRiP segmentation is on azide signal . Quantitation of number of DRiP foci enriched for polyUb ( > 2 ) is shown ; n = 590 - 2114 ; statistical significance via 2 ‐ Student ' s t ‐ ; p < 10 - 10 . Scale bars : 10 µm .

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DRiPs and polyUb (FK1) protein distribution in cells treated as described in (A). Automated DRiP segmentation is based on azide signal. Quantitation of the number of DRiP foci enriched for polyUb (>2) is shown; n = 590 - 2114; statistical significance via 2‐tailed Student's t‐test; p < 10-10. Scale bars: 10 µm.


Immunoblotting on extracts from HeLa lipofected with non - targeting siRNA control or a specific siRNA against Ubc9 . A dilution curve of control sample is shown . TUBA4A was used as loading control . Quantitation of number of PML - NBs / nucleus from treated as described in ( D ) . Statistical significance via 2 ‐ Student ' s t ‐ . number analyzed : 192 - 430 ; p < 10 - 10 . Automated PML - NB segmentation is on PML signal .

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Immunoblotting on protein extracts from HeLa cells lipofected with non-targeting siRNA control or a specific siRNA against Ubc9. A dilution curve of the control sample is shown. TUBA4A was used as loading control. Quantitation of the number of PML-NBs/nucleus from cells treated as described in (D). Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 192 - 430; p < 10-10. Automated PML-NB segmentation is based on PML signal.


lipofected for 72 h with non - targeting siRNA control or a specific siRNA against Ubc9 were treated with MG132 ( 10 µM ) OP - puro ( 25 µM ) for 4 h . Automated quantitation of number of PML - NBs / nucleus is reported . Statistical significance via 2 ‐ Student ' s t ‐ . number analyzed : 151 - 225 ; p < 10 - 10 ( see panel G ) .

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Cells lipofected for 72 h with non-targeting siRNA control or a specific siRNA against Ubc9 were treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Automated quantitation of the number of PML-NBs/nucleus is reported. Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 151 - 225; p < 10-10 (see panel G).


Quantitation of number of PML - NBs / nucleus from treated as described in ( D ) . Statistical significance via 2 ‐ Student ' s t ‐ . number analyzed : 192 - 430 ; p < 10 - 10 . Automated PML - NB segmentation is on PML signal . Representative pictures showing that Ubc9 - depleted cannot efficiently induce self - assembly of PML - NBs upon treatment with MG132 ( 10 µM ) OP - puro ( 25 µM ) for 4 h . Scale bars : 10 µm .

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Quantitation of the number of PML-NBs/nucleus from cells treated as described in (D). Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 192 - 430; p < 10-10. Automated PML-NB segmentation is based on PML signal. Representative pictures showing that Ubc9-depleted cells cannot efficiently induce the self-assembly of PML-NBs upon treatment with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Scale bars: 10 µm.


PolyUb ( FK1 ) labelling in PML - GFP HeLa Kyoto that were either left untreated or treated with MG132 ( 10 µM ) OP - puro ( 25 µM ) for 4 h . were either immediately fixed or let to recover for 5 h in drug - free medium ( Control ) , with VER ( 40 µM ) or Eeyarestatin I ( 5 µM ) . Scale bars : 10 µm . Quantitation of % of with polyUb ( FK1 ) foci shown in ( A ) . Number of counted / condition : 310 - 1258 in three independent experiments ; statistical significance via One - way ANOVA ; p = 0 . 0003 or 10 - 6 , ± s . e . m .

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PolyUb protein (FK1) labelling in PML-GFP HeLa Kyoto cells that were either left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Cells were either immediately fixed or let to recover for 5 h in drug-free medium (Control), with VER (40 µM) or Eeyarestatin I (5 µM). Scale bars: 10 µm. Quantitation of the % of cells with polyUb (FK1) nuclear foci shown in (A). Number of cells counted/condition: 310 - 1258 in three independent experiments; statistical significance via One-way ANOVA; p = 0.0003 or 10-6, ± s.e.m.


Quantitation of number of PML - NBs enriched for polyUb ( > 2 ) ; n = 611 - 2612 ; statistical significance via One - way ANOVA ; p < 10 - 10 . Automated PML - NB segmentation is on PML signal .

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Quantitation of the number of PML-NBs enriched for polyUb (>2); n = 611 - 2612; statistical significance via One-way ANOVA; p < 10-10. Automated PML-NB segmentation is based on PML signal.


Quantitation of % of with polyUb ( FK1 ) sequestered in foci . HeLa were treated with OP - puro ( 25 µM ) heat shock ( HS ) at 42 ˚ C for 2 h . Then , were either immediately fixed or let to recover for 3 h in drug - free medium ( control ) , with VER ( 40 µM ) or Eeyarestatin I ( 5 µM ) . Number of counted / condition : 593 - 1016 in three independent experiments ; statistical significance via One - way ANOVA ; p = 10 - 3 , ± s . e . m .

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Quantitation of the % of cells with polyUb proteins (FK1) sequestered in nuclear foci. HeLa cells were treated with OP-puro (25 µM) and heat shock (HS) at 42˚C for 2 h. Then, cells were either immediately fixed or let to recover for 3 h in drug-free medium (control), with VER (40 µM) or Eeyarestatin I (5 µM). Number of cells counted/condition: 593 - 1016 in three independent experiments; statistical significance via One-way ANOVA; p = 10-3, ± s.e.m.


Quantitation of number of PML - NBs enriched for polyUb ( > 2 ) in HeLa treated as described in ( D ) but with a recovery time of 2 h ; n = 1155 - 3283 ; statistical significance via One - way ANOVA ; p < 10 - 10 . Automated PML - NB segmentation is on PML signal .

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Quantitation of the number of PML-NBs enriched for polyUb (>2) in HeLa cells treated as described in (D) but with a recovery time of 2 h; n = 1155 - 3283; statistical significance via One-way ANOVA; p < 10-10. Automated PML-NB segmentation is based on PML signal.


Amylo - Glo of PML - GFP HeLa Kyoto that were subjected to following treatments : untreated ( Control ) ; MG132 ( 10 µM ) OP - puro ( 25 µM ) for 4 h , alone or combined with CHX ( 50 μg / ml ) . Where indicated were allowed to recover for 5 hrs in drug - free medium ( Control ) or with VER ( 40 µM ) . Confocal showing Amylo - Glo PML - GFP . Scale bars : 5 µm .

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Amylo-Glo staining of PML-GFP HeLa Kyoto cells that were subjected to the following treatments: untreated (Control); MG132 (10 µM) and OP-puro (25 µM) for 4 h, alone or combined with CHX (50 μg/ml). Where indicated cells were allowed to recover for 5 hrs in drug-free medium (Control) or with VER (40 µM). Confocal images showing Amylo-Glo and PML-GFP. Scale bars: 5 µm.


Amylo - Glo of PML - GFP HeLa Kyoto that were subjected to heat shock ( HS ) at 42 ˚ C for 2 hrs . Confocal showing Amylo - Glo PML - GFP . Scale bars : 5 µm .

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Amylo-Glo staining of PML-GFP HeLa Kyoto cells that were subjected to heat shock (HS) at 42˚C for 2 hrs. Confocal images showing Amylo-Glo and PML-GFP. Scale bars: 5 µm.


PML - GFP HeLa Kyoto were lipofected with a cDNA encoding for mCherry - VHL . 24 h post - transfection , were left untreated or treated with MG132 ( 10 µM ) OP - puro ( 25 µM ) for 4 h . Where indicated ( OP - puro + MG132 + CHX ) , translation was inhibited during stress with cycloheximide ( CHX ; 50 μg / ml ) . In lower panel , were allowed to recover for 5 h in drug - free medium ( Control ) , or with VER ( 40 µM ) . subcellular distribution of PML - GFP mCherry - VHL was studied by live - confocal imaging . Representative pictures are shown . Scale bars : 5 µm . Quantitation of recovery after bleach of PML - GFP . PML - GFP HeLa Kyoto overexpressing mCherry - VHL for 24 h treated as described in ( A ) were analyzed . mean of 19 - 23 FRAP curves fitting curves are shown in black red respectively . In gray , SD is shown .

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PML-GFP HeLa Kyoto cells were lipofected with a cDNA encoding for mCherry-VHL. 24 h post-transfection, cells were left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Where indicated (OP-puro + MG132 + CHX), translation was inhibited during stress with cycloheximide (CHX; 50 μg/ml). In the lower panel, cells were allowed to recover for 5 h in drug-free medium (Control), or with VER (40 µM). The subcellular distribution of PML-GFP and mCherry-VHL was studied by live-cell confocal imaging. Representative pictures are shown. Scale bars: 5 µm. Quantitation of the fluorescence intensity recovery after bleach of PML-GFP. PML-GFP HeLa Kyoto overexpressing mCherry-VHL for 24 h and treated as described in (A) were analyzed. The mean of 19-23 FRAP curves and the fitting curves are shown in black and red respectively. In gray, the SD is shown.


Intranuclear distribution of 20S proteasomes , HSP70 , VCP DRiPs in HeLa treated with MG132 ( 10 µM ) OP - puro ( 25 µM ) for 4 h allowed to recover for 5 h in drug - free medium ( control ) , with VER ( 40 µM ) , Eeyarestatin I ( 5 µM ) or MG132 ( 10 µM ) . Scale bars : 5 µm .

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Intranuclear distribution of 20S proteasomes, HSP70, VCP and DRiPs in HeLa cells treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h and allowed to recover for 5 h in drug-free medium (control), with VER (40 µM), Eeyarestatin I (5 µM) or MG132 (10 µM). Scale bars: 5 µm.


GFP - PSMA7 HeLa Kyoto were left untreated or treated as described in A . were subjected to FRAP to analyze GFP - PSMA7 mobility . Quantitation of three populations of GFP - PSMA7 molecules is shown : fast moving , slow moving immobile ; statistical significance via One - way ANOVA ; n = 12 - 14 ± .

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GFP-PSMA7 HeLa Kyoto cells were left untreated or treated as described in A. Cells were subjected to FRAP to analyze GFP-PSMA7 mobility. Quantitation of three populations of GFP-PSMA7 molecules is shown: fast moving, slow moving and immobile; statistical significance via One-way ANOVA; n = 12 - 14 ± SEM.


Subcellular distribution of 53BP1 DRiPs in HeLa that were left untreated or treated with MG132 ( 10 µM ) OP - puro ( 25 µM ) for 4 h , followed by 5h recovery in drug - free medium ( + rec . control ) , with VER ( 40 µM ; + rec . VER ) or Eeyarestatin I ( 5 µM ; + rec . EeyI ) . Where indicated , were treated with MG132 , VER or EeyI alone for 5 h . Scale bars : 20 µm . Quantitation of % of with 53BP1 foci . were treated as described in ( A ) divided in three categories , on number of 53BP1 foci / nucleus : 0 , 1 - 2 > 3 . Number of counted / condition : 1112 - 1537 in three independent experiments ; statistical significance via One - way ANOVA ; p < 0 . 01 .

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Subcellular distribution of 53BP1 and DRiPs in HeLa cells that were left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h, followed by 5h recovery in drug-free medium (+ rec. control), with VER (40 µM; + rec. VER) or Eeyarestatin I (5 µM; + rec. EeyI). Where indicated, cells were treated with MG132, VER or EeyI alone for 5 h. Scale bars: 20 µm. Quantitation of the % of cells with 53BP1 foci. Cells were treated as described in (A) and divided in three categories, based on the number of 53BP1 foci/nucleus: 0, 1-2 and > 3. Number of cells counted/condition: 1112 - 1537 in three independent experiments; statistical significance via One-way ANOVA; p < 0.01.


HeLa were lipofected with cDNAs encoding for HA - Ubiquitin , Flag - Ubiquitin or Flag - VCP 24 h post - transfection , were either left untreated or treated with MG132 ( 10 µM ) OP - puro ( 25 µM ) . were processed for of HA , Flag endogenous 53BP1 . * indicates that do not express HA - or Flag - Ubiquitin show no rescue of 53BP1 foci formation after stress . Scale bars : 20 µm . Quantitation of % of with 53BP1 foci . were treated as described in ( C ) divided in three categories , on number of 53BP1 foci / nucleus : 0 , 1 - 2 > 3 . Number of counted / condition : 710 - 938 in three independent experiments ; statistical significance via One - way ANOVA ; p < 0 . 01 .

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HeLa cells were lipofected with cDNAs encoding for HA-Ubiquitin, Flag-Ubiquitin or Flag-VCP 24 h post-transfection, cells were either left untreated or treated with MG132 (10 µM) and OP-puro (25 µM). Cells were processed for staining of HA, Flag and endogenous 53BP1. * indicates cells that do not express HA- or Flag-Ubiquitin and show no rescue of 53BP1 foci formation after stress. Scale bars: 20 µm. Quantitation of the % of cells with 53BP1 foci. Cells were treated as described in (C) and divided in three categories, based on the number of 53BP1 foci/nucleus: 0, 1-2 and > 3. Number of cells counted/condition: 710 - 938 in three independent experiments; statistical significance via One-way ANOVA; p < 0.01.


Colony formation . number of colonies surviving 10 days after plating is shown . Control or treated with MG132 ( 10 µM ) puromycin ( 5 µg / ml ) , followed by 5h recovery in drug - free medium ( + rec . control ) , with VER ( 40 µM ; + rec . VER ) or Eeyarestatin I ( 5 µM ; + rec . EeyI ) are shown . Where indicated , were treated with VER or EeyI alone for 5 h . Number of colonies counted / condition : up to 526 in three independent experiments ; statistical significance via One - way ANOVA ; p < 0 . 03 , ± s . e . m .

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Colony formation assay. The number of colonies surviving 10 days after plating is shown. Control cells or cells treated with MG132 (10 µM) and puromycin (5 µg/ml), followed by 5h recovery in drug-free medium (+ rec. control), with VER (40 µM; + rec. VER) or Eeyarestatin I (5 µM; + rec. EeyI) are shown. Where indicated, cells were treated with VER or EeyI alone for 5 h. Number of colonies counted/condition: up to 526 in three independent experiments; statistical significance via One-way ANOVA; p < 0.03, ± s.e.m.


( b ) SAR405 inhibits function of Vps34 in GFP - FYVE . HeLa stably transfected with GFP - FYVE were incubated with DMSO ( left ) or with 1 μM SAR405 ( right ) for 2 h . were fixed , nuclei were stained using Hoechst 33342 ( blue ) . was analyzed using an imaging cytometer ( X40 ) . One representative image out of three independent experiments is depicted . Scale bars , 20 μm .

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(b) SAR405 inhibits the function of Vps34 in the GFP-FYVE cellular assay. HeLa cells stably transfected with GFP-FYVE were incubated with DMSO (left) or with 1 μM SAR405 (right) for 2 h. Cells were fixed, and nuclei were stained using Hoechst 33342 (blue). Fluorescence was analyzed using an imaging cytometer (X40). One representative image out of three independent experiments is depicted. Scale bars, 20 μm.


( c ) SAR405 does not prevent phosphorylation of Akt S6 . U87MG were treated with SAR405 or PI103 for 2 h at indicated concentrations . effect of compounds on phosphorylated Akt ( p - Akt ) Ser473 Thr308 residues , total Akt on phosphorylated S6 ( Ser240 Ser244 residues ) total S6 was measured using western blotting . One representative experiment from at least two independent experiments is shown . Full - blots are shown in Supplementary Figure 6 .

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(c) SAR405 does not prevent phosphorylation of Akt and S6. U87MG cells were treated with SAR405 or PI103 for 2 h at the indicated concentrations. The effect of compounds on phosphorylated Akt (p-Akt) Ser473 and Thr308 residues, total Akt and on phosphorylated S6 (Ser240 and Ser244 residues) and total S6 protein was measured using western blotting. One representative experiment from at least two independent experiments is shown. Full-length blots are shown in Supplementary Figure 6.


( a ) SAR405 induces late endosome - lysosome swelling . RKO were treated with DMSO ( upper panels ) or with 10 μM SAR405 for 16 h ( lower panels ) . late endosomes - lysosomes compartment was assessed using Lysotracker green ( left ) or Alexa 488 - conjugated anti - Lamp1 antibodies ( right ) , as indicated . Nuclei were stained using Hoechst 33342 ( blue ) . One representative image chosen from three independent experiments , showing imaged using a Zeiss microscope ( X40 ) , is shown for each condition . Arrows indicate accumulation of swollen late endosome - lysosomes . Scale bars , 20 μm .

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(a) SAR405 induces late endosome-lysosome swelling. RKO cells were treated with DMSO (upper panels) or with 10 μM SAR405 for 16 h (lower panels). The late endosomes-lysosomes compartment was assessed using Lysotracker green (left) or Alexa 488-conjugated anti-Lamp1 antibodies (right), as indicated. Nuclei were stained using Hoechst 33342 (blue). One representative image chosen from three independent experiments, showing fluorescence imaged using a Zeiss microscope (X40), is shown for each condition. Arrows indicate accumulation of swollen late endosome-lysosomes. Scale bars, 20 μm.


( b ) SAR405 triggers accumulation of p62 . RKO were treated with indicated of Vps34 inhibitor for 24 h . effect on p62 biomarker was analyzed by western blotting . GAPDH was used as a loading control . results are representative of at least two independent experiments . Full - blots are shown in Supplementary Figure 14 .

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(b) SAR405 triggers accumulation of p62. RKO cells were treated with the indicated concentration of Vps34 inhibitor for 24 h. The effect on the p62 biomarker was analyzed by western blotting. GAPDH was used as a loading control. The results are representative of at least two independent experiments. Full-length blots are shown in Supplementary Figure 14.


( c ) SAR405 affects maturation of cathepsin D ( cat D ) . RKO were treated at indicated concentrations of SAR405 for 24 h . different forms of cathepsin D ( PPCD , pre - pro - catD ) were analyzed by western blotting . GAPDH was used as a loading control . results are representative of at least two independent experiments . Full - blots are shown in Supplementary Figure 16 .

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(c) SAR405 affects the maturation of cathepsin D (cat D). RKO cells were treated at the indicated concentrations of SAR405 for 24 h. The different forms of cathepsin D (PPCD, pre-pro-catD) were analyzed by western blotting. GAPDH was used as a loading control. The results are representative of at least two independent experiments. Full-length blots are shown in Supplementary Figure 16.


( a ) GFP - LC3 HeLa were cultured in fed conditions or starved using EBSS plus 10 μM hydroxychloroquine treated with Vps34 inhibitor . were fixed , nuclei were stained using Hoechst 33342 ( blue ) . One representative image , acquired with an iCyte imaging cytometer , for three independent experiments is depicted ( X40 ) . arrows indicate GFP - LC3 spots , which correspond to autophagosomes . Scale bars , 10 μm . ( b ) Histograms show of positive GFP , measured using an imaging cytometer for different compound concentrations on GFP - LC3 HeLa . Black gray histograms correspond to fed starved conditions , respectively . For GFP , values were normalized to 1 for fed conditions . Data show mean ± s . d . for three independent experiments .

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(a) GFP-LC3 HeLa cells were cultured in fed conditions or starved using EBSS plus 10 μM hydroxychloroquine and treated with Vps34 inhibitor. Cells were fixed, and nuclei were stained using Hoechst 33342 (blue). One representative image, acquired with an iCyte imaging cytometer, for three independent experiments is depicted (X40). White arrows indicate GFP-LC3 spots, which correspond to the autophagosomes. Scale bars, 10 μm. (b) Histograms show the percentage of positive cells and GFP intensity, measured using an imaging cytometer for different compound concentrations on GFP-LC3 HeLa cells. Black and gray histograms correspond to fed and starved conditions, respectively. For GFP intensity, the values were normalized to 1 for fed conditions. Data show mean ± s.d. for three independent experiments.


( c ) Wild - type HeLa H1299 lines were cultured either in fed or starved conditions with EBSS 10 μM hydroxychloroquine ( HCQ ) , as indicated . were treated at same time with SAR405 at different concentrations . LC3 - II was measured by western blotting , GAPDH was used as a loading control . One representative experiment for three independent experiments is shown . Full - blots are shown in Supplementary Figure 18 . ( d ) Histograms show LC3 - II / GAPDH for each condition in two lines . Black gray histograms correspond to fed condition EBSS conditions , respectively . addition of hydroxychloroquine at 10 μM is indicated . Values were normalized to 1 for fed conditions . Data show mean ± s . d . for three independent experiments .

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(c) Wild-type HeLa and H1299 cell lines were cultured either in fed or starved conditions with EBSS and 10 μM hydroxychloroquine (HCQ), as indicated. Cells were treated at the same time with SAR405 at different concentrations. LC3-II was measured by western blotting, and GAPDH was used as a loading control. One representative experiment for three independent experiments is shown. Full-length blots are shown in Supplementary Figure 18. (d) Histograms show the LC3-II/GAPDH ratio for each condition in the two cell lines. Black and gray histograms correspond to fed condition and EBSS conditions, respectively. The addition of hydroxychloroquine at 10 μM is indicated. Values were normalized to 1 for fed conditions. Data show mean ± s.d. for three independent experiments.


( a ) GFP - LC3 H1299 were treated with 1 μM of catalytic mTOR inhibitor AZD8055 in fed conditions with DMSO ( left ) or with 1 μM SAR405 ( right ) . were fixed , nuclei were stained using Hoechst 33342 ( blue ) . One representative image of out of three independent experiments , acquired using an iCyte imaging cytometer , is shown ( X40 ) . arrows indicate GFP - LC3 spots , which correspond to autophagosomes . Scale bars , 10 μm . ( b ) Histograms show of positive ( top ) GFP ( bottom ) , as measured using an imaging cytometer . Treatment of GFP - LC3 H1299 in fed conditions with 1 μM of AZD8055 different concentrations of SAR405 is indicated . For GFP , values were normalized to 1 for fed conditions . Data show mean ± s . d . for three independent experiments .

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(a) GFP-LC3 H1299 cells were treated with 1 μM of the catalytic mTOR inhibitor AZD8055 in fed conditions with DMSO (left) or with 1 μM SAR405 (right). Cells were fixed, and nuclei were stained using Hoechst 33342 (blue). One representative image of cells out of three independent experiments, acquired using an iCyte imaging cytometer, is shown (X40). White arrows indicate the GFP-LC3 spots, which correspond to the autophagosomes. Scale bars, 10 μm. (b) Histograms show the percentage of positive cells (top) and the GFP intensity (bottom), as measured using an imaging cytometer. Treatment of GFP-LC3 H1299 in fed conditions with 1 μM of AZD8055 and different concentrations of SAR405 is indicated. For GFP intensity, the values were normalized to 1 for fed conditions. Data show mean ± s.d. for three independent experiments.


( c ) needed to reach 40 % inhibition ( IC40 ) of proliferation is indicated . top histograms indicate IC40 of SAR405 as a single agent in combination with everolimus in three lines . bottom histograms indicate IC40 of everolimus as a single agent in combination with SAR405 . IC40 values for combination of two compounds were determined using rays with an effective fraction ∼ 0 . 5 , corresponding to compounds that are in equipotent proportion in mixture ( Supplementary Fig . 20 ) . absolute IC40 determination of both compounds is represented as geometric mean from two or three independent experiments .

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(c) The concentration needed to reach 40% inhibition (IC40) of cell proliferation is indicated. The top histograms indicate the IC40 of SAR405 as a single agent and in combination with everolimus in the three cell lines. The bottom histograms indicate the IC40 of everolimus as a single agent and in combination with SAR405. The IC40 values for the combination of the two compounds were determined using rays with an effective fraction ∼0.5, corresponding to compounds that are in equipotent proportion in the mixture (Supplementary Fig. 20). The absolute IC40 determination of both compounds is represented as the geometric mean from two or three independent experiments.


D Cruciform unfolding with increasing WRN concentrations cruciform consisting of random inverted repeats ( random - IR ) . Top , quantitation of cruciform unfolding . amount of linear from " No " lane was subtracted from other samples . Averages shown ; n = 3 technical replicates ; error bars , .

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D Cruciform unfolding assay with increasing WRN concentrations and cruciform consisting of random inverted repeats (random-IR). Top, quantitation of cruciform unfolding. The amount of linear DNA from the "No protein" lane was subtracted from all other samples. Averages shown; n = 3 technical replicates; error bars, SEM.


E Cruciform unfolding assays as in ( D ) with WRN , WRN K577M , WRN E84A ( used at 10 nM ) , carried out either in presence or absence of ATP , or with ATP - ƔS . Averages shown ; n ≥ 3 technical replicates ; error bars , .

O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O

E Cruciform unfolding assays as in (D) with WRN, WRN K577M, and WRN E84A (all used at 10 nM), carried out either in the presence or absence of ATP, or with ATP-ƔS. Averages shown; n ≥ 3 technical replicates; error bars, SEM.


G Representative cruciform unfolding assays with increasing concentrations of WRN , together with human RPA ( hRPA , 30 nM ) , yeast S . cerevisiae RPA ( yRPA , 30 nM ) , or human mitochondrial SSB ( hmtSSB , 50 nM ) . pUC19 with random - IR cruciform structure was used as a substrate . H Quantitation of assays such as in ( G ) . amount of linear from " No " lane was subtracted from other samples . Averages shown ; n ≥ 3 technical replicates ; error bars , .

O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Representative cruciform unfolding assays with increasing concentrations of WRN, together with human RPA (hRPA, 30 nM), yeast S. cerevisiae RPA (yRPA, 30 nM), or human mitochondrial SSB (hmtSSB, 50 nM). pUC19 with the random-IR cruciform structure was used as a substrate. H Quantitation of assays such as in (G). The amount of linear DNA from the "No protein" lane was subtracted from all other samples. Averages shown; n ≥ 3 technical replicates; error bars, SEM.