figf1 ( a ) Representative images of rat liver ( FAO ) cells immunostained with TSC2 , TSC1 or Rheb ( green ) and PMP70 ( peroxisome marker ) or LAMP1 ( lysosome marker ) ( red ) antibodies . Scale bars , 10 μm . ( b ) Pearson ' s correlation coefficient for TSC1 or TSC2 co - localization with PMP70 or LAMP1 calculated using Imaris software . Quantification was performed on 8 - 12 cells from each of the 4 independent experiments , giving rise to a total of 40 cells . All error bars represent s . e . m . , * * * P0 . 001 . ( c ) Representative images using HeLa cells transfected with Myc - TSC1 and Flag - TSC2 wild type ( WT ) and stained with anti - Flag ( green ) and anti - Myc ( red ) antibodies . Scale bar , 10 μm . ( d ) Representative images of HepG2 cells transfected with control or TSC2 siRNA immunostained for TSC2 ( green ) and PMP70 ( red ) . Individual boundaries are shown to identify cells with TSC2 knockdown ( white ) versus cells that retain TSC2 ( yellow ) . Scale bars , 10 μm . ( e ) Corresponding immunoblots for HepG2 cells transfected with control or TSC2 siRNA showing the extent of knockdown ( average over population of cells ) . ( f ) HepG2 cells from d were analysed for Pearson ' s correlation coefficient of TSC2 co - localization with PMP70 using Imaris software . Quantification was performed on 8 - 12 cells from each of the 4 independent experiments , giving rise to a total of 40 cells . All error bars represent s . e . m . , * * * P0 . 001 . Uncropped images of western blots are shown in Supplementary Fig . S6 . Source data for the statistical analysis are shown in Supplementary Table S1 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1(a) Representative images of rat liver (FAO) cells immunostained with TSC2, TSC1 or Rheb (green) and PMP70 (peroxisome marker) or LAMP1 (lysosome marker) (red) antibodies. Scale bars, 10 μm. (b) Pearson's correlation coefficient for TSC1 or TSC2 co-localization with PMP70 or LAMP1 calculated using Imaris software. Quantification was performed on 8-12 cells from each of the 4 independent experiments, giving rise to a total of 40 cells. All error bars represent s.e.m., ***P0.001.(c) Representative images using HeLa cells transfected with Myc-TSC1 and Flag-TSC2 wild type (WT) and stained with anti-Flag (green) and anti-Myc (red) antibodies. Scale bar, 10 μm.(d) Representative images of HepG2 cells transfected with control or TSC2 siRNA immunostained for TSC2 (green) and PMP70 (red). Individual boundaries are shown to identify cells with TSC2 knockdown (white) versus cells that retain TSC2 (yellow). Scale bars, 10 μm. (e) Corresponding immunoblots for HepG2 cells transfected with control or TSC2 siRNA showing the extent of knockdown (average over population of cells). (f) HepG2 cells from d were analysed for Pearson's correlation coefficient of TSC2 co-localization with PMP70 using Imaris software. Quantification was performed on 8-12 cells from each of the 4 independent experiments, giving rise to a total of 40 cells. All error bars represent s.e.m., ***P0.001. Uncropped images of western blots are shown in Supplementary Fig. S6. Source data for the statistical analysis are shown in Supplementary Table S1.


figf2 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2(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.(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.


figf3 ( a ) Western analysis of whole - cell extracts ( WCE ) , membrane ( M ) , cytosolic ( C ) and peroxisome ( P ) fractions from parental and MCF - 7 cells stably expressing constitutively active myristoylated AKT ( myr - AKT ) immunoblotted for TSC2 , LDH , catalase and PMP70 . Light and dark represent short and long autoradiographic exposures , respectively . ( b ) Western analysis of whole - cell extracts , membrane , cytosolic and peroxisome fractions from MCF - 7 cells stimulated with insulin ( 200 nM ) for 30 min after 1 h of serum starvation and immunoblotted for TSC2 , LDH , catalase and PMP70 . Light and dark represent short and long autoradiographic exposures , respectively . ( c ) Representative western analysis of HEK293 whole - cell extracts , cytosolic and peroxisome fractions , immunoblotted for phospho - TSC2 ( S939 ; inactive ) , TSC2 and phospho - AKT ( S473 and T308 ; activated ) . LDH and catalase were used as markers for the cytosolic and peroxisome fractions , respectively . ( d ) Proteinase K protection assays performed in the presence or absence of Triton X - 100 to disrupt peroxisomes on equal masses of peroxisome fractions ( HEK293 cells ) collected at the indicated time points . The lysates were immunoblotted for TSC2 , TSC1 , Rheb , catalase and PMP70 . Uncropped images of western blots are shown in Supplementary Fig . S6 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(a) Western analysis of whole-cell extracts (WCE), membrane (M), cytosolic (C) and peroxisome (P) fractions from parental and MCF-7 cells stably expressing constitutively active myristoylated AKT (myr-AKT) immunoblotted for TSC2, LDH, catalase and PMP70. Light and dark represent short and long autoradiographic exposures, respectively.(b) Western analysis of whole-cell extracts, membrane, cytosolic and peroxisome fractions from MCF-7 cells stimulated with insulin (200 nM) for 30 min after 1 h of serum starvation and immunoblotted for TSC2, LDH, catalase and PMP70. Light and dark represent short and long autoradiographic exposures, respectively.(c) Representative western analysis of HEK293 whole-cell extracts, cytosolic and peroxisome fractions, immunoblotted for phospho-TSC2 (S939; inactive), TSC2 and phospho-AKT (S473 and T308; activated). LDH and catalase were used as markers for the cytosolic and peroxisome fractions, respectively.(d) Proteinase K protection assays performed in the presence or absence of Triton X-100 to disrupt peroxisomes on equal masses of peroxisome fractions (HEK293 cells) collected at the indicated time points. The lysates were immunoblotted for TSC2, TSC1, Rheb, catalase and PMP70. Uncropped images of western blots are shown in Supplementary Fig. S6.


figf4 ( a ) Representative merged images using GFP - LC3 MCF - 7 cells expressing Flag - TSC1 , Flag - TSC2 or both Flag - TSC1 and Flag - TSC2 showing GFP - LC3 ( green ) puncta . Scale bars , 10 μm . ( b ) Quantification of GFP - LC3 puncta was performed and the results are represented as the average puncta fluorescence per cell ( ± s . e . m . , n = 3 independent experiments ) from 100 cells per experiment as shown in a . * * * P0 . 001 ; NS , not significant . ( c ) Representative immunocytochemistry images using MCF - 7 cells transfected with an mRFP - GFP - LC3 construct . Cells treated with 0 . 4 mM H2O2 were analysed at 0 h ( Control ) , 1 h , 3 h and 6 h . Scale bars , 10 μm . ( d ) Quantification of autophagosomes ( AP , GFP - LC3 ) and autolysosomes ( AL , RFP - LC3 ) per cell in different conditions as shown in Fig . 4c . The results are represented as the average puncta fluorescence per cell ( ± s . e . m . , n = 3 independent experiments ) from 100 cells per experiment . * * P0 . 01 ; * * * P0 . 001 . ( e ) Western analysis of FAO cells treated with 50 μM Wy - 14643 ( WY ) or vehicle ( DMSO ) for PPAR - alpha - inducible proteins ( EHHADH and ACAA1 ) , mTORC1 signalling proteins ( ( pS6 ( S235 / 236 ) , S6 , pS6K ( T389 ) and S6K ) ) and autophagy markers ( LC3 and p62 ) . ( f ) Representative images of FAO cells treated with vehicle ( DMSO ) or 50 μM Wy - 14643 ( WY ) for 1 h , with superoxide production detected using dihydroethidium ( DHE ) . Scale bars , 30 μm . ( g ) Representative images of LC3 puncta ( green ) in FAO cells following treatment with 50 μM Wy - 14643 ( WY ) or vehicle ( DMSO ) for the indicated time periods . Scale bars , 10 μm . ( h ) Quantification of LC3 puncta per cell in response to Wy - 14643 ( WY ) or vehicle ( DMSO ) . The results are represented as the average LC3 puncta fluorescence per cell ( ± s . e . m . , n = 3 independent experiments ) from 100 cells per experiment as shown in Fig . 4a . * P0 . 05 ; * * P0 . 01 . ( i ) Transmission electron microscopy of FAO cells treated with vehicle ( DMSO ) or 50 μM Wy - 14643 ( WY ) for 24 , 48 and 72 h . Peroxisomes and autophagosomes are indicated with red and yellow arrows , respectively . Scale bars , 500 nm . Uncropped images of western blots are shown in Supplementary Fig . S6 . Source data for the statistical analysis are shown in Supplementary Table S1 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(a) Representative merged images using GFP-LC3 MCF-7 cells expressing Flag-TSC1, Flag-TSC2 or both Flag-TSC1 and Flag-TSC2 showing GFP-LC3 (green) puncta. Scale bars, 10 μm. (b) Quantification of GFP-LC3 puncta was performed and the results are represented as the average puncta fluorescence per cell (±s.e.m., n = 3 independent experiments) from 100 cells per experiment as shown in a. ***P0.001; NS, not significant.(c) Representative immunocytochemistry images using MCF-7 cells transfected with an mRFP-GFP-LC3 construct. Cells treated with 0.4 mM H2O2 were analysed at 0 h (Control), 1 h, 3 h and 6 h. Scale bars, 10 μm. (d) Quantification of autophagosomes (AP, GFP-LC3) and autolysosomes (AL, RFP-LC3) per cell in different conditions as shown in Fig. 4c. The results are represented as the average puncta fluorescence per cell (±s.e.m., n = 3 independent experiments) from 100 cells per experiment. **P0.01;***P0.001.(e) Western analysis of FAO cells treated with 50 μM Wy-14643 (WY) or vehicle (DMSO) for PPAR-alpha-inducible proteins (EHHADH and ACAA1), mTORC1 signalling proteins ((pS6 (S235/236), S6, pS6K (T389) and S6K)) and autophagy markers (LC3 and p62). (f) Representative images of FAO cells treated with vehicle (DMSO) or 50 μM Wy-14643 (WY) for 1 h, with superoxide production detected using dihydroethidium (DHE). Scale bars, 30 μm.(g) Representative images of LC3 puncta (green) in FAO cells following treatment with 50 μM Wy-14643 (WY) or vehicle (DMSO) for the indicated time periods. Scale bars, 10 μm. (h) Quantification of LC3 puncta per cell in response to Wy-14643 (WY) or vehicle (DMSO). The results are represented as the average LC3 puncta fluorescence per cell (±s.e.m., n = 3 independent experiments) from 100 cells per experiment as shown in Fig. 4a. *P0.05; **P0.01.(i) Transmission electron microscopy of FAO cells treated with vehicle (DMSO) or 50 μM Wy-14643 (WY) for 24, 48 and 72 h. Peroxisomes and autophagosomes are indicated with red and yellow arrows, respectively. Scale bars, 500 nm. Uncropped images of western blots are shown in Supplementary Fig. S6. Source data for the statistical analysis are shown in Supplementary Table S1.


figf5 ( a ) Representative images of Zellweger cells ( GM13267 ) showing endogenous TSC2 , TSC1 and Rheb ( green ) co - localization with PMP70 ( red ) . Scale bars , 15 μm . ( b ) Western analysis of human fibroblasts obtained from a Zellweger ( GM13267 ) or a corresponding control patient ( with Ehlers - Danlos syndrome ( GM15871 ) ) treated with the indicated doses of H2O2 for 1 h . mTORC1 signalling was assessed by western analysis for pS6K ( T389 ) , S6K , pS6 ( S235 / 236 ) , S6 , p4E - BP1 ( T37 / 46 ) , 4E - BP1 , pATM ( S1981 ) , ATM , pAMPK ( T172 ) , AMPK , p62 and LC3 . ( c ) Western analysis of human fibroblast ( GM13427 ) cells pre - incubated with 3 mM N - acetyl cysteine ( NAC ; ROS scavenger ) for 1 h before being treated with 0 . 4 mM H2O2 for 1 h using anti - pS6K ( T389 ) , S6K , p62 and LC3 antibodies . ( d ) Representative western analysis using cell extracts from human fibroblasts obtained from a Zellweger patient ( GM13267 ) or control fibroblasts ( GM15871 ) treated with amino - acid - free media for 60 min , and stimulated with a mixture of amino acids for 10 min . mTOR signalling was monitored using anti - pS6K ( T389 ) , S6K , pS6 ( S235 / 236 ) , S6 , p4E - BP1 ( T37 / 46 ) and 4E - BP1 antibodies . Uncropped images of western blots are shown in Supplementary Fig . S6 .

O B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a) Representative images of Zellweger cells (GM13267) showing endogenous TSC2, TSC1 and Rheb (green) co-localization with PMP70 (red). Scale bars, 15 μm.(b) Western analysis of human fibroblasts obtained from a Zellweger (GM13267) or a corresponding control patient (with Ehlers-Danlos syndrome (GM15871)) treated with the indicated doses of H2O2 for 1 h. mTORC1 signalling was assessed by western analysis for pS6K (T389), S6K, pS6 (S235/236), S6, p4E-BP1 (T37/46), 4E-BP1, pATM (S1981), ATM, pAMPK (T172), AMPK, p62 and LC3.(c) Western analysis of human fibroblast (GM13427) cells pre-incubated with 3 mM N-acetyl cysteine (NAC; ROS scavenger) for 1 h before being treated with 0.4 mM H2O2 for 1 h using anti-pS6K (T389), S6K, p62 and LC3 antibodies.(d) Representative western analysis using cell extracts from human fibroblasts obtained from a Zellweger patient (GM13267) or control fibroblasts (GM15871) treated with amino-acid-free media for 60 min, and stimulated with a mixture of amino acids for 10 min. mTOR signalling was monitored using anti-pS6K (T389), S6K, pS6 (S235/236), S6, p4E-BP1(T37/46) and 4E-BP1 antibodies. Uncropped images of western blots are shown in Supplementary Fig. S6.


figf6 ( 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 . ( 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 . ( 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 . ( 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf6(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.(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.(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.(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.


figf7 ( 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 . ( 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 . ( 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 .

O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf7(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.(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.(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.


Figure 1 ( A - B ) Spinal cord sections from 5 - month - old WT and Tmem106b - / - mice were stained with anti - neurofascin , anti - LAMP1 and anti - Cath D antibodies . Accumulation of LAMP1 and CathD positive vacuoles at the distal end of AIS was observed in Tmem106b - / - motor neurons . Representative images from three independent mice were shown . Scale bar = 10 µm . n = 3 . Data presented as mean ± SEM . Unpaired Student ' s t test . * * * * , p < 0 . 0001 . ( C - F ) Western blot analysis of p62 , ubiquitin ( Ub ) , TDP - 43 and p - TDP - 43 in RIPA - and urea - soluble fractions from brain ( C and E ) and spinal cord ( D and F ) of 16 - month - old WT and Tmem106b - / - mice . n = 5 . Data presented as mean ± SEM . Unpaired Student ' s t test . * , p < 0 . 05 , * * * , p < 0 . 001 , * * * * , p < 0 . 0001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A-B) Spinal cord sections from 5-month-old WT and Tmem106b-/- mice were stained with anti- neurofascin, anti- LAMP1 and anti-Cath D antibodies. Accumulation of LAMP1 and CathD positive vacuoles at the distal end of AIS was observed in Tmem106b-/- motor neurons. Representative images from three independent mice were shown. Scale bar = 10 µm. n=3. Data presented as mean ± SEM. Unpaired Student's t test. ****, p<0.0001.(C-F) Western blot analysis of p62, ubiquitin (Ub), TDP-43 and p-TDP-43 in RIPA- and urea-soluble fractions from brain (C and E) and spinal cord (D and F) of 16-month-old WT and Tmem106b-/- mice. n=5. Data presented as mean ± SEM. Unpaired Student's t test. *, p<0.05, ***, p<0.001, ****, p<0.0001.


Figure 2 ( A ) Tmem106b - / - Grn - / - mice do not show any changes in body weight . Body weight of 5 - month - old mice of indicated genotypes were measured and analyzed . n = 6 - 10 . ( B Tmem106b - / - Grn - / - mice show reduced activities in the open field test . 4 . 5 - month - old mice were used in the study . C ) Tmem106b - / - Grn - / - mice show reduced activities in the open field test . 4 . 5 - month - old mice were used in the study . Total movement was quantified . n = 8 - 10 . Data presented as mean ± SEM . One - way ANOVA tests with Bonferroni ' s multiple comparisons : * * * * , p < 0 . 0001 . ( D Tmem106b - / - Grn - / - mice show abnormal hindlimb clasping behavior . 5 - month - old mice were used in the study . E ) Tmem106b - / - Grn - / - mice show abnormal hindlimb clasping behavior . 5 - month - old mice were used in the study . Severity score was quantified . n = 8 - 10 . Data presented as mean ± SEM . One - way ANOVA tests with Bonferroni ' s multiple comparisons : * * * , p < 0 . 001 , * * * * , p < 0 . 0001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Tmem106b-/-Grn-/- mice do not show any changes in body weight. Body weight of 5-month-old mice of indicated genotypes were measured and analyzed. n=6-10.(B Tmem106b-/-Grn-/- mice show reduced activities in the open field test. 4.5-month-old mice were used in the study.C) Tmem106b-/-Grn-/- mice show reduced activities in the open field test. 4.5-month-old mice were used in the study. Total movement was quantified. n=8-10. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ****, p<0.0001.(D Tmem106b-/-Grn-/- mice show abnormal hindlimb clasping behavior. 5-month-old mice were used in the study.E) Tmem106b-/-Grn-/- mice show abnormal hindlimb clasping behavior. 5-month-old mice were used in the study. Severity score was quantified. n= 8-10. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001.


Figure 3 ( A , B ) Spinal cord sections ( C1 - C4 ) from 5 - month - old mice of indicated genotypes were stained with indicated antibodies ( A ) and the numbers of NeuN , or CHAT positive cells , as well as IBA1 and GFAP intensities were quantified ( B ) . 3 - 5 sections / mouse were used for quantification . n = 3 . Data presented as mean ± SEM . One - way ANOVA tests with Bonferroni ' s multiple comparisons : * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 , * * * * , p < 0 . 0001 . Scale bar : 20 µm for CHAT staining , 200 µm for other images .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A,B) Spinal cord sections (C1-C4) from 5-month-old mice of indicated genotypes were stained with indicated antibodies (A) and the numbers of NeuN, or CHAT positive cells, as well as IBA1 and GFAP intensities were quantified (B). 3-5 sections/mouse were used for quantification. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001. Scale bar: 20 µm for CHAT staining, 200 µm for other images.


Figure 4 ( A - D ) Western blot analysis of NeuN , PSD95 , SYN , GFAP , TMEM106B , PGRN and GAPDH in spinal cord ( C5 - C8 ) ( A , B ) and brain ( C , D ) lysates from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A-D) Western blot analysis of NeuN, PSD95, SYN, GFAP, TMEM106B, PGRN and GAPDH in spinal cord (C5-C8) (A, B) and brain (C, D) lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice.


Figure 5 ( A , B ) Retinal sections from 5 - month - old mice were stained with indicated antibodies . Thickness of each layer , number of Brn3a positive cells ( retinal ganglion cells ) per section and length of outer segment as indicated by opsin staining are quantified . Data information : ONL : outer nuclear layer ; INL : Inner nuclear layer ; OPL : outer plexiform layer ; IPL : inner plexiform layer ; GCL : ganglion cell layer . ( C Increase in apoptosis in Grn - / - and Tmem106b - / - Grn - / - retina as indicated by TUNEL assay . TUNEL positive signals per section were quantified . Data information : ONL : outer nuclear layer ; INL : Inner nuclear layer ; OPL : outer plexiform layer ; IPL : inner plexiform layer ; GCL : ganglion cell layer . D ) Increase in apoptosis in Grn - / - and Tmem106b - / - Grn - / - retina as indicated by TUNEL assay . TUNEL positive signals per section were quantified . ( E Increase in Müller glial activation in Tmem106b - / - Grn - / - retina as indicated by GFAP staining . GFAP intensity per section was quantified and normalized to WT control . Data information : ONL : outer nuclear layer ; INL : Inner nuclear layer ; OPL : outer plexiform layer ; IPL : inner plexiform layer ; GCL : ganglion cell layer . F ) Increase in Müller glial activation in Tmem106b - / - Grn - / - retina as indicated by GFAP staining . GFAP intensity per section was quantified and normalized to WT control . ( G , H ) Increase in microglia activation in Grn - / - and Tmem106b - / - Grn - / - retina as indicated by IBA1 staining . The number of IBA1 positive cells per section was quantified . Data information : ONL : outer nuclear layer ; INL : Inner nuclear layer ; OPL : outer plexiform layer ; IPL : inner plexiform layer ; GCL : ganglion cell layer .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A, B) Retinal sections from 5-month-old mice were stained with indicated antibodies. Thickness of each layer, number of Brn3a positive cells (retinal ganglion cells) per section and length of outer segment as indicated by opsin staining are quantified. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer.(C Increase in apoptosis in Grn-/- and Tmem106b-/-Grn-/- retina as indicated by TUNEL assay. TUNEL positive signals per section were quantified. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer.D) Increase in apoptosis in Grn-/- and Tmem106b-/-Grn-/- retina as indicated by TUNEL assay. TUNEL positive signals per section were quantified.(E Increase in Müller glial activation in Tmem106b-/-Grn-/- retina as indicated by GFAP staining. GFAP intensity per section was quantified and normalized to WT control. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer.F) Increase in Müller glial activation in Tmem106b-/-Grn-/- retina as indicated by GFAP staining. GFAP intensity per section was quantified and normalized to WT control.(G, H) Increase in microglia activation in Grn-/- and Tmem106b-/-Grn-/- retina as indicated by IBA1 staining. The number of IBA1 positive cells per section was quantified. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer.


Figure 6 ( A Heatmap illustrating gene expression changes in WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - cortex samples isolated from 2 . 7 - month - old mice . n = 3 . Genes with FDR p ≤ 0 . 05 , LogFC ≥ 0 . 5 or ≤ 0 . 5 between WT and Tmem106b - / - Grn - / - groups are plotted based on hierarchical clustering analysis . Data information : Inflammation genes are highlighted in magenta and lysosomal genes are highlighted in blue . B ) Heatmap illustrating gene expression changes in WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - spinal cord samples isolated from 2 . 7 - month - old mice . n = 3 . Genes with FDR p ≤ 0 . 05 , LogFC ≥ 0 . 5 or ≤ 0 . 5 between WT and Tmem106b - / - Grn - / - groups are plotted based on hierarchical clustering analysis . Data information : Inflammation genes are highlighted in magenta and lysosomal genes are highlighted in blue . ( C ) Heatmap illustrating gene expression changes in the inflammation and lysosome pathways in WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - spinal cord samples . Data information : Inflammation genes are highlighted in magenta and lysosomal genes are highlighted in blue .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A Heatmap illustrating gene expression changes in WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- cortex samples isolated from 2.7-month-old mice. n=3. Genes with FDR p≤0.05, LogFC≥0.5 or ≤0.5 between WT and Tmem106b-/-Grn-/- groups are plotted based on hierarchical clustering analysis. Data information: Inflammation genes are highlighted in magenta and lysosomal genes are highlighted in blue.B) Heatmap illustrating gene expression changes in WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- spinal cord samples isolated from 2.7-month-old mice. n=3. Genes with FDR p≤0.05, LogFC≥0.5 or ≤0.5 between WT and Tmem106b-/-Grn-/- groups are plotted based on hierarchical clustering analysis. Data information: Inflammation genes are highlighted in magenta and lysosomal genes are highlighted in blue.(C) Heatmap illustrating gene expression changes in the inflammation and lysosome pathways in WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- spinal cord samples. Data information: Inflammation genes are highlighted in magenta and lysosomal genes are highlighted in blue.


Figure 7Western blot analysis of Galectin - 3 and GPNMB in the spinal cord lysates from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . n = 3 . Data presented as mean ± SEM . One - way ANOVA tests with Bonferroni ' s multiple comparisons : * * * , p < 0 . 001 , * * * * , p < 0 . 0001 . Western blot analysis of Galectin - 3 and GPNMB in the brain lysates from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . n = 3 . Data presented as mean ± SEM . One - way ANOVA tests with Bonferroni ' s multiple comparisons : * * * , p < 0 . 001 , * * * * , p < 0 . 0001 . ( E ) Immunostaining of Galectin - 3 , CathD and CD68 in spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . Scale bar = 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Western blot analysis of Galectin-3 and GPNMB in the spinal cord lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001.Western blot analysis of Galectin-3 and GPNMB in the brain lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001.(E) Immunostaining of Galectin-3, CathD and CD68 in spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Scale bar = 10 µm.


Figure 8 ( A - B ) Lipofuscin accumulation in the retina : the number of auto fluorescent puncta per section was quantified . ONL : outer nuclear layer ; INL : Inner nuclear layer ; OPL : outer plexiform layer ; IPL : inner plexiform layer ; GCL : ganglion cell layer . n = 3 - 6 . Scale bar = 50 µm . TMEM106B and PGRN deletion results in increased autofluorescence in the spinal cord of 5 - month - old mice . n = 3 . Scale bar = 10 µm . TMEM106B and PGRN deletion results in increased autofluorescence in the thalamus of 5 - month - old mice . n = 3 . Scale bar = 10 µm . ( G , H ) Western blot analysis of lysosomal proteins in the spinal cord lysates from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . Asterisk indicates non - specific bands . n = 3 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A-B) Lipofuscin accumulation in the retina: the number of auto fluorescent puncta per section was quantified. ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer. n=3-6. Scale bar = 50 µm.TMEM106B and PGRN deletion results in increased autofluorescence in the spinal cord of 5-month-old mice. n=3. Scale bar = 10 µm.TMEM106B and PGRN deletion results in increased autofluorescence in the thalamus of 5-month-old mice. n=3. Scale bar = 10 µm.(G, H) Western blot analysis of lysosomal proteins in the spinal cord lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Asterisk indicates non-specific bands. n=3.


Figure 9 ( A ) Spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - and Tmem106b - / - Grn - / - mice were stained with anti - neurofascin , anti - LAMP1 and anti - CathD antibodies . Representative images from three independent mice were shown . Scale bar = 10 µm . ( B ) Quantification of LAMP1 positive vacuoles ( > 2 µm ) for experiments in ( A ) . 4 - 6 sections / spinal cord from 3 mice were analyzed for each genotype . Data presented as mean ± SEM . Unpaired Student ' s t test : * * * * , p < 0 . 0001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9(A) Spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/- and Tmem106b-/-Grn-/- mice were stained with anti-neurofascin, anti-LAMP1 and anti-CathD antibodies. Representative images from three independent mice were shown. Scale bar = 10 µm. (B) Quantification of LAMP1 positive vacuoles (> 2 µm) for experiments in (A). 4-6 sections/spinal cord from 3 mice were analyzed for each genotype. Data presented as mean ± SEM. Unpaired Student's t test: ****, p<0.0001.


Figure 10 ( A Immunostaining of GFAP with LAMP1 and Cath D in the spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . Scale bar = 5 µm . B ) Immunostaining of IBA1 with LAMP1 and Cath D in the spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . Scale bar = 5 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 10(A Immunostaining of GFAP with LAMP1 and Cath D in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Scale bar = 5 µm.B) Immunostaining of IBA1 with LAMP1 and Cath D in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Scale bar = 5 µm.


Figure 11 ( A ) Accumulation of autophagosomes and lysosomes in the spinal cord sections from a 5 - month - old Tmem106b - / - Grn - / - mouse as shown by transmission electron microscope analysis . Scale bar = 1µm . Arrows : autophagosome ; arrow heads : lysosomes ; ( a ) : axon . ( B ) Accumulation of myelin debris and amorphous inclusions in the microglia in the spinal cord sections from a 5 - month - old Tmem106b - / - Grn - / - mouse as shown by transmission electron microscope analysis . Scale bar = 1µm . Arrows : myelin debris ; arrow heads : dark amorphous inclusions ; ( m ) : microglia . ( C ) Western blot analysis of LC3 - I and LC3 - II in the spinal cord lysates from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . n = 3 . Data presented as mean ± SEM . One - way ANOVA tests with Bonferroni ' s multiple comparisons : * * * * , p < 0 . 0001 . ( D Immunostaining of TFE3 and IBA1 in the spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . E ) Immunostaining of TFE3 and IBA1 in the spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . Nuclear signals of TFE3 in IBA1 positive microglia were quantified . n = 3 . Data presented as mean ± SEM . One - way ANOVA tests with Bonferroni ' s multiple comparisons : * * * , p < 0 . 001 , * * * * , p < 0 . 0001 . Scale bar = 10 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 11(A) Accumulation of autophagosomes and lysosomes in the spinal cord sections from a 5-month-old Tmem106b-/-Grn-/- mouse as shown by transmission electron microscope analysis. Scale bar = 1µm. Arrows: autophagosome; arrow heads: lysosomes; (a): axon.(B) Accumulation of myelin debris and amorphous inclusions in the microglia in the spinal cord sections from a 5-month-old Tmem106b-/-Grn-/- mouse as shown by transmission electron microscope analysis. Scale bar = 1µm. Arrows: myelin debris; arrow heads: dark amorphous inclusions; (m): microglia.(C) Western blot analysis of LC3-I and LC3-II in the spinal cord lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ****, p<0.0001.(D Immunostaining of TFE3 and IBA1 in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice.E) Immunostaining of TFE3 and IBA1 in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Nuclear signals of TFE3 in IBA1 positive microglia were quantified. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001. Scale bar = 10 µm.


Figure 12 ( A , B ) Western blot analysis of p62 , ubiquitin ( Ub ) , TDP - 43 and p - TDP - 43 in RIPA - and urea - soluble fractions from spinal cord ( C5 - C8 ) and brain of 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . Spinal cord : n = 3 ; brain : n = 4 . Data presented as mean ± SEM . One - way ANOVA tests with Bonferroni ' s multiple comparisons : * , p < 0 . 05 ; * * , p < 0 . 01 * * * , p < 0 . 001 , * * * * , p < 0 . 0001 . ( C , D ) Immunostaining of p62 and ubiquitin in the spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . The number of p62 or Ub positive puncta was quantified . n = 3 . Data presented as mean ± SEM . One - way ANOVA tests with Bonferroni ' s multiple comparisons : * * * , p < 0 . 001 , * * * * , p < 0 . 0001 . Scale bar = 10 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 12(A, B) Western blot analysis of p62, ubiquitin (Ub), TDP-43 and p-TDP-43 in RIPA- and urea-soluble fractions from spinal cord (C5-C8) and brain of 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Spinal cord: n=3; brain: n=4. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: *, p<0.05; **, p<0.01***, p<0.001, ****, p<0.0001.(C, D) Immunostaining of p62 and ubiquitin in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. The number of p62 or Ub positive puncta was quantified. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001. Scale bar = 10 µm.


Figure 1 ( A ) The expression pattern of FOXM1 in fetal human cortex at GW15 . VZ , ventricular zone ; ISVZ , inner subventricular zone ; OSVZ , outer subventricular zone . Right panels show higher magnification images . Arrows show that FOXM1 + cells were colocalized with SOX2 + cells . Scale bars , 100μm ( left ) ; 50μm ( right ) . ( B ) Staining for NESTIN and FOXM1 in human neural progenitor cells cultured in vitro . Down panels show higher magnification images . Scale bars , 50μm ( up ) ; 10μm ( down ) . ( D ) Staining for Pax6 in E15 . 5 mice after IUE at E13 . 5 . Arrowheads indicate Pax6 + cell in enlarged SVZ . Right panels show higher magnification images . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bars , 50μm ( left ) ; 20μm ( right ) . ( G ) Staining for upper neuron marker Cux1 in P3 mice after IUE at E13 . 5 . Arrowhead and asterisk indicate deformation of the cortical surface at regions containing electroporated cells . Right panels show higher magnification images . Arrows show GFP + Cux1 + double - positive cells . Scale bars , 100μm ( left ) ; 20μm ( right ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) The expression pattern of FOXM1 in fetal human cortex at GW15. VZ, ventricular zone; ISVZ, inner subventricular zone; OSVZ, outer subventricular zone. Right panels show higher magnification images. Arrows show that FOXM1+cells were colocalized with SOX2+ cells. Scale bars, 100μm (left); 50μm (right).(B) Staining for NESTIN and FOXM1 in human neural progenitor cells cultured in vitro. Down panels show higher magnification images. Scale bars, 50μm(up); 10μm(down).(D) Staining for Pax6 in E15.5 mice after IUE at E13.5. Arrowheads indicate Pax6+ cell in enlarged SVZ. Right panels show higher magnification images. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bars,50μm(left); 20μm (right).(G) Staining for upper neuron marker Cux1 in P3 mice after IUE at E13.5. Arrowhead and asterisk indicate deformation of the cortical surface at regions containing electroporated cells. Right panels show higher magnification images. Arrows show GFP+ Cux1+ double-positive cells. Scale bars,100μm(left); 20μm (right).


Figure 2 ( A ) Representative images of whole brains from wild - type ( WT ) and hFOXM1f / + - NesCre ( hFOXM1 - cKI ) mice pups at P0 . Scale bar , 100μm . ( B ) Quantification of cortical width and length at P0 ( WT n = 7 ; hFOXM1 - cKI n = 7 ; two - tailed unpaired t - test , P = 0 . 0361for width , P = 0 . 2866 for length ) . ( C ) Quantification of brain / body weight at P0 ( WT n = 9 ; hFOXM1 - cKI n = 9 ; two - tailed unpaired t - test , P = 0 . 0004 ) . ( D ) Quantification of cortex thickness at P0 ( WT n = 7 ; hFOXM1 - cKI n = 11 ; two - tailed unpaired t - test , P = 0 . 0002 ) . ( E ) ( E ) Staining for Satb2 and Ctip2 in P0 WT and hFOXM1 - cKI mice folding structure . Asterisks indicate gyrus and arrows indicate sulcus structures . A1 , A2 , B1and B2 ( DAPI ) are high - magnification images . Scale bars , 200μm ( left ) ; 100μm ( right ) . ( F ) Quantification of GI of WT and hFOXM1 - cKI mice pups at P0 . The quantification of the GI was analyzed in pre - selected hFOXM1 - cKI mice with folding . ( n = 6 each group ; two - tailed u unpaired t - test , P = 0 . 0010 ) . ( H ) Graph shows the number of Satb2 + cells in 4 × 104µm2 at P0 ( WT n = 5 ; hFOXM1 - cKI n = 5 ; two - tailed unpaired t - test , P = 0 . 0086 ) . ( I ) Graph shows the number of Ctip2 + cells in 4 × 104µm2 at P0 ( WT n = 5 ; hFOXM1 - cKI n = 5 ; two - tailed unpaired t - test , P = 0 . 0025 ) . ( ( K ) Staining for Cux1 in P0 WT and hFOXM1 - cKI mice . Scale bar , 100μm . ( I ) Graph shows the number of Cux1 + cells in 4 × 104µm2 at P0 ( WT n = 3 ; hFOXM1 - cKI n = 3 ; two - tailed unpaired t - test , P = 0 . 0451 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Representative images of whole brains from wild-type (WT) and hFOXM1f/+-NesCre (hFOXM1-cKI) mice pups at P0. Scale bar,100μm. (B) Quantification of cortical width and length at P0 (WT n=7; hFOXM1-cKI n=7; two-tailed unpaired t-test, P= 0.0361for width, P= 0.2866 for length). (C) Quantification of brain/body weight at P0 (WT n=9; hFOXM1-cKI n=9; two-tailed unpaired t-test, P= 0.0004). (D) Quantification of cortex thickness at P0 (WT n=7; hFOXM1-cKI n=11; two-tailed unpaired t-test, P= 0.0002). (E)(E) Staining for Satb2 and Ctip2 in P0 WT and hFOXM1-cKI mice folding structure. Asterisks indicate gyrus and arrows indicate sulcus structures. A1, A2, B1and B2 (DAPI) are high-magnification images. Scale bars, 200μm(left); 100μm(right). (F) Quantification of GI of WT and hFOXM1-cKI mice pups at P0. The quantification of the GI was analyzed in pre-selected hFOXM1-cKI mice with folding. (n = 6 each group; two-tailed u unpaired t-test, P= 0.0010).(H) Graph shows the number of Satb2+cells in 4×104µm2 at P0 (WT n=5; hFOXM1-cKI n=5; two-tailed unpaired t-test, P= 0.0086). (I) Graph shows the number of Ctip2 +cells in 4×104µm2 at P0 (WT n=5; hFOXM1-cKI n=5; two-tailed unpaired t-test, P= 0.0025). ((K) Staining for Cux1 in P0 WT and hFOXM1-cKI mice. Scale bar,100μm. (I) Graph shows the number of Cux1+cells in 4×104µm2 at P0 (WT n=3; hFOXM1-cKI n=3; two-tailed unpaired t-test, P= 0.0451). D


Figure 3 ( A ) Staining for Pax6 and p - Vim in E15 WT and hFOXM1 - cKI mice . The right A and B are high - magnification images . Arrows indicate double positive of Pax6 + p - Vim + cells with cells with a basal process ( arrowheads ) . Scale bars , 50μm ( left ) ; 5μm ( right ) . ( B ) Graph shows the percentage of Pax6 + cells in enlarged SVZ ( n = 6 each group ; two - tailed unpaired t - test , P = 0 . 0002 ) . ( C ) Graph shows the number of Pax6 + p - Vim + cells in 4x10 ^ 4 μm2 in enlarged SVZ ( n = 6 each group ; two - tailed unpaired t - test , P = 0 . 0003 ) . ( D ( D ) Staining for Sox2 in E15 WT and hFOXM1 - cKI mice . Scale bar , 50μm . ( E ) Graph shows the number of Sox2 + cells in 4x104 μm2 in enlarged SVZ ( WT n = 3 ; hFOXM1 - cKI n = 4 ; two - tailed unpaired t - test , P = 0 . 0292 ) . ( ( F ) Staining for Tbr2 in E15 WT and hFOXM1 - cKI mice . Scale bar , 50μm . ( G ) Graph shows the number of Tbr2 + cells in 4x104 μm2 in enlarged SVZ ( WT n = 6 ; hFOXM1 - cKI n = 9 ; two - tailed unpaired t - test , P = 0 . 0003 ) . ( ( H ) Staining for HOPX in E15 WT and hFOXM1 - cKI mice . Scale bar , 50μm . ( I ) Graph shows the number of HOPX + cells in 4 × 104 μm2 in enlarged SVZ ( n = 4 each group ; two - tailed unpaired t - test , P = 0 . 0044 ) . ( ( J ) Staining for Ki67 in E15 WT and hFOXM1 - cKI mice . Scale bar , 50μm . ( L ) The experimental scheme and staining for BrdU and EdU in E15 WT and hFOXM1 - cKI mice . White arrows represent BrdU and EdU double - positive cells . Scale bars , 50μm ( left ) ; 20μm ( right ) . ( M ) Graph shows the percentage of BrdU and EdU double - positive cells in E15 WT and hFOXM1 - cKI mice ( n = 6 each group ; two - tailed unpaired t - test , P = 0 . 0413 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Staining for Pax6 and p-Vim in E15 WT and hFOXM1-cKI mice. The right A and B are high-magnification images. Arrows indicate double positive of Pax6+ p-Vim+ cells with cells with a basal process (arrowheads). Scale bars, 50μm (left); 5μm (right). (B) Graph shows the percentage of Pax6+ cells in enlarged SVZ (n = 6 each group; two-tailed unpaired t-test, P=0.0002). (C) Graph shows the number of Pax6+ p-Vim+ cells in 4x10^4 μm2 in enlarged SVZ (n = 6 each group; two-tailed unpaired t-test, P=0.0003). (D(D) Staining for Sox2 in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm. (E) Graph shows the number of Sox2+ cells in 4x104 μm2 in enlarged SVZ (WT n=3; hFOXM1-cKI n=4; two-tailed unpaired t-test, P=0.0292). ((F) Staining for Tbr2 in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm. (G) Graph shows the number of Tbr2+ cells in 4x104 μm2 in enlarged SVZ (WT n=6; hFOXM1-cKI n=9; two-tailed unpaired t-test, P=0.0003). ((H) Staining for HOPX in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm. (I) Graph shows the number of HOPX+ cells in 4×104 μm2 in enlarged SVZ (n = 4 each group; two-tailed unpaired t-test, P= 0.0044). ((J) Staining for Ki67 in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm.(L) The experimental scheme and staining for BrdU and EdU in E15 WT and hFOXM1-cKI mice. White arrows represent BrdU and EdU double-positive cells. Scale bars, 50μm(left);20μm(right). (M) Graph shows the percentage of BrdU and EdU double-positive cells in E15 WT and hFOXM1-cKI mice (n = 6 each group; two-tailed unpaired t-test, P=0.0413). D


Figure 4 ( B ) Cell immunostaining for HOPX in control , hFOXM1 - FL and mutants in mice primary neural progenitors 3 days following lentiviral infection . White arrows represent GFP HOPX double - positive cells . Scale bar , 50μm . ( C ) Graph shows the percentage of HOPX + cells among GFP + cells ( Control n = 3 ; hFOXM1 - FL n = 6 ; hFLΔexon9 n = 5 ; one - way ANOVA , F ( 2 , 12 ) = 29 . 13 , control vs . hFOXM1 , P = 0 . 0002 ; control vs . hFOXM1Δexon9 , P = 0 . 9080 ) . ( D ) Cell immunostaining for HOPX in control , mFoxm1 - FL and mutants in mice primary neural progenitors 3 days following lentiviral infection . White arrows represent GFP HOPX double - positive cells . Scale bar , 50μm . ( E ) ( E ) Graph shows the percentage of HOPX + cells among GFP + cells ( Control n = 4 ; mFoxm1 - FL n = 6 ; mFL + h exon9 n = 4 ; one - way ANOVA , F ( 2 , 11 ) = 8 . 163 , control vs . mFoxm1 , P = 0 . 4747 , control vs . mFoxm1 + h exon9 , P = 0 . 0066 ) . ( ( J ) Staining for Tbr2 in E15 . 5 mice after IUE at E13 . 5 . Down panels show higher magnification images . White arrows represent GFP and TBR2 double - positive cells . Scale bars , 50μm . ( K ) Graph shows the percentage of GFP + Tbr2 + / GFP + in enlarged SVZ ( n = 3 each group ; one - way ANOVA , F ( 3 , 8 ) = 8 . 787 , P = 0 . 0065 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) Cell immunostaining for HOPX in control, hFOXM1-FL and mutants in mice primary neural progenitors 3 days following lentiviral infection. White arrows represent GFP HOPX double-positive cells. Scale bar, 50μm. (C) Graph shows the percentage of HOPX+ cells among GFP+ cells (Control n=3; hFOXM1-FL n=6; hFLΔexon9 n=5; one-way ANOVA, F (2, 12) = 29.13, control vs. hFOXM1, P=0.0002; control vs. hFOXM1Δexon9, P=0.9080). (D) Cell immunostaining for HOPX in control, mFoxm1-FL and mutants in mice primary neural progenitors 3 days following lentiviral infection. White arrows represent GFP HOPX double-positive cells. Scale bar, 50μm. (E) (E) Graph shows the percentage of HOPX+ cells among GFP+ cells (Control n=4; mFoxm1-FL n=6; mFL+h exon9 n=4; one-way ANOVA, F (2, 11) = 8.163, control vs. mFoxm1, P= 0.4747, control vs. mFoxm1+h exon9, P= 0.0066). ((J) Staining for Tbr2 in E15.5 mice after IUE at E13.5. Down panels show higher magnification images. White arrows represent GFP and TBR2 double-positive cells. Scale bars, 50μm. (K) Graph shows the percentage of GFP+Tbr2+/GFP+ in enlarged SVZ (n = 3 each group; one-way ANOVA, F (3,8) =8.787, P=0.0065). D


Figure 5 ( C ) Volcano plots shows the gene expression states . The yellow dots are the up - regulated genes , and the blue dots are the down - regulated genes . The Lin28a gene ( red dot ) is significantly up - regulated . ( D ) Western blot analysis for Lin28a in cortex of E15 WT and hFOXM1 - cKI mice . β - actin was detected as loading control . ( F ) Staining for Sox2 in E15 . 5 mice after IUE at E13 . 5 . Arrowheads indicate Sox2 + cell in enlarged SVZ . Scale bar , 50μm . ( G ) Graph shows the percentage of Sox2 + cells among GFP + cells in enlarged SVZ ( n = 3 each group ; two - tailed unpaired t - test , P = 0 . 0154 ) . ( H ) Graph shows the percentage of Sox2 + cells among GFP + cells ( n = 3 each group ; two - tailed unpaired t - test , P = 0 . 0018 ) . ( I ) LIN28A - shRNA1 rescues the neuronal progenitor cell increase in hFOXM1 - cKI mice . Brain slice staining for Sox2 two days after IUE at E13 . Scale bar , 50μm . ( J ) Graph shows the percentage of Sox2 + cells among GFP + cells ( n = 3 each group ; one - way

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(C) Volcano plots shows the gene expression states. The yellow dots are the up-regulated genes, and the blue dots are the down-regulated genes. The Lin28a gene (red dot) is significantly up-regulated.(D) Western blot analysis for Lin28a in cortex of E15 WT and hFOXM1-cKI mice. β-actin was detected as loading control.(F) Staining for Sox2 in E15.5 mice after IUE at E13.5. Arrowheads indicate Sox2+ cell in enlarged SVZ. Scale bar, 50μm. (G) Graph shows the percentage of Sox2+ cells among GFP+ cells in enlarged SVZ (n = 3 each group; two-tailed unpaired t-test, P=0.0154). (H) Graph shows the percentage of Sox2+ cells among GFP+ cells (n = 3 each group; two-tailed unpaired t-test, P=0.0018).(I) LIN28A-shRNA1 rescues the neuronal progenitor cell increase in hFOXM1-cKI mice. Brain slice staining for Sox2 two days after IUE at E13. Scale bar, 50μm. (J) Graph shows the percentage of Sox2+ cells among GFP+ cells (n=3 each group; one-way A


Figure 6 ( B ) Representative cases of FOXM1 + / + and FOXM1 - / - NPCs clones . Scale bar , 20μm . ( C ) Graph shows the number of cells per clone in FOXM1 + / + and FOXM1 - / - human NPCs ( FOXM1 + / + n = 12 ; FOXM1 - / - n = 13 ; at least three biological replicates . two - tailed unpaired t - test , P = 0 . 0014 ) . ( ( D ) Overexpression of FOXM1 or LIN28A rescues the human neural progenitor cell reduction in FOXM1 - / - cell lines . Western blot analysis for SOX2 , PAX6 and HOPX . β - actin was detected as loading control . ( E ) Statistics of relative intensity of SOX2 , PAX6 and HOPX ( PAX6 : n = 3 biological replicates ; one - way ANOVA , F ( 2 , 6 ) = 8 . 716 , FOXM1 - / - ( Control ) vs . FOXM1 - / - ( hFOXM1 ) , P = 0 . 0173 , FOXM1 + / + ( Control ) vs . FOXM1 - / - ( hFOXM1 ) , P = 0 . 0476 . SOX2 : n = 5 biological replicates ; one - way ANOVA , F ( 2 , 12 ) = 14 . 72 , FOXM1 + / + ( Control ) vs . FOXM1 - / - ( Control ) , P = 0 . 0015 , FOXM1 + / + ( Control ) vs . FOXM1 - / - ( hFOXM1 ) , P = 0 . 9906 . HOPX : n = 4 biological replicates , one - way ANOVA F ( 2 , 9 ) = 25 . 28 , FOXM1 + / + ( Control ) vs . FOXM1 - / - ( Control ) , P = 0 . 0018 , FOXM1 + / + ( Control ) vs . FOXM1 - / - ( hFOXM1 ) , P = 0 . 2211 ) . (

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(B) Representative cases of FOXM1+/+ and FOXM1-/- NPCs clones. Scale bar, 20μm. (C) Graph shows the number of cells per clone in FOXM1+/+ and FOXM1-/- human NPCs (FOXM1+/+ n=12; FOXM1-/- n=13; at least three biological replicates. two-tailed unpaired t-test, P= 0.0014). ((D) Overexpression of FOXM1 or LIN28A rescues the human neural progenitor cell reduction in FOXM1-/- cell lines. Western blot analysis for SOX2, PAX6 and HOPX. β-actin was detected as loading control. (E) Statistics of relative intensity of SOX2, PAX6 and HOPX (PAX6: n=3 biological replicates; one-way ANOVA, F (2,6) = 8.716, FOXM1-/-(Control) vs. FOXM1-/-(hFOXM1), P= 0.0173, FOXM1+/+(Control) vs. FOXM1-/-(hFOXM1), P= 0.0476. SOX2: n=5 biological replicates; one-way ANOVA, F (2, 12) = 14.72, FOXM1+/+(Control) vs. FOXM1-/-(Control), P= 0.0015, FOXM1+/+(Control) vs. FOXM1-/-(hFOXM1), P=0.9906. HOPX: n=4 biological replicates, one-way ANOVA F (2, 9) = 25.28, FOXM1+/+(Control) vs. FOXM1-/- (Control), P= 0.0018, FOXM1+/+(Control) vs. FOXM1-/-(hFOXM1), P= 0.2211). (


Figure 1B ) Single point activity assays of USP1WT and deletion mutants on Ub - Rho show signifi cantly increased activity in mutants where insert L1 and L3 are both deleted . C ) Single point activity assays of USP1WT and deletion mutants ( ± UAF1 ) on Ub - Rho show the loss in hyper - activation of USP1ΔL1L3 upon addition of UAF1 . D ) Michaelis - Menten analysis of USP1 deletion mutants against ubiquitin - rhodamine ( Ub - Rho ) shows that USP1ΔL1L3 has significantly higher activity compared to WT and other mutants ( n = 2 , biological replicates ) . E ) Michaelis - Menten analysis of USP1 deletion mutants ( + UAF1 ) against Ub - Rho shows that all deletions mutants have similar catalytic activity ( n = 2 , biological replicates ) . F ) Comparing activity of USP1WT and USP1ΔL1L3 on a peptide substrate i . e . LRGG - AMC ( 100µM ) . USP1ΔL1L3 cleaves the peptide substrate more efficiently compared to USP1WT ( n = 2 , technical replicates ) . G ) Comparing activity of USP1WT and USP1ΔL1L3 ( + UAF1 ) on a peptide substrate i . e . LRGG - AMC ( 100µM ) . Addition of UAF1 allows cleavage of LRGG - AMC by both USP1WT and USP1ΔL1L3 ( n = 2 , technical replicates ) .

O B-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B) Single point activity assays of USP1WT and deletion mutants on Ub-Rho show significantly increased activity in mutants where insert L1 and L3 are both deleted.C) Single point activity assays of USP1WT and deletion mutants (± UAF1) on Ub-Rho show the loss in hyper-activation of USP1ΔL1L3 upon addition of UAF1.D) Michaelis-Menten analysis of USP1 deletion mutants against ubiquitin-rhodamine (Ub-Rho) shows that USP1ΔL1L3 has significantly higher activity compared to WT and other mutants (n=2, biological replicates).E) Michaelis-Menten analysis of USP1 deletion mutants (+UAF1) against Ub-Rho shows that all deletions mutants have similar catalytic activity (n=2, biological replicates).F) Comparing activity of USP1WT and USP1ΔL1L3 on a peptide substrate i.e. LRGG-AMC (100µM). USP1ΔL1L3 cleaves the peptide substrate more efficiently compared to USP1WT (n=2, technical replicates).G) Comparing activity of USP1WT and USP1ΔL1L3 (+UAF1) on a peptide substrate i.e. LRGG-AMC (100µM). Addition of UAF1 allows cleavage of LRGG-AMC by both USP1WT and USP1ΔL1L3 (n=2, technical replicates).


Figure 2A ) Quantification of gel - based activity assays where the activity of USP1WT and deletion mutants ( + UAF1 ) on PCNA - UbTAMRA is compared . The USP1ΔL1 mutant has reduced activity on PCNA - UbTAMRA compared to the other USP1 variants tested ( n = 2 , biological replicates ) . C ) Quantification of gel - based activity assays where the activity of USP1 PIP mutants ( + UAF1 ) on PCNA - UbTAMRA is compared . Both the PIP mutants have reduced activity on PCNA - Ub compared to USP1WT ( n = 2 , biological replicates ) . D ) FP based binding assays of USP1 mutants ( + UAF1 ) with PCNA - UbTAMRA show reduced binding of USP1ΔL1 and USP1PIP1 compared to USP1WT ( n = 2 , technical replicates ) . E ) Quantification of gel - based activity assays where the activity of USP1WT + UAF1 on PCNA - UbWT is compared with PCNA - UbPIM1 shows that PCNA - UbWT is cleaved much faster compared to PCNA - UbPIM1 ( n = 2 , technical replicates ( example shown here from 1 of 2 biological replicates ) ) F ) Comparing activity of USP1WT and USP1PIP1 ( + UAF1 ) on PCNA - UbWT and PCNA - UbPIM1 in gel - based assays shows that the activity of USP1WT is severely reduced by the PIM1 mutation in PCNA , whereas USP1PIP1 activity is not further affected indicating that PIP1 and PIM mutation affect the same interaction ( n = 2 , technical replicates ( example shown here from 1 of 2 biological replicates ) ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A) Quantification of gel-based activity assays where the activity of USP1WT and deletion mutants (+UAF1) on PCNA-UbTAMRA is compared. The USP1ΔL1 mutant has reduced activity on PCNA-UbTAMRA compared to the other USP1 variants tested (n=2, biological replicates).C) Quantification of gel-based activity assays where the activity of USP1 PIP mutants (+UAF1) on PCNA-UbTAMRA is compared. Both the PIP mutants have reduced activity on PCNA-Ub compared to USP1WT (n=2, biological replicates).D) FP based binding assays of USP1 mutants (+ UAF1) with PCNA-UbTAMRA show reduced binding of USP1ΔL1 and USP1PIP1 compared to USP1WT (n=2, technical replicates).E) Quantification of gel-based activity assays where the activity of USP1WT+UAF1 on PCNA-UbWT is compared with PCNA-UbPIM1 shows that PCNA-UbWT is cleaved much faster compared to PCNA-UbPIM1 (n=2, technical replicates (example shown here from 1 of 2 biological replicates))F) Comparing activity of USP1WT and USP1PIP1 (+UAF1) on PCNA-UbWT and PCNA-UbPIM1 in gel-based assays shows that the activity of USP1WT is severely reduced by the PIM1 mutation in PCNA, whereas USP1PIP1 activity is not further affected indicating that PIP1 and PIM mutation affect the same interaction (n=2, technical replicates (example shown here from 1 of 2 biological replicates)).


Figure 3A ) Schematic representation of loading and purification of PCNA - Ub on nicked circular DNA . The DNA - loaded PCNA - Ub elutes in the void of the SEC column and this sample is collected and used for studying activity of USP1 on DNA - loaded PCNA - Ub . B ) Michaelis - Menten analysis of USP1 + UAF1 with and without DNA ( 65bp dsDNA ) shows that DNA binding alone has no effect on USP1 activity ( n = 2 , technical replicates ( example shown here from 1 of 2 biological replicates ) ) . C ) Coomassie stained gel of in vitro activity assay showing increased activity of USP1 - UAF1 on DNA - loaded PCNA - Ub compared to PCNA - Ub ( - DNA ) and PCNA - Ub ( + DNA , + RFC and not ATP ) . D ) Quantification of gel - based activity assays showing enhanced activity of USP1 + UAF1 on DNA - loaded PCNA - Ub compared to free PCNA - Ub ( n = 3 , technical replicates ( example shown here from 1 of 2 biological replicates ) ) . E ) Quantification of gel - based activity assays showing enhanced activity of USP1 on DNA - loaded PCNA - Ub compared to free PCNA - Ub ( n = 3 , technical replicates ( example shown here from 1 of 2 biological replicates ) ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A) Schematic representation of loading and purification of PCNA-Ub on nicked circular DNA. The DNA-loaded PCNA-Ub elutes in the void of the SEC column and this sample is collected and used for studying activity of USP1 on DNA-loaded PCNA-Ub.B) Michaelis-Menten analysis of USP1+UAF1 with and without DNA (65bp dsDNA) shows that DNA binding alone has no effect on USP1 activity (n=2, technical replicates (example shown here from 1 of 2 biological replicates)).C) Coomassie stained gel of in vitro activity assay showing increased activity of USP1-UAF1 on DNA-loaded PCNA-Ub compared to PCNA-Ub (-DNA) and PCNA-Ub (+DNA, +RFC and not ATP).D) Quantification of gel-based activity assays showing enhanced activity of USP1+UAF1 on DNA-loaded PCNA-Ub compared to free PCNA-Ub (n=3, technical replicates (example shown here from 1 of 2 biological replicates)).E) Quantification of gel-based activity assays showing enhanced activity of USP1 on DNA-loaded PCNA-Ub compared to free PCNA-Ub (n=3, technical replicates (example shown here from 1 of 2 biological replicates)).


Figure 4A ) Gel based quantification of USP1 - UAF1 activity on PCNA - Ub ( DNA loaded and free ) for three different concentration of enzyme and substrate . B ) FP based activity assays on free PCNA - Ub ( three concentrations of USP1 and PCNA - Ub ) and activity of USP1 on increasing concentrations of Ub - Rho .

O B-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A) Gel based quantification of USP1-UAF1 activity on PCNA-Ub (DNA loaded and free) for three different concentration of enzyme and substrate.B) FP based activity assays on free PCNA-Ub (three concentrations of USP1 and PCNA-Ub) and activity of USP1 on increasing concentrations of Ub-Rho.


Figure 5A ) Comparison of USP1WT and deletion mutants ( + UAF1 ) for activity on DNA - loaded PCNA - Ub ( solid lines ) and free PCNA - Ub ( dashed lines ) shows no increase in USP1ΔL1 activity on the loaded substrate . Left panel : Quantification of gel - based activity assays showing percentage of cleaved PCNA - Ub at the mentioned time points ( n = 2 , biological replicates ) , Right panel : Quantification of the activation fold observed in USP1WT and mutants on DNA - loaded PCNA - Ub versus free PCNA - Ub ( n = 2 , biological replicates ) . B ) Multiple sequence alignment of USP1 insert L1 which highlights the DNA binding region and the conservation of the positively charged residues across species . Three groups of positively charged residues ( * * * ) are mutated separately and tested for binding to double stranded DNA ( 65bp ) using SPR which shows the importance of this region for DNA binding of USP1 . C ) Comparison of USP1DM1 , USP1PIP1 and USP1PIP1 + DM1 ( + UAF1 ) activity on DNA - loaded PCNA - Ub ( solid lines ) and free PCNA - Ub ( dashed lines ) shows reduced increase in activity of these mutants compared to USP1WT . Left panel : Quantification of gel based activity assays showing percentage of cleaved PCNA - Ub at the mentioned time points ( n = 2 , technical replicates ) , Right panel : Quantification of the activation fold observed in USP1WT , USP1PIP1 , USP1DM1 and USP1PIP1 + DM1 on DNA - loaded PCNA - Ub versus free PCNA - Ub ( n = 2 , technical replicates ) . D ) EMSA based DNA binding experiment shows that USP1PIP1 and USP1WT has similar DNA binding capability while USP1DM1 has lost its ability to bind DNA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A) Comparison of USP1WT and deletion mutants (+UAF1) for activity on DNA-loaded PCNA-Ub (solid lines) and free PCNA-Ub (dashed lines) shows no increase in USP1ΔL1 activity on the loaded substrate. Left panel: Quantification of gel-based activity assays showing percentage of cleaved PCNA-Ub at the mentioned time points (n=2, biological replicates), Right panel: Quantification of the activation fold observed in USP1WT and mutants on DNA-loaded PCNA-Ub versus free PCNA-Ub (n=2, biological replicates).B) Multiple sequence alignment of USP1 insert L1 which highlights the DNA binding region and the conservation of the positively charged residues across species. Three groups of positively charged residues (***) are mutated separately and tested for binding to double stranded DNA (65bp) using SPR which shows the importance of this region for DNA binding of USP1.C) Comparison of USP1DM1, USP1PIP1 and USP1PIP1+DM1 (+UAF1) activity on DNA-loaded PCNA-Ub (solid lines) and free PCNA-Ub (dashed lines) shows reduced increase in activity of these mutants compared to USP1WT. Left panel: Quantification of gel based activity assays showing percentage of cleaved PCNA-Ub at the mentioned time points (n=2, technical replicates), Right panel: Quantification of the activation fold observed in USP1WT, USP1PIP1, USP1DM1 and USP1PIP1+DM1 on DNA-loaded PCNA-Ub versus free PCNA-Ub (n=2, technical replicates).D) EMSA based DNA binding experiment shows that USP1PIP1 and USP1WT has similar DNA binding capability while USP1DM1 has lost its ability to bind DNA.


Figure 1ABCE1IIEA efficiently splits 70S ribosomes in the presence of AMP - PNP and aRF1 / aPelota . The 30S population contains a stoichiometric ratio of ABCE1 and ribosomal proteins , forming the post - splitting complex . rps : small subunit ribosomal proteins .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1ABCE1IIEA efficiently splits 70S ribosomes in the presence of AMP-PNP and aRF1/aPelota. The 30S population contains a stoichiometric ratio of ABCE1 and ribosomal proteins, forming the post-splitting complex. rps: small subunit ribosomal proteins.


Figure 2Yeast survival of ABCE1 variants ( S . solfataricus colored , S . cerevisiae in gray ) . Most residues connecting to 30S in the post - SC show a growth defect when exchanged for a small one ( alanine ) or a negative charge ( glutamate ) . + + no effect , + growth defect , - lethal .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Yeast survival of ABCE1 variants (S. solfataricus colored, S. cerevisiae in gray). Most residues connecting to 30S in the post-SC show a growth defect when exchanged for a small one (alanine) or a negative charge (glutamate). ++ no effect, + growth defect, - lethal.


Figure 3Mutations in the α - helices of hinge 2 prevent 30S binding while the Y592A / Y593A ( C - terminus ) and L353Y ( A - loop in NBSII ) exchanges do not influence ribosome binding . 70S splitting efficiency normalized to wildtype . Hinge 2 mutations Y592A / Y593A , R565E and S580E display strongly impaired splitting activity . Unspecific ribosome dissociation level as determined in control experiments in the absence of ABCE1 is marked by the dotted line . ATP turnover per ABCE1 is not affected in all tested mutants .

O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Mutations in the α-helices of hinge 2 prevent 30S binding while the Y592A/Y593A (C-terminus) and L353Y (A-loop in NBSII) exchanges do not influence ribosome binding.70S splitting efficiency normalized to wildtype. Hinge 2 mutations Y592A/Y593A, R565E and S580E display strongly impaired splitting activity. Unspecific ribosome dissociation level as determined in control experiments in the absence of ABCE1 is marked by the dotted line. ATP turnover per ABCE1 is not affected in all tested mutants.