( E ) Kaplan Meier plots from survival analyses of human breast cancers patient samples ( GSE18229 ) showing relapse free survival ( RFS ) based on the combined expression of the 9 selected ZEB1 / YAP / AP - 1 activated targets HR : hazard ratio . p - value from log - rank test . ( F ) Kaplan Meier plots from survival analyses of human ER - / PR - breast cancers showing distant metastasis free survival ( DMFS ) based on the combined expression of the 9 selected ZEB1 / YAP / AP - 1 activated targets HR : hazard ratio . p - value from log - rank test .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-DISEASE I-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-DISEASE I-DISEASE I-DISEASE I-DISEASE I-DISEASE I-DISEASE I-DISEASE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O

(E) Kaplan Meier plots from survival analyses of human breast cancers patient samples (GSE18229) showing relapse free survival (RFS) based on the combined expression of the 9 selected ZEB1/YAP/AP-1 activated targets HR: hazard ratio. p-value from log-rank test. (F) Kaplan Meier plots from survival analyses of human ER-/PR- breast cancers showing distant metastasis free survival (DMFS) based on the combined expression of the 9 selected ZEB1/YAP/AP-1 activated targets HR: hazard ratio. p-value from log-rank test.


( 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 .

O O O O O O B-ORGANISM B-TISSUE O B-CELL_LINE O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-SUBCELLULAR O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD 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

(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 .

O O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O

(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 .

O O O O O O B-CELL_LINE O O O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

(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 , b ) Subcellular fractionation of FAO ( a ) 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 .

O O O O O O O O B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O

(a,b) Subcellular fractionation of FAO (a) 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.


( a , b ) Subcellular fractionation of 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 .

O O O O O O O O B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O

(a,b) Subcellular fractionation of 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 .

O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(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 ) 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 .

O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O

(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 .

O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-TISSUE O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O

(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 .

O O O O O O O B-CELL_LINE O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O

(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 .

O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O B-CELL_LINE O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(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 ) 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 .

O O O O O O O B-GENEPROD O B-GENEPROD B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD 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

(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 .

O O O O B-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE 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 B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD 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 O O

(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 .

O O O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O

(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 .

O O O O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR 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

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

O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O

(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 .

O O O O O O B-ORGANISM B-CELL_TYPE 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 B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O

(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 .

O O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O

(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 .

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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 ) 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 cells expressing Flag - TSC1 and TSC2 - TSC2 wild type , Flag - TSC2 mutants or the Flag - TSC2 GAP mutant ( L1624P ) , for the 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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 ) .

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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).


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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 ) ± all - trans RA ( 10 μM ) for 3 days and subjected to Oil red O staining . 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 analysis of cell lysates from AGN193109 ± RA - treated HSCs in ( G ) . * * P = 0 . 003 , paired t - test ( 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 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 .

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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 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 .

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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 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 ) .

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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 analysis of whole - cell lysates from albumin - and / 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 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 .

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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.


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 .

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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 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 .

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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 concentration , 0 . 15 μM ) for 30 min , washed , and 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 the two different STRA6 - specific siRNAs , and after 48 h , cells were incubated with R - III for 30 min and 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 and CCl4 - , CCl4 / albumin - , CCl4 / RBP - , and 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 and Sirius red , and 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 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 ) .

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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 analysis on liver extracts from the 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 analysis of α - SMA on representative liver sections from the 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 and CCl4 - and CCl4 / R - III - treated mice were immunohistochemically stained for TGF - β and F4 / 80 . Scale bar , 50 μm .

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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 , and α - SMA . His - positive signals largely overlapped with desmin / α - SMA staining . 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 and CCl4 - and 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 the livers ( μg / g liver ) . * P = 0 . 034 , two - sample t - test ( 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 , and 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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.


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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 levels in Hdac1 - proficient and - deficient cell 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 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 .

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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 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 .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY 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 B-SMALL_MOLECULE O O

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 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 .

O O O B-GENEPROD O B-GENEPROD B-CELL_LINE I-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O

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 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 .

O O B-SUBCELLULAR O B-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O O

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 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 .

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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 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 ) .

O O O B-GENEPROD O B-GENEPROD B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O

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 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 .

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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 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 .

O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

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 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 .

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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 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 .

B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O

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 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 ) .

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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 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 ) .

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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 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 .

B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD 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

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 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 .

O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD B-CELL_LINE O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-EXP_ASSAY O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O

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 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 .

B-SMALL_MOLECULE O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

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 proteins ( FK1 ) and PML - NBs in PML - GFP HeLa cells treated with HS at 42 ˚ C for 2 h . Scale bars : 5 µm .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD B-CELL_LINE O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY 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

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 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 .

O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD B-CELL_LINE O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY 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

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 cells left untreated or treated with MG132 ( 10 µM ) and 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 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 .

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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 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 .

B-CELL_LINE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O

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 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 .

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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 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 .

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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.


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 ) .

O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR 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

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 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 .

O O O O O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR 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 B-GENEPROD O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O

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 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 .

B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD B-CELL_LINE I-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY 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

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 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 .

O O O O O B-GENEPROD O B-SUBCELLULAR O O B-SMALL_MOLECULE 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 B-GENEPROD O B-SUBCELLULAR O O O O B-GENEPROD O O

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 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 .

O O O O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O B-CELL_LINE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O B-EXP_ASSAY 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

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 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 .

O O O O O B-GENEPROD O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O B-CELL_LINE 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 B-GENEPROD O B-SUBCELLULAR O O O O B-GENEPROD O O

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 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 .

B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE 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 B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O O O

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 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 .

B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O O O

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.