H . Survival frequencies after DSB induction at LYS2 locus , in strains where SIR3 is expressed from its native , pADH1 or pGPD promoters respectively and in which SAE2 is expressed or not from a high copy number 2µ plasmid . Fold increase in Sir3 protein by pAHD1 or pGPD is indicated . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

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H. Survival frequencies after DSB induction at LYS2 locus, in strains where SIR3 is expressed from its native, pADH1 or pGPD promoters respectively and in which SAE2 is expressed or not from a high copy number 2µ plasmid. Fold increase in Sir3 protein by pAHD1 or pGPD is indicated. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


A . Representative images of Sir3 - mCherry and Sae2 - GFP signal in WT and SIR3 overexpressing cells . Scale bars are 2 μm .

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A. Representative images of Sir3-mCherry and Sae2-GFP signal in WT and SIR3 overexpressing cells. Scale bars are 2 μm.


B . Sir3 - binding at TEL6R in untagged , WT , sir3Δ cells or in cells overexpressing Sir3 ( oeSir3 ) . Binding is probed by ChIP - qPCR 0 . 2 ( red arrows ) and 1kb ( blue arrows ) from telomeres and at the OGG1 control locus using antibodies against Sae2 - GFP . The mean of three independent biological replicates is shown and error bars correspond to the variation between replicates ( SEM ) .

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B. Sir3-binding at TEL6R in untagged, WT, sir3Δ cells or in cells overexpressing Sir3 (oeSir3). Binding is probed by ChIP-qPCR 0.2 (red arrows) and 1kb (blue arrows) from telomeres and at the OGG1 control locus using antibodies against Sae2-GFP. The mean of three independent biological replicates is shown and error bars correspond to the variation between replicates (SEM).


C . Co - immunoprecipitation between Sir3 and Sae2 - GFP from cells overexpressing Sir3 , analysed by Western blot with anti - GFP and anti - Sir3 antibodies . D . Co - immunoprecipitation between Sir3 and Sae2 - GFP from WT cells using antibodies against Sae2 - GFP , analysed by Western blot .

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C. Co-immunoprecipitation between Sir3 and Sae2-GFP from cells overexpressing Sir3, analysed by Western blot with anti-GFP and anti-Sir3 antibodies. D. Co-immunoprecipitation between Sir3 and Sae2-GFP from WT cells using antibodies against Sae2-GFP, analysed by Western blot.


D . Yeast two - hybrid interaction analysis between Sae2C and Sir3SaID domains in WT or sir4Δ cells . Growth on - His + 3AT and blue coloration on X - gal indicate an interaction .

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D. Yeast two-hybrid interaction analysis between Sae2C and Sir3SaID domains in WT or sir4Δ cells. Growth on -His + 3AT and blue coloration on X-gal indicate an interaction.


B . Representative images of two hybrid assays in the yKD1991 strain testing the interaction of the WT or the mutant SIR3SaID fragment isolated from the screen with SAE2C or SIR4C . C . Representative images of two hybrid assays testing the interaction of the WT or the mutant SIR3SaIDT557I fragment with SAE2C or SIR4C .

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B. Representative images of two hybrid assays in the yKD1991 strain testing the interaction of the WT or the mutant SIR3SaID fragment isolated from the screen with SAE2C or SIR4C. C. Representative images of two hybrid assays testing the interaction of the WT or the mutant SIR3SaIDT557I fragment with SAE2C or SIR4C.


A . Survival frequencies after DSB induction at LYS2 locus in WT or sae2Δ strains where the Sir3SaID or Sir3SaIDT557I domains are overexpressed from a GPD promoter at the SIR3 locus . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

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A. Survival frequencies after DSB induction at LYS2 locus in WT or sae2Δ strains where the Sir3SaID or Sir3SaIDT557I domains are overexpressed from a GPD promoter at the SIR3 locus. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


B . Representative images of Sae2 - GFP in WT cells and in cells overexpressing either full - length Sir3 or the sir3SaID domain . Scale bars are 2 μm .

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B. Representative images of Sae2-GFP in WT cells and in cells overexpressing either full-length Sir3 or the sir3SaID domain. Scale bars are 2 μm.


F . Representative silver - stained gels of in vitro GST - pulldown of GST or GST - Sir3SaID , GST - sir3 - T557ISaID and Sae2C purified peptides . Control : Sae2C ( 300 ng , lane 6 ) .

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F. Representative silver-stained gels of in vitro GST-pulldown of GST or GST-Sir3SaID, GST-sir3-T557ISaID and Sae2C purified peptides. Control: Sae2C (300 ng, lane 6).


G . Co - immunoprecipitation between Sae2 - GFP and Sir3 from untagged , Sae2 - GFP WT cells , and Sae2 - GFP cells overexpressing WT Sir3 ( oeSir3 , WT ) , Sae2 - GFP sir3Δ or Sae2 - GFP overexpressing the sir3 - T557I mutant ( oeSir3 , T557I ) using antibodies against Sae2 - GFP , analysed by Western blot with anti - GFP and anti - Sir3 antibodies .

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G. Co-immunoprecipitation between Sae2-GFP and Sir3 from untagged, Sae2-GFP WT cells, and Sae2-GFP cells overexpressing WT Sir3 (oeSir3, WT), Sae2-GFP sir3Δ or Sae2-GFP overexpressing the sir3-T557I mutant (oeSir3, T557I) using antibodies against Sae2-GFP, analysed by Western blot with anti-GFP and anti-Sir3 antibodies.


A . Survival frequencies after DSB induction at LYS2 locus in the indicated strains . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

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A. Survival frequencies after DSB induction at LYS2 locus in the indicated strains. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


C . Survival frequencies after DSB induction at LYS2 locus in the indicated strains . Insertion of the strong TEF1p promoter upstream of the SIR4 ORF leads to Sir4 overexpression . Insertion of the ADH1p promoter upstream of SIR3 leads to mild Sir3 overexpression . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

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C. Survival frequencies after DSB induction at LYS2 locus in the indicated strains. Insertion of the strong TEF1p promoter upstream of the SIR4 ORF leads to Sir4 overexpression. Insertion of the ADH1p promoter upstream of SIR3 leads to mild Sir3 overexpression. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


E . Representative images of two hybrid assays testing the interaction between the full - length Sir3 and full - length Sae2 proteins in WT cells expressing or not high levels of Sir4 ( oeSir4 and WT respectively ) .

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E. Representative images of two hybrid assays testing the interaction between the full-length Sir3 and full-length Sae2 proteins in WT cells expressing or not high levels of Sir4 (oeSir4 and WT respectively).


A . Relative fold enrichment of Sae2 at 0 . 2 kb from I - SceI site was evaluated by qPCR after ChIP with anti - GFP antibodies . The error bars indicate the variation between at least three biological replicas ( SEM ) . B . Relative fold enrichment of Sae2 at 0 . 2 kb from TEL6R after DSB induction at the LYS2 I - SceI site was evaluated by qPCR after ChIP with anti - GFP antibodies . The error bars indicate the variation between at least three biological replicas ( SEM ) . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

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A. Relative fold enrichment of Sae2 at 0.2 kb from I-SceI site was evaluated by qPCR after ChIP with anti-GFP antibodies. The error bars indicate the variation between at least three biological replicas (SEM). B. Relative fold enrichment of Sae2 at 0.2 kb from TEL6R after DSB induction at the LYS2 I-SceI site was evaluated by qPCR after ChIP with anti-GFP antibodies. The error bars indicate the variation between at least three biological replicas (SEM). Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


D . Survival frequencies after DSB induction at LYS2 locus in sae2 - E267E mutant cells overexpressing or not full - length Sir3 ( oeSir3 ) . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

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D. Survival frequencies after DSB induction at LYS2 locus in sae2-E267E mutant cells overexpressing or not full-length Sir3 (oeSir3). Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


E . Survival curves of mice challenged with SARS - CoV - 2 Wuhan - D614G at 104 PFU , after a primo - infection by BANAL - 236 or Wuhan - 372 viruses at 103 or 104 PFU . Survival of K18 - hACE2 naive mice infected by SARS - CoV - 2 Wuhan - D614G at 104 PFU is presented as a reference ( yellow ) .

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E. Survival curves of mice challenged with SARS-CoV-2 Wuhan-D614G at 104 PFU, after a primo-infection by BANAL-236 or Wuhan-372 viruses at 103 or 104 PFU. Survival of K18-hACE2 naive mice infected by SARS-CoV-2 Wuhan-D614G at 104 PFU is presented as a reference (yellow).


F . Histopathological analysis , 3 days post - inoculation , of the lung of K18 - hACE2 mice infected with 104 PFU of BANAL - 236 ( a - d ) or SARS - CoV - 2 Wuhan - 372 ( e , f ) viruses . Interstitial pneumonia characterized by interstitial inflammation , often centered on blood vessels ( perivasculitis , black arrows ) or bronchi / bronchioles ( black arrowheads ) , and endothelial cell injury and inflammation ( endothelitis ) . Lesions were globally more severe after Wuhan - 372 infection ( n = 4 ; all of moderate severity ) than after BANAL - 236 infection ( n = 4 ; 3 / 4 of minimal severity ( a - c ) and 1 / 4 of moderate severity ( d ) ) . Scale bar : 100 µ . Low magnification images are provided in Figure S3 .

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F. Histopathological analysis, 3 days post-inoculation, of the lung of K18-hACE2 mice infected with 104 PFU of BANAL-236 (a-d) or SARS-CoV-2 Wuhan-372 (e,f) viruses. Interstitial pneumonia characterized by interstitial inflammation, often centered on blood vessels (perivasculitis, black arrows) or bronchi/bronchioles (black arrowheads), and endothelial cell injury and inflammation (endothelitis). Lesions were globally more severe after Wuhan-372 infection (n=4; all of moderate severity) than after BANAL-236 infection (n=4; 3/4 of minimal severity (a-c) and 1/4 of moderate severity (d)). Scale bar: 100 µ. Low magnification images are provided in Figure S3.


Neutralizing antibody titer ( expressed in ED50 ) of mice primo - infected at 103 ( left ) or 104 ( right ) PFU by BANAL - 236 ( red ) or SARS - CoV - 2 Wuhan - 372 ( blue ) viruses . The antibody response against the Wuhan - D614G challenge strain of mice primo - infected by BANAL - 236 is presented as a dashed line . PNT means pseudo - neutralization .

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Neutralizing antibody titer (expressed in ED50) of mice primo-infected at 103 (left) or 104 (right) PFU by BANAL-236 (red) or SARS-CoV-2 Wuhan-372 (blue) viruses. The antibody response against the Wuhan-D614G challenge strain of mice primo-infected by BANAL-236 is presented as a dashed line. PNT means pseudo-neutralization.


B . Effects of infection by BANAL - 236 virus in non - human primates . Animal # MF1 is presented in green and animal # MF2 in purple . Clinical score , body weight variation ( in % ) , CT scores ( compared to Wuhan - 372 ) and neutralizing antibodies are measured from 10 days before infection to 43 days post - infection .

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B. Effects of infection by BANAL-236 virus in non-human primates. Animal #MF1 is presented in green and animal #MF2 in purple. Clinical score, body weight variation (in %), CT scores (compared to Wuhan-372) and neutralizing antibodies are measured from 10 days before infection to 43 days post-infection.


C . Viral load expressed in log10 copies of genomic RNA ( solid line ) or sub - genomic RNA ( dashed line ) per mL of tracheal , nasopharyngeal , BAL and rectal fluids . Lower limit of quantification is presented in red for both genomic ( solid line ) and sub - genomic ( dashed line ) RNA , and limits of detection are presented in green for both genomic ( solid line ) and sub - genomic ( dashed line ) RNA . Results were compared to historical data of SARS - CoV - 2 Wuhan - 372 virus inoculated at 106 or 105 PFU ( presented as the mean virus load + / - 95 % CI expressed in log10 copies of genomic RNA / mL ) .

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C. Viral load expressed in log10 copies of genomic RNA (solid line) or sub-genomic RNA (dashed line) per mL of tracheal, nasopharyngeal, BAL and rectal fluids. Lower limit of quantification is presented in red for both genomic (solid line) and sub-genomic (dashed line) RNA, and limits of detection are presented in green for both genomic (solid line) and sub-genomic (dashed line) RNA. Results were compared to historical data of SARS-CoV-2 Wuhan-372 virus inoculated at 106 or 105 PFU (presented as the mean virus load +/- 95% CI expressed in log10 copies of genomic RNA/mL).


( A - C ) Results of Pseudoneutralisation ( PNT ) , Luciferase immunoprecipitation ( LIPS ) and LuLISA tests are shown . Pre - pandemic French sera ( black ) were used as negative controls and Laotian sera samples from confirmed SARS - CoV - 2 infection ( green ) were used as cross - reacting positive controls . Laotian sera samples collected in the general population before 2019 ( blue , n = 100 ) or late 2020 ( gray , n = 100 ) or in people exposed to bats ( purple , n = 74 ) were tested for BANAL - 236 ( A ) , BANAL - 52 ( B ) and BANAL - 103 ( C ) antibody responses . The ANOVA non - parametric Kruskal - Wallis test was conducted to compare each sub - population to the reference French population ( * P < 0 . 05 ; * * P < 0 . 005 ; * * * P < 0 . 0005 ; * * * * P < 0 . 0001 ) . The red bars represent the median .

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(A-C) Results of Pseudoneutralisation (PNT), Luciferase immunoprecipitation (LIPS) and LuLISA tests are shown. Pre-pandemic French sera (black) were used as negative controls and Laotian sera samples from confirmed SARS-CoV-2 infection (green) were used as cross-reacting positive controls. Laotian sera samples collected in the general population before 2019 (blue, n=100) or late 2020 (gray, n=100) or in people exposed to bats (purple, n=74) were tested for BANAL-236 (A), BANAL-52 (B) and BANAL-103 (C) antibody responses. The ANOVA non-parametric Kruskal-Wallis test was conducted to compare each sub-population to the reference French population (* P<0.05; **P<0.005; ***P<0.0005; ****P<0.0001). The red bars represent the median.


( A ) - ( D ) Hepatocytes accumulate apical bulkheads upon bile duct ligation ( BDL ) and MDR2 KO . ( A ) - ( B ) Overview images of ( A ) murine liver tissue after control surgery or bile duct ligation ( BDL ) for 24h ( N = 3 biological replicates ) , or ( B ) control and MDR2 KO mice after 12 weeks ( N = 3 biological replicates ) . Immunofluorescence for the apical membrane marker CD13 ( magenta ) , F - Actin ( green ) and nuclei ( grey ) . Representative images are shown . Scale bar 10 µm . ( C ) - ( D ) High - resolution imaging of individual bile canaliculi in murine liver tissue ( C ) after control and 24h BDL surgery , or ( D ) in control and MDR2 KO mice , stained for F - Actin ( green ) . Representative images are shown . Scale bar 2 µm . Apical bulkheads are highlighted with red arrowheads .

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(A)- (D) Hepatocytes accumulate apical bulkheads upon bile duct ligation (BDL) and MDR2 KO. (A)-(B) Overview images of (A) murine liver tissue after control surgery or bile duct ligation (BDL) for 24h (N=3 biological replicates), or (B) control and MDR2 KO mice after 12 weeks (N=3 biological replicates). Immunofluorescence for the apical membrane marker CD13 (magenta), F-Actin (green) and nuclei (grey). Representative images are shown. Scale bar 10 µm. (C)-(D) High-resolution imaging of individual bile canaliculi in murine liver tissue (C) after control and 24h BDL surgery, or (D) in control and MDR2 KO mice, stained for F-Actin (green). Representative images are shown. Scale bar 2 µm. Apical bulkheads are highlighted with red arrowheads.


Apical bulkheads in the adult liver resemble those described in the embryonic liver . ( E ) Transmission electron microscopy ( TEM ) longitudinal 80 nm - thin serial section on liver tissue with BDL for 24h shows the ultrastructure characteristic of apical bulkheads . Representative images are shown . Scale bar 1 µm . I - III represent consecutive sections with 460 nm distance showing four apical bulkheads ( B1 - B4 ) sealed by electron - dense tight junctions . Scale bar 1 µm .

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Apical bulkheads in the adult liver resemble those described in the embryonic liver. (E) Transmission electron microscopy (TEM) longitudinal 80 nm-thin serial section on liver tissue with BDL for 24h shows the ultrastructure characteristic of apical bulkheads. Representative images are shown. Scale bar 1 µm. I-III represent consecutive sections with 460 nm distance showing four apical bulkheads (B1-B4) sealed by electron-dense tight junctions. Scale bar 1 µm.


Hepatocytes form aberrantly dilated canaliculi formed by > 3 cells after BDL . ( I ) Transmission electron microscopy ( TEM ) in liver tissue after 24h BDL . Scale bar 10 µm . Bile canaliculi ( BC ) can be severely dilated similar to the size of sinusoids ( S ) and are formed by multiple hepatocytes ( see inset A , scale bar 1 µm ) . Apical bulkheads ( marked with red arrowheads ) are visible . Individual hepatocytes are part of a liver cell rosette with one part of their apical membrane while they form normal canaliculi with apical bulkheads at another pole ( see inset A , cell 3 and 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hepatocytes form aberrantly dilated canaliculi formed by >3 cells after BDL. (I) Transmission electron microscopy (TEM) in liver tissue after 24h BDL. Scale bar 10 µm. Bile canaliculi (BC) can be severely dilated similar to the size of sinusoids (S) and are formed by multiple hepatocytes (see inset A, scale bar 1 µm). Apical bulkheads (marked with red arrowheads) are visible. Individual hepatocytes are part of a liver cell rosette with one part of their apical membrane while they form normal canaliculi with apical bulkheads at another pole (see inset A, cell 3 and 4).


( A ) - ( B ) Hepatocytes accumulate apical bulkheads in the liver tissue of primary sclerosing cholangitis ( PSC ) patients ( N = 3 control patients and N = 4 PSC patients ) . ( A ) Overview images in liver tissue of control and PSC patients . Immunofluoresence for the apical membrane marker BSEP ( magenta ) , F - Actin ( green ) and nuclei ( grey ) . Scale bar 10 µm . ( B ) High - resolution imaging of F - Actin ( green ) showing individual bile canaliculi in control or PSC liver tissue . Red arrowheads mark apical bulkheads . Scale bar 2 µm .

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(A)-(B) Hepatocytes accumulate apical bulkheads in the liver tissue of primary sclerosing cholangitis (PSC) patients (N=3 control patients and N=4 PSC patients). (A) Overview images in liver tissue of control and PSC patients. Immunofluoresence for the apical membrane marker BSEP (magenta), F-Actin (green) and nuclei (grey). Scale bar 10 µm. (B) High-resolution imaging of F-Actin (green) showing individual bile canaliculi in control or PSC liver tissue. Red arrowheads mark apical bulkheads. Scale bar 2 µm.


( C ) Different lumen morphologies of a typical bile canaliculus ( left column ) and bile duct ( middle column ) in control patients and rosette ( right column ) in PSC patients . Immunofluorescence for BSEP ( magenta ) and F - Actin ( green ) . Shows also differences dependent on the imaging plane i . e . transversal ( top row ) or longitudinal ( bottom row ) cut of the lumen . White dashed lines mark the lumen . Scale bar 2 µm .

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(C) Different lumen morphologies of a typical bile canaliculus (left column) and bile duct (middle column) in control patients and rosette (right column) in PSC patients. Immunofluorescence for BSEP (magenta) and F-Actin (green). Shows also differences dependent on the imaging plane i.e. transversal (top row) or longitudinal (bottom row) cut of the lumen. White dashed lines mark the lumen. Scale bar 2 µm.


( F ) Liver cell rosettes in PSC patients are formed by hepatocyte - like cells ( N = 3 control patients and N = 4 PSC patients ) . Immunofluoresence for BSEP ( magenta ) , F - Actin ( green ) and nuclei ( grey ) in healthy parenchyma ( left ) and bile duct ( middle ) in control patients and liver cell rosettes in PSC patients ( right ) . Scale bar 10 µm .

O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Liver cell rosettes in PSC patients are formed by hepatocyte-like cells (N=3 control patients and N=4 PSC patients). Immunofluoresence for BSEP (magenta), F-Actin (green) and nuclei (grey) in healthy parenchyma (left) and bile duct (middle) in control patients and liver cell rosettes in PSC patients (right). Scale bar 10 µm.


( A ) - ( D ) Liver cell rosettes in PSC patients do not acquire other markers of bile duct cells or intermediate hepatobiliary cells . Immunofluoresence for different bile duct cell or intermediate hepatobiliary cell type marker ( magenta ) , F - Actin ( green ) and nuclei ( grey ) in healthy parenchyma ( left ) and bile duct ( middle ) in control patients and liver cell rosettes ( right ) in PSC patients ( N = 3 control patients and N = 4 PSC patients ) . Scale bar 10 µm . ( A ) SOX9 , ( B ) Pan - CK , Representative images are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O

(A)-(D)Liver cell rosettes in PSC patients do not acquire other markers of bile duct cells or intermediate hepatobiliary cells. Immunofluoresence for different bile duct cell or intermediate hepatobiliary cell type marker (magenta), F-Actin (green) and nuclei (grey) in healthy parenchyma (left) and bile duct (middle) in control patients and liver cell rosettes (right) in PSC patients (N=3 control patients and N=4 PSC patients). Scale bar 10 µm. (A) SOX9, (B) Pan-CK, Representative images are shown.


( A ) - ( D ) Liver cell rosettes in PSC patients do not acquire other markers of bile duct cells or intermediate hepatobiliary cells . Immunofluoresence for different bile duct cell or intermediate hepatobiliary cell type marker ( magenta ) , F - Actin ( green ) and nuclei ( grey ) in healthy parenchyma ( left ) and bile duct ( middle ) in control patients and liver cell rosettes ( right ) in PSC patients ( N = 3 control patients and N = 4 PSC patients ) . Scale bar 10 µm . TROP2 CD133 / PROM1 . Representative images are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O

(A)-(D)Liver cell rosettes in PSC patients do not acquire other markers of bile duct cells or intermediate hepatobiliary cells. Immunofluoresence for different bile duct cell or intermediate hepatobiliary cell type marker (magenta), F-Actin (green) and nuclei (grey) in healthy parenchyma (left) and bile duct (middle) in control patients and liver cell rosettes (right) in PSC patients (N=3 control patients and N=4 PSC patients). Scale bar 10 µm. TROP2 CD133/PROM1. Representative images are shown.


3D reconstruction of the bile canaliculi network reveals fundamental changes in the network architecture of PSC patients . ( A ) Top panel : 3D reconstructed bile canaliculi network across full central vein to portal vein ( CV - PV ) axes in control ( left ) and PSC ( right ) patients . Bottom panel : Projection of the 3D reconstruction . Colours correspond to the mean bile canaliculi diameter in µm . Scale bar 100 µm . ( B ) - ( I ) Quantification of bile canaliculi network characteristics from the 3D reconstruction . Represented are mean values with standard deviation of control and PSC patients at different zones across the CV - PV axis . 0 represents the zone surrounding the CV and 10 the area surrounding the PV . N = 3 with 1x axis / patient for control patients and N = 4 with 3x axis / patient for PSC patients . Paired t - test * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . ( B ) bile canaliculi radius [ µm ] , ( C ) volume fraction [ % ] , ( D ) surface area / volume ( µm2 / mm3 ) , ( E ) connectivity ( 1 / # disjointed bile canaliculi ) , ( F ) branches crossing regions [ # / mm2 ] . ( G ) junctions / length [ # /

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

3D reconstruction of the bile canaliculi network reveals fundamental changes in the network architecture of PSC patients. (A) Top panel: 3D reconstructed bile canaliculi network across full central vein to portal vein (CV-PV) axes in control (left) and PSC (right) patients. Bottom panel: Projection of the 3D reconstruction. Colours correspond to the mean bile canaliculi diameter in µm. Scale bar 100 µm. (B)-(I) Quantification of bile canaliculi network characteristics from the 3D reconstruction. Represented are mean values with standard deviation of control and PSC patients at different zones across the CV-PV axis. 0 represents the zone surrounding the CV and 10 the area surrounding the PV. N=3 with 1x axis/patient for control patients and N=4 with 3x axis/patient for PSC patients. Paired t-test *P < 0.05, **P < 0.01, ***P < 0.001. (B) bile canaliculi radius [µm], (C) volume fraction [%], (D) surface area/volume (µm2/mm3), (E) connectivity (1/# disjointed bile canaliculi), (F) branches crossing regions [#/mm2]. (G) junctions/length [#/µm


( A ) Liver cell rosettes occur in early - and late - stage PSC patients and in end - stage alcoholic liver disease ( ALD ) patients . Immunofluorescence of Fibronectin ( magenta ) , F - actin ( green ) and nuclei ( grey ) in early - stage PSC ( N = 3 ) , late - stage PSC ( N = 5 ) and end - stage ALD ( N = 5 ) patients showing liver cell rosettes . Representative images are shown . Scale bar 50 µm . Examples of rosettes are labelled with a red asterisk and examples of sinusoids are labelled with a red arrowhead . ( B ) Similar numbers of liver cell rosettes in early - stage and end - stage PSC patients . Quantification of the total number of rosettes in early - stage PSC ( N = 3 ) , late - stage PSC ( N = 5 ) and end - stage ALD ( N = 5 ) patients . Unpaired t - test * P < 0 . 05 . ( C ) Similar mean diameter of analysed liver cell rosettes in different liver diseases . Quantification of the mean rosette diameter in early - stage PSC ( N = 3 ) , late - stage PSC ( N = 5 ) and end - stage ALD ( N = 5 ) . Unpaired t - test . Data information : Boxplot shows the 25th and 75th percentiles and the central band shows the median value . Whiskers extend to the min and max values .

O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Liver cell rosettes occur in early- and late-stage PSC patients and in end-stage alcoholic liver disease (ALD) patients. Immunofluorescence of Fibronectin (magenta), F-actin (green) and nuclei (grey) in early-stage PSC (N=3), late-stage PSC (N=5) and end-stage ALD (N=5) patients showing liver cell rosettes. Representative images are shown. Scale bar 50 µm. Examples of rosettes are labelled with a red asterisk and examples of sinusoids are labelled with a red arrowhead. (B) Similar numbers of liver cell rosettes in early-stage and end-stage PSC patients. Quantification of the total number of rosettes in early-stage PSC (N=3), late-stage PSC (N=5) and end-stage ALD (N=5) patients. Unpaired t-test *P < 0.05. (C) Similar mean diameter of analysed liver cell rosettes in different liver diseases. Quantification of the mean rosette diameter in early-stage PSC (N=3), late-stage PSC (N=5) and end-stage ALD (N=5). Unpaired t-test. Data information: Boxplot shows the 25th and 75th percentiles and the central band shows the median value. Whiskers extend to the min and max values.


( C ) - ( D ) DCA treatment induces a cholestatic response in primary hepatocytes . Gene expression analysis of genes involved in bile acid metabolism and transport in primary hepatocytes treated with DMSO or 200 µM DCA for 16h . Represented are mean Cq values normalized to Gapdh expression ( N = 4 biological replicates ) . One sample t - test * P < 0 . 05 , * * * P < 0 . 001 . ( C ) Abcb11 / Bsep , ( D ) Cyp7a1 / Cyp7 . Data information : Boxplots show the 25th and 75th percentiles and the central band shows the median value . Whiskers extend to the min and max values .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) -(D) DCA treatment induces a cholestatic response in primary hepatocytes. Gene expression analysis of genes involved in bile acid metabolism and transport in primary hepatocytes treated with DMSO or 200 µM DCA for 16h. Represented are mean Cq values normalized to Gapdh expression (N=4 biological replicates). One sample t-test *P < 0.05, ***P < 0.001. (C) Abcb11/Bsep, (D) Cyp7a1/Cyp7. Data information: Boxplots show the 25th and 75th percentiles and the central band shows the median value. Whiskers extend to the min and max values.


( I - J ) DCA treatment in 3D hepatocyte organoids also induce canalicular dilation and rosette - like canaliculi and Sox9 expression . Hepatocyte organoids were treated with DMSO or 200 µM DCA for 24h ( N = 3 biological replicates ) . ( I ) Maximum projection of full organoids stained for F - actin ( green ) . Scale bar 50 µm . Inset A and B . Maximum projection of zoomed - in parts of the bile canaliculi network in DMSO - treated ( A ) and DCA - treated ( B ) hepatocyte organoids stained with F - actin ( green ) , nuclei ( white ) and the tight junction protein ZO - 1 ( yellow ) . Scale bar 10 µm . ( J ) High - resolution microscopy of individual bile canaliculi in DMSO - treated and DCA - treated hepatocyte organoids stained for F - actin ( green ) . Red arrows indicate apical bulkheads . Scale bar 2 µm .

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(I-J) DCA treatment in 3D hepatocyte organoids also induce canalicular dilation and rosette-like canaliculi and Sox9 expression. Hepatocyte organoids were treated with DMSO or 200 µM DCA for 24h (N=3 biological replicates). (I) Maximum projection of full organoids stained for F-actin (green). Scale bar 50 µm. Inset A and B. Maximum projection of zoomed-in parts of the bile canaliculi network in DMSO-treated (A) and DCA-treated (B) hepatocyte organoids stained with F-actin (green), nuclei (white) and the tight junction protein ZO-1 (yellow). Scale bar 10 µm. (J) High-resolution microscopy of individual bile canaliculi in DMSO-treated and DCA-treated hepatocyte organoids stained for F-actin (green). Red arrows indicate apical bulkheads. Scale bar 2 µm.


( K ) Maximum projection of F - actin ( green ) and nuclei ( white ) , and the bile duct cell marker Sox9 ( magenta ) in DMSO - treated and DCA - treated hepatocyte organoids ( N = 3 biological replicates ) . Untreated bile duct cell organoids serve as positive control . Scale bar 50 µm .

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(K) Maximum projection of F-actin (green) and nuclei (white), and the bile duct cell marker Sox9 (magenta) in DMSO-treated and DCA-treated hepatocyte organoids (N=3 biological replicates). Untreated bile duct cell organoids serve as positive control.Scale bar 50 µm.


( A ) Rosette - like canaliculi with reduced number of apical bulkheads can emerge from usual canaliculi with accumulated apical bulkheads . Timelapse imaging with brightfield phase - contrast of primary hepatocytes either untreated ( top row ) , DMSO - treated ( middle row ) or DCA - treated ( bottom row ) . An image was acquired every 15 minutes . Representative images are shown ( N = 3 biological replicates ) . Inserts show magnification of bile canaliculi with red arrows pointing towards apical bulkheads . Red stars mark areas in between apical bulkheads that markedly bulge out . Scale bar 10 µm .

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(A)Rosette-like canaliculi with reduced number of apical bulkheads can emerge from usual canaliculi with accumulated apical bulkheads. Timelapse imaging with brightfield phase-contrast of primary hepatocytes either untreated (top row), DMSO-treated (middle row) or DCA-treated (bottom row). An image was acquired every 15 minutes. Representative images are shown (N=3 biological replicates). Inserts show magnification of bile canaliculi with red arrows pointing towards apical bulkheads. Red stars mark areas in between apical bulkheads that markedly bulge out. Scale bar 10 µm.


( B ) Pharmacological inhibition of canalicular water influx can prevent canalicular dilation upon DCA treatment . Confocal microscopy of CD13 ( magenta ) , F - actin ( green ) and nuclei ( white ) in primary hepatocytes treated with DMSO and 200 µM DCA with 200 µM phloretin and 1 µM ouabain for 16h . Scale bar 10 µm . Representative images are shown .

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(B) Pharmacological inhibition of canalicular water influx can prevent canalicular dilation upon DCA treatment. Confocal microscopy of CD13 (magenta), F-actin (green) and nuclei (white) in primary hepatocytes treated with DMSO and 200 µM DCA with 200 µM phloretin and 1 µM ouabain for 16h. Scale bar 10 µm. Representative images are shown.


( C ) Quantification of the mean bile canaliculi volume in µm3of primary hepatocyte treated with DMSO and 200 µM DCA with 200 µM Phloretin and 1 µM Ouabain for 16h ( N = 4 biological replicates ) . Unpaired t - test * * * P < 0 . 001 . Data information : Boxplots show the 25th and 75th percentiles and the central band shows the median value . Whiskers extend to the min and max values .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the mean bile canaliculi volume in µm3of primary hepatocyte treated with DMSO and 200 µM DCA with 200 µM Phloretin and 1 µM Ouabain for 16h (N=4 biological replicates). Unpaired t-test ***P < 0.001. Data information: Boxplots show the 25th and 75th percentiles and the central band shows the median value. Whiskers extend to the min and max values.


( D ) - ( E ) Pharmacological inhibitors do not affect key bile acid signalling pathways . Gene expression analysis of different genes involved in bile acid signalling in primary hepatocytes treated with DMSO or 200 µM DCA with 200 µM Phloretin and 1 µM Ouabain for 16h . Represented are mean Cq values normalized to Gapdh expression and whiskers show min and max values ( N = 5 biological replicates ) . Unpaired t - test . ( D ) Cyp7a1 / Cyp7 , ( E ) Nr0b2 / Shp . Data information : Boxplots show the 25th and 75th percentiles and the central band shows the median value . Whiskers extend to the min and max values .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D)- (E) Pharmacological inhibitors do not affect key bile acid signalling pathways. Gene expression analysis of different genes involved in bile acid signalling in primary hepatocytes treated with DMSO or 200 µM DCA with 200 µM Phloretin and 1 µM Ouabain for 16h. Represented are mean Cq values normalized to Gapdh expression and whiskers show min and max values (N=5 biological replicates). Unpaired t-test. (D) Cyp7a1/Cyp7, (E) Nr0b2/Shp. Data information: Boxplots show the 25th and 75th percentiles and the central band shows the median value. Whiskers extend to the min and max values.


( C ) The 3 . 1 Å 2Fo - Fc map ( 1 . 2 σ ) around the DNA of the final refined ATPγS - MjMR - DNA complex structure .

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(C) The 3.1 Å 2Fo-Fc map (1.2 σ) around the DNA of the final refined ATPγS-MjMR-DNA complex structure.


( D ) A ribbon representation of the 3 . 1 Å ATPγS - MjMR - DNA structure showing the Mre11 nuclease , capping , and C - terminal three helix - bundle domain ( light pink ) , Rad50 molecules ( green and light blue ) , and the DNA ( yellow and orange ) . ATPγS is shown in spheres . See Movie EV 1 for the movement of the complex .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) A ribbon representation of the 3.1 Å ATPγS-MjMR-DNA structure showing the Mre11 nuclease, capping, and C-terminal three helix-bundle domain (light pink), Rad50 molecules (green and light blue), and the DNA (yellow and orange). ATPγS is shown in spheres. See Movie EV 1 for the movement of the complex.


( A ) A ribbon diagram showing the overall DNA recognition by the ATPγS - Rad50 dimer .

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(A) A ribbon diagram showing the overall DNA recognition by the ATPγS-Rad50 dimer.


( B ) Close - up view of the interaction between the internal segment ( major and minor grooves ) of the DNA strand and the edge of lobe I of Rad50A ( green ) . Loop α1 - α2 wedge faces the minor groove between the 10th and 13 ' th phosphates of the template ( orange ) and non - template ( yellow ) strands , respectively . ( C ) Close - up view of the interaction between the DNA strand and the α1 - α2 loop in an orientation different from Fig 2B .

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(B) Close-up view of the interaction between the internal segment (major and minor grooves) of the DNA strand and the edge of lobe I of Rad50A (green). Loop α1-α2 wedge faces the minor groove between the 10th and 13' th phosphates of the template (orange) and non-template (yellow) strands, respectively. (C) Close-up view of the interaction between the DNA strand and the α1-α2 loop in an orientation different from Fig 2B.


( B , C ) DNA binding analysis for the Interactions between WT or four MjMRcd mutants and a closed circular dsDNA ( φx174RFII ) in the absence ( B ) or presence ( C ) of AMP - PNP / Mg2 + . Each protein sample was incubated with φx174RFII dsDNA ( 3 . 5 nM ) for 30 min on 4 ° C . The following molar ratio of protein : DNA is used in Fig 3B - 3E ; lane 2 , 50 : 1 ; lane 3 , 100 : 1 ; lane 4 , 200 : 1 ; lane 5 , 500 : 1 ; lane 6 , 1000 : 1 ; lane 7 , 2000 : 1 . We note that some MRcd variants may form a second form of the complex with DNA .

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(B, C) DNA binding analysis for the Interactions between WT or four MjMRcd mutants and a closed circular dsDNA (φx174RFII) in the absence (B) or presence (C) of AMP-PNP/Mg2+. Each protein sample was incubated with φx174RFII dsDNA (3.5 nM) for 30 min on 4°C. The following molar ratio of protein: DNA is used in Fig 3B-3E; lane 2, 50:1; lane 3, 100:1; lane 4, 200:1; lane 5, 500:1; lane 6, 1000:1; lane 7, 2000:1. We note that some MRcd variants may form a second form of the complex with DNA.


( D , E ) Interactions of the WT or five full - length TmMR mutants with φx174RFII were examined in the absence ( D ) or presence of AMP - PNP / Mg2 + ( E ) . Reaction conditions are same as those in Fig 3B .

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(D, E) Interactions of the WT or five full-length TmMR mutants with φx174RFII were examined in the absence (D) or presence of AMP-PNP/Mg2+ (E). Reaction conditions are same as those in Fig 3B.


( F ) Nuclease activities of free MjMre11 ( lane 3 , 4 ) , nucleotide unbound - ( 5 , 6 ) and - bound MjMRcd complex ( 7 , 8 ) towards a hairpin DNA . Each protein sample was incubated with a hairpin DNA ( 10 nM ) in a 1 : 10 or 1 : 20 molar ratio ( protein : DNA ) for 30 min on 55 ° C . The error bars for the quantified values of uncleaved substrates on the right panel are calculated from the standard deviation from three repetitions of each experiment .

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(F) Nuclease activities of free MjMre11 (lane 3, 4), nucleotide unbound- (5, 6) and -bound MjMRcd complex (7, 8) towards a hairpin DNA. Each protein sample was incubated with a hairpin DNA (10 nM) in a 1:10 or 1:20 molar ratio (protein: DNA) for 30 min on 55°C. The error bars for the quantified values of uncleaved substrates on the right panel are calculated from the standard deviation from three repetitions of each experiment.


( G ) Nuclease activities of free TmMre11 alone ( lane 3 , 4 ) , nucleotide unbound - ( lane 5 , 6 ) and - bound ( lane 7 , 8 ) full - length TmMR complex towards a hairpin DNA .

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(G) Nuclease activities of free TmMre11 alone (lane 3, 4), nucleotide unbound- (lane 5, 6) and -bound (lane 7, 8) full-length TmMR complex towards a hairpin DNA.


( A ) Superposition of the ATPγS - MjMR - DNA complex ( green ) and apo - MjMR ( 3AV0 , magenta ) structures . Alignment was done using their NBDs ( 1 . 3 Å rmsd in the positions of 311 Cα atoms ) . The coiled - coils of the DNA - bound Rad50 molecules are shifted ( arrow ) to more parallel orientation . The entire structures can be superimposed with a 1 . 9 Å rmsd in 692 Cα positions .

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(A) Superposition of the ATPγS-MjMR-DNA complex (green) and apo-MjMR (3AV0, magenta) structures. Alignment was done using their NBDs (1.3 Å rmsd in the positions of 311 Cα atoms). The coiled-coils of the DNA-bound Rad50 molecules are shifted (arrow) to more parallel orientation. The entire structures can be superimposed with a 1.9 Å rmsd in 692 Cα positions.


( B , C ) Comparison of the local structures between the apo ( B ) and the DNA - bound MjMR complex ( C ) . Upon DNA binding , helix α6 translates to DNA backbone , resulting in the tighter packing of Leu155 and Leu156 against a hydrophobic pocket formed by lobe I and II . Arrows indicate the direction of the helix movement by DNA binding . See Fig EV5 for a close - up view .

(B, C) Comparison of the local structures between the apo (B) and the DNA-bound MjMR complex (C). Upon DNA binding, helix α6 translates to DNA backbone, resulting in the tighter packing of Leu155 and Leu156 against a hydrophobic pocket formed by lobe I and II. Arrows indicate the direction of the helix movement by DNA binding. See Fig EV5 for a close-up view.


( A ) Stereo view of the superposition of the ADP - MjRad50 ( 3AUX ) complex ( black ) onto the ATPγS - MjMR - DNA complex ( green and blue ) by aligning their lobe II domains . The α1 - α2 loop that could collide with parts of the DNA molecule is indicated . ( B ) Aligning the lobe II domains of two structures in Fig 5A shows that rotation of the lobe I opens the gate for Mre11 , which could allow the access of the unwound DNA . Color schemes are same as those in Fig 5A .

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(A) Stereo view of the superposition of the ADP-MjRad50 (3AUX) complex (black) onto the ATPγS-MjMR-DNA complex (green and blue) by aligning their lobe II domains. The α1-α2 loop that could collide with parts of the DNA molecule is indicated. (B) Aligning the lobe II domains of two structures in Fig 5A shows that rotation of the lobe I opens the gate for Mre11, which could allow the access of the unwound DNA. Color schemes are same as those in Fig 5A.


( C ) DNA - unwinding activities of TmMR . Lane 2 , no protein ; lanes 3 and 4 , no ATP ; lanes 5 and 6 , with ATPγS ; lanes 7 and 8 , with ATP . The DNA : protein ratio was 1 : 2 for lanes 3 , 5 , and 7 , and 1 : 5 for lanes 4 , 6 , and 8 . Plotted data represent the mean of three experiments , with error bars indicating one standard deviation . A graph of the quantified data is shown in the lower panel .

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(C) DNA-unwinding activities of TmMR. Lane 2, no protein; lanes 3 and 4, no ATP; lanes 5 and 6, with ATPγS; lanes 7 and 8, with ATP. The DNA: protein ratio was 1:2 for lanes 3, 5, and 7, and 1:5 for lanes 4, 6, and 8. Plotted data represent the mean of three experiments, with error bars indicating one standard deviation. A graph of the quantified data is shown in the lower panel.


( D ) Comparison of the ATP - dependent DNA unwinding activities of WT and mutant TmMR proteins . Lane 2 , no protein ; lane 3 and 4 , WT TmMR ; lane 5 and 6 , Rad50 ( Δ54 - 56 ) mutant ; lane 7 and 8 , R87E mutant ; lane 9 and 10 , K95E mutant ; lane 11 and 12 , R94E / K95E mutant ; lane 13 and 14 , K115E mutant . For each TmMR , reaction contains 1 : 2 or 1 : 5 ratio of DNA : protein .

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(D) Comparison of the ATP-dependent DNA unwinding activities of WT and mutant TmMR proteins. Lane 2, no protein; lane 3 and 4, WT TmMR; lane 5 and 6, Rad50 (Δ54-56) mutant; lane 7 and 8, R87E mutant; lane 9 and 10, K95E mutant; lane 11 and 12, R94E/K95E mutant; lane 13 and 14, K115E mutant. For each TmMR, reaction contains 1:2 or 1:5 ratio of DNA: protein.


( A ) Endonuclease activities of the TmMR WT and mutant proteins towards a duplex DNA in the absence ( left panel ) or presence of ATP ( right panel ) . Major products are marked with dots . The 5 ' - 32P - label is indicated by a star . See Appendix Fig S4A and S4B for the quantified values .

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(A) Endonuclease activities of the TmMR WT and mutant proteins towards a duplex DNA in the absence (left panel) or presence of ATP (right panel). Major products are marked with dots. The 5'-32P-label is indicated by a star. See Appendix Fig S4A and S4B for the quantified values.


( B ) Quantification of a bubble DNA binding by WT or mutant TmMR in the presence or absence of AMP - PNP . See Appendix Fig S3E and S3F for the original data .

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(B) Quantification of a bubble DNA binding by WT or mutant TmMR in the presence or absence of AMP-PNP. See Appendix Fig S3E and S3F for the original data.


( C ) Endonuclease activities of the WT and mutant TmMR proteins towards a bubble DNA molecule in the absence ( left panel ) or presence of ATP ( right panel ) . See Appendix Fig S4C and S4D for the quantified values .

(C) Endonuclease activities of the WT and mutant TmMR proteins towards a bubble DNA molecule in the absence (left panel) or presence of ATP (right panel). See Appendix Fig S4C and S4D for the quantified values.


( D ) The effects of single or multiple point mutations in the DNA - contacting or neighboring residues of S . cerevisiae rad50 on the sensitivity to chemical drugs . Sensitivities of rad50 expressing the mutants towards indicated concentrations of CPT , PHL , and MMS were examined . ( E ) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats . See Appendix Table S1 for the quantified values .

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(D) The effects of single or multiple point mutations in the DNA-contacting or neighboring residues of S. cerevisiae rad50 on the sensitivity to chemical drugs. Sensitivities of rad50 expressing the mutants towards indicated concentrations of CPT, PHL, and MMS were examined. (E) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats. See Appendix Table S1 for the quantified values.


( E ) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats . See Appendix Table S1 for the quantified values .

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(E) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats. See Appendix Table S1 for the quantified values.


( A - C ) Extra - mitochondrial puncta formation of CHCHD2T61I in MEFs . Chchd2KO cells were transfected with the CHCHD2WT - HA and CHCHD2T61I - HA plasmids . At the indicated times , cells were fixed and stained with anti - HA and anti - Tom20 antibodies , and observed by confocal microscopy . In ( A ) , representative images are shown . Magnified images of the areas within the dashed squares are shown in the insets . Representative images at lower magnification are shown in Appendix Fig . S1D . ( B , C ) Quantification of cells displaying mitochondrial CHCHD2 and extra - mitochondrial CHCHD2 puncta ( n ≥ 100 cells in each experiment ) . In ( B , C , data are shown as the mean ± SD ( n = 3 ) . Comparisons were performed using unpaired two - tailed Student t - tests and one - way ANOVA followed by the Tukey post - hoc tests . * * p < 0 .

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

(A-C) Extra-mitochondrial puncta formation of CHCHD2T61I in MEFs. Chchd2KO cells were transfected with the CHCHD2WT-HA and CHCHD2T61I-HA plasmids. At the indicated times, cells were fixed and stained with anti-HA and anti-Tom20 antibodies, and observed by confocal microscopy. In (A), representative images are shown. Magnified images of the areas within the dashed squares are shown in the insets. Representative images at lower magnification are shown in Appendix Fig. S1D. (B, C) Quantification of cells displaying mitochondrial CHCHD2 and extra-mitochondrial CHCHD2 puncta (n ≥ 100 cells in each experiment). In (B, C, data are shown as the mean ± SD (n = 3). Comparisons were performed using unpaired two-tailed Student t-tests and one-way ANOVA followed by the Tukey post-hoc tests. **p < 0.01


( D , E ) Extra - mitochondrial puncta formation of CHCHD2T61I in Neuro2a cells . Neuro2a cells were transfected with the CHCHD2WT - HA and CHCHD2T61I - HA plasmids for 4 hr , then cultured with medium containing 2 % FBS and 10 µM retinoic acid for neuronal differentiation . At 28 hr after transfection , cells were fixed and stained with anti - HA and anti - Tom20 antibodies . In ( D ) , representative images are shown . Magnified images of the areas within the dashed squares are shown in the insets . Representative images at lower magnification are shown ( E ) Quantification of cells displaying mitochondrial CHCHD2 and extra - mitochondrial CHCHD2 puncta ( n ≥ 100 cells in each experiment ) . In E , data are shown as the mean ± SD ( n = 3 ) . Comparisons were performed using unpaired two - tailed Student t - tests and one - way ANOVA followed by the Tukey post - hoc tests . * * p < 0 .

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(D, E) Extra-mitochondrial puncta formation of CHCHD2T61I in Neuro2a cells. Neuro2a cells were transfected with the CHCHD2WT-HA and CHCHD2T61I-HA plasmids for 4 hr, then cultured with medium containing 2% FBS and 10 µM retinoic acid for neuronal differentiation. At 28 hr after transfection, cells were fixed and stained with anti-HA and anti-Tom20 antibodies. In (D), representative images are shown. Magnified images of the areas within the dashed squares are shown in the insets. Representative images at lower magnification are shown (E) Quantification of cells displaying mitochondrial CHCHD2 and extra-mitochondrial CHCHD2 puncta (n ≥ 100 cells in each experiment). In E, data are shown as the mean ± SD (n = 3). Comparisons were performed using unpaired two-tailed Student t-tests and one-way ANOVA followed by the Tukey post-hoc tests. **p < 0.01


( F ) Neuro2a cells were treated as described in ( D ) , and cell lysates were fractionated into cytosol , organelles ( including mitochondria ) , nuclei , and cytoskeleton ( including insoluble matter ) . The expression of each protein was analyzed by western blotting using an anti - HA antibody . Cox4 was used as a control . ( G ) A semiquantitative analysis of protein expression in ( F ) is shown . In G ) , data are shown as the mean ± SD ( n = 3 ) . Comparisons were performed using unpaired two - tailed Student t - tests and one - way ANOVA followed by the Tukey post - hoc tests . * * p < 0 .

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(F) Neuro2a cells were treated as described in (D), and cell lysates were fractionated into cytosol, organelles (including mitochondria), nuclei, and cytoskeleton (including insoluble matter). The expression of each protein was analyzed by western blotting using an anti-HA antibody. Cox4 was used as a control. (G) A semiquantitative analysis of protein expression in (F) is shown. In G), data are shown as the mean ± SD (n = 3). Comparisons were performed using unpaired two-tailed Student t-tests and one-way ANOVA followed by the Tukey post-hoc tests. **p < 0.01


Neuro2a cells were transfected with the CHCHD2WT - HA and CHCHD2T61I - HA plasmids for 4 hr , and then cultured in medium containing 2 % FBS and 10 µM retinoic acid to induce neuronal differentiation . At 28 hr after transfection , cells were fixed and stained with an anti - HA antibody and ProteoStat protein aggregation dye ( A , B ) , anti - HA and anti - Nefl antibodies ( C , D ) , anti - HA and anti - p - Nefl473 antibodies ( E , F ) , In ( A , C , E , representative images are shown . Magnified images of the areas within the dashed squares are shown in the inset . Arrowheads indicate colocalized puncta between CHCHD2T61I - HA and aggresomes or the indicated proteins . Representative images at lower magnification are shown ( B , D , F , The amount of aggresomes ( B ) , Nefl ( D ) , p - Nefl473 ( F ) was measured as the fluorescence intensity per cell ( n = 30 cells in each experiment ) . Red bars indicate mean values . In ( B , D , F , comparisons were performed using unpaired two - tailed Student t - tests . * p < 0 . 05 . * * p < 0 .

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Neuro2a cells were transfected with the CHCHD2WT-HA and CHCHD2T61I-HA plasmids for 4 hr, and then cultured in medium containing 2% FBS and 10 µM retinoic acid to induce neuronal differentiation. At 28 hr after transfection, cells were fixed and stained with an anti-HA antibody and ProteoStat protein aggregation dye (A, B), anti-HA and anti-Nefl antibodies (C, D), anti-HA and anti-p-Nefl473 antibodies (E, F), In (A, C, E, representative images are shown. Magnified images of the areas within the dashed squares are shown in the inset. Arrowheads indicate colocalized puncta between CHCHD2T61I-HA and aggresomes or the indicated proteins. Representative images at lower magnification are shown (B, D, F, The amount of aggresomes (B), Nefl (D), p-Nefl473 (F) was measured as the fluorescence intensity per cell (n  = 30 cells in each experiment). Red bars indicate mean values. In (B, D, F, comparisons were performed using unpaired two-tailed Student t-tests. *p < 0.05. **p < 0.01


Neuro2a cells were transfected with the CHCHD2WT - HA and CHCHD2T61I - HA plasmids for 4 hr , and then cultured in medium containing 2 % FBS and 10 µM retinoic acid to induce neuronal differentiation . At 28 hr after transfection , cells were fixed and stained with anti - HA and anti - p - α - Synuclein129 ( p - α - Syn129 ) antibodies ( G , H ) . In G ) , representative images are shown . Magnified images of the areas within the dashed squares are shown in the inset . Arrowheads indicate colocalized puncta between CHCHD2T61I - HA and the indicated proteins . Representative images at lower magnification are shown H ) The amount of p - α - Syn129 ( H ) was measured as the fluorescence intensity per cell ( n = 30 cells in each experiment ) . Red bars indicate mean values . In H , comparisons were performed using unpaired two - tailed Student t - tests . * p < 0 . 05 . * * p < 0 .

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Neuro2a cells were transfected with the CHCHD2WT-HA and CHCHD2T61I-HA plasmids for 4 hr, and then cultured in medium containing 2% FBS and 10 µM retinoic acid to induce neuronal differentiation. At 28 hr after transfection, cells were fixed and stained with anti-HA and anti-p-α-Synuclein129 (p-α-Syn129) antibodies (G, H). In G), representative images are shown. Magnified images of the areas within the dashed squares are shown in the inset. Arrowheads indicate colocalized puncta between CHCHD2T61I-HA and the indicated proteins. Representative images at lower magnification are shown H) The amount of p-α-Syn129 (H) was measured as the fluorescence intensity per cell (n  = 30 cells in each experiment). Red bars indicate mean values. In H, comparisons were performed using unpaired two-tailed Student t-tests. *p < 0.05. **p < 0.01


In ( I ) , cell lysates were harvested at 28 hr , and the expression of each protein was analyzed by western blotting . ( J ) A semiquantitative analysis of protein expression in ( I ) is shown . Data are shown as the mean ± SD ( n = 3 ) . In J ) , comparisons were performed using unpaired two - tailed Student t - tests . * p < 0 . 05 . * * p < 0 .

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In (I), cell lysates were harvested at 28 hr, and the expression of each protein was analyzed by western blotting. (J) A semiquantitative analysis of protein expression in (I) is shown. Data are shown as the mean ± SD (n = 3). In J), comparisons were performed using unpaired two-tailed Student t-tests. *p < 0.05. **p < 0.01


( E , F ) Interaction between CHCHD2T61I and Csnk1e / d . Neuro2a cells were transfected with the CHCHD2WT - HA and CHCHD2T61I - HA plasmids together with pmax - GFP ( to detect transfected cells ) , and were differentiated into neuronal cells at 4 hr after transfection . At 28 hr after transfection , cells were fixed and the PLA was performed using anti - HA and anti - Csnk1e / d antibodies and Duolink PLA reagents . In ( E ) , PLA signals were clearly observed in cells expressing CHCHD2T61I - HA , but were faint and localized in the cytosol in cells expressing CHCHD2WT - HA . Representative images at lower magnification are shown In ( F ) , the number of PLA signals were counted ( n = 50 cells in each experiment ) . Red bars indicate mean values . In F ) , comparisons were performed using the unpaired two - tailed Student t - test and one - way ANOVA followed by the Tukey post - hoc test . * p < 0 . 05 . * * p < 0 . 01 . NS : not

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(E, F) Interaction between CHCHD2T61I and Csnk1e/d. Neuro2a cells were transfected with the CHCHD2WT-HA and CHCHD2T61I-HA plasmids together with pmax-GFP (to detect transfected cells), and were differentiated into neuronal cells at 4 hr after transfection. At 28 hr after transfection, cells were fixed and the PLA was performed using anti-HA and anti-Csnk1e/d antibodies and Duolink PLA reagents. In (E), PLA signals were clearly observed in cells expressing CHCHD2T61I-HA, but were faint and localized in the cytosol in cells expressing CHCHD2WT-HA. Representative images at lower magnification are shown In (F), the number of PLA signals were counted (n  = 50 cells in each experiment). Red bars indicate mean values. In F), comparisons were performed using the unpaired two-tailed Student t-test and one-way ANOVA followed by the Tukey post-hoc test. *p < 0.05. **p < 0.01. NS: not significant


( D - I ) Neuro2a cells were transfected with the CHCHD2T61I - HA plasmid together with the indicated siRNAs , and were differentiated into neuronal cells after 4 hr . At 28 hr after transfection , cells were fixed and stained with anti - HA , anti - p - Nefl473 , and anti - p - α - Syn129 antibodies , or ProteoStat aggresome detection dye . Similar experiments were performed by the addition of PF - 670462 ( 10 µM ) after 4 hr of transfection instead of gene silencing . In ( D , F , H ) , representative images are shown . Magnified images of the areas within the dashed squares are shown in the insets . Arrowheads indicate colocalized puncta between CHCHD2T61I - HA and the indicated phosphorylated proteins or aggresomes . In ( E , G , I ) , the amount of p - Nefl473 ( E ) , p - α - Syn129 ( G ) , and aggresomes ( I ) was measured as the fluorescence intensity per cell ( n = 30 cells in each experiment ) . Red bars indicate mean values . In E , G , I ) , comparisons were performed using one - way ANOVA followed by the Tukey post - hoc tests or unpaired two - tailed Student t - test . * p < 0 . 05 ; * * p < 0 . 01 . NS : not

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-I) Neuro2a cells were transfected with the CHCHD2T61I-HA plasmid together with the indicated siRNAs, and were differentiated into neuronal cells after 4 hr. At 28 hr after transfection, cells were fixed and stained with anti-HA, anti-p-Nefl473, and anti-p-α-Syn129 antibodies, or ProteoStat aggresome detection dye. Similar experiments were performed by the addition of PF-670462 (10 µM) after 4 hr of transfection instead of gene silencing. In (D, F, H), representative images are shown. Magnified images of the areas within the dashed squares are shown in the insets. Arrowheads indicate colocalized puncta between CHCHD2T61I-HA and the indicated phosphorylated proteins or aggresomes. In (E, G, I), the amount of p-Nefl473 (E), p-α-Syn129 (G), and aggresomes (I) was measured as the fluorescence intensity per cell (n = 30 cells in each experiment). Red bars indicate mean values. In E, G, I), comparisons were performed using one-way ANOVA followed by the Tukey post-hoc tests or unpaired two-tailed Student t-test. *p < 0.05; **p < 0.01. NS: not significant


Reduction in TH signals in the SNpc of knock - in mice . In ( D ) , brain cryosections were immunostained with the dopaminergic cell marker TH . Representative images of the SNpc and ventral tegmental area ( VTA ) are shown . Dashed lines indicate the SNpc and VTA regions . Bars = 200 µm . In ( E ) , TH signals in the SNpc ( average fluorescence intensity per region ) are shown as the mean ± SD ( n = 3 ) . In E ) , comparisons were performed using one - way ANOVA followed by the Tukey post - hoc tests . * p < 0 . 05 ; * * p < 0 . 01 . NS : not

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Reduction in TH signals in the SNpc of knock-in mice. In (D), brain cryosections were immunostained with the dopaminergic cell marker TH. Representative images of the SNpc and ventral tegmental area (VTA) are shown. Dashed lines indicate the SNpc and VTA regions. Bars = 200 µm. In (E), TH signals in the SNpc (average fluorescence intensity per region) are shown as the mean ± SD (n = 3). In E), comparisons were performed using one-way ANOVA followed by the Tukey post-hoc tests. *p < 0.05; **p < 0.01. NS: not significant


Reduction in TH signals in the SNpc of knock - in mice . In ( F ) , cryosections of the SNpc were immunostained with anti - Chchd2 and anti - TH antibodies . Arrowheads indicate Chchd2T61I puncta in TH - positive cells . Dashed lines indicate cell shapes . ( G - K ) Extra - mitochondrial aggresome formation by p - Nefl473 , p - α - Syn129 , and Csnk1e / d in the SNpc of knock - in mice . Cryosections of the SNpc were immunostained with anti - Chchd2 and anti - Ant1 / 2 ( G ) , anti - p - Nefl473 ( H ) , anti - p - α - Syn129 ( I ) , and anti - Csnk1e / d antibodies ( J ) , and with ProteoStat dye ( K ) . Ant1 / 2 are mitochondrial membrane proteins and arrowheads indicate extra - mitochondrial Chchd2T61I ( G ) . In ( H - K ) , arrowheads indicate the colocalization of puncta with Chchd2T61I and the indicated proteins or aggresomes . Dashed lines indicate cell shapes .

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Reduction in TH signals in the SNpc of knock-in mice. In (F), cryosections of the SNpc were immunostained with anti-Chchd2 and anti-TH antibodies. Arrowheads indicate Chchd2T61I puncta in TH-positive cells. Dashed lines indicate cell shapes. (G-K) Extra-mitochondrial aggresome formation by p-Nefl473, p-α-Syn129, and Csnk1e/d in the SNpc of knock-in mice. Cryosections of the SNpc were immunostained with anti-Chchd2 and anti-Ant1/2 (G), anti-p-Nefl473 (H), anti-p-α-Syn129 (I), and anti-Csnk1e/d antibodies (J), and with ProteoStat dye (K). Ant1/2 are mitochondrial membrane proteins and arrowheads indicate extra-mitochondrial Chchd2T61I (G). In (H-K), arrowheads indicate the colocalization of puncta with Chchd2T61I and the indicated proteins or aggresomes. Dashed lines indicate cell shapes.


( L ) Isolated midbrain and diencephalon lysates were subjected to western blotting .

(L) Isolated midbrain and diencephalon lysates were subjected to western blotting.


( F - J ) The same experiments as Fig . 5G - K were performed using PF - 670462 - infused and vehicle - infused Chchd2T61I knock - in mice .

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(F-J) The same experiments as Fig. 5G-K were performed using PF-670462-infused and vehicle-infused Chchd2T61I knock-in mice.


( A ) Photos of the midbrain of a control subject ( left ) and PD patient harboring the CHCHD2T61I mutation ( right ) . The dashed lines indicate the SNpc . A portion of the midbrain of the control subject was lost when slicing .

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(A) Photos of the midbrain of a control subject (left) and PD patient harboring the CHCHD2T61I mutation (right). The dashed lines indicate the SNpc. A portion of the midbrain of the control subject was lost when slicing.


( B - G ) Colocalization of extra - mitochondrial CHCHD2T61I with CSNK1E / D , p - NEFL472 , and p - α - SYN129 in the brain of a PD patient harboring the CHCHD2T61I mutation . Each brain section was deparaffinized and immunostained with anti - CHCHD2 and anti - ANT1 / 2 ( B ) , anti - CSNK1E / D ( C ) , anti - p - NEFL472 ( D , E ) , or anti - p - α - SYN129 antibodies ( F , G ) . Arrowheads indicate extra - mitochondrial CHCHD2T61I puncta ( B ) , and puncta showing colocalization of CHCHD2T61I with CSNK1E / D ( C ) , p - NEFL472 ( D ) , and p - α - SYN129 ( F ) . Arrows indicate weaker colocalization between CHCHD2T61I and the indicated proteins in the control brain ( C , D , F ) . Dashed lines indicate cell shapes . In ( E , G ) , the amount of p - NEFL472 ( E ) and p - α - SYN129 ( G ) was measured by the average fluorescence intensity per cell ( n = 30 cells in each experiment ) . Red bars indicate mean values . Comparisons were performed using the unpaired two - tailed Student t - tests . * * p < 0 .

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(B-G) Colocalization of extra-mitochondrial CHCHD2T61I with CSNK1E/D, p-NEFL472, and p-α-SYN129 in the brain of a PD patient harboring the CHCHD2T61I mutation. Each brain section was deparaffinized and immunostained with anti-CHCHD2 and anti-ANT1/2 (B), anti-CSNK1E/D (C), anti-p-NEFL472 (D, E), or anti-p-α-SYN129 antibodies (F, G). Arrowheads indicate extra-mitochondrial CHCHD2T61I puncta (B), and puncta showing colocalization of CHCHD2T61I with CSNK1E/D (C), p-NEFL472 (D), and p-α-SYN129 (F). Arrows indicate weaker colocalization between CHCHD2T61I and the indicated proteins in the control brain (C, D, F). Dashed lines indicate cell shapes. In (E, G), the amount of p-NEFL472 (E) and p-α-SYN129 (G) was measured by the average fluorescence intensity per cell (n  = 30 cells in each experiment). Red bars indicate mean values. Comparisons were performed using the unpaired two-tailed Student t-tests. **p < 0.01


iPSCs were prepared from a healthy control and a PD patient harboring the CHCHD2T61I mutation . An isogenic control iPSC line was generated by the correction of the gene mutation Then , these iPSC cells were differentiated into dopaminergic ( DA ) neurons , and analyzed by immunofluorescence using anti - CHCHD2 ( A - E ) , anti - ANT1 / 2 ( A ) , anti - CSNK1E / D ( B ) , anti - p - NEFL472 ( C ) , and anti - p - α - SYN129 antibodies ( D ) Arrowheads indicate extra - mitochondrial CHCHD2T61I ( A ) , puncta showing colocalization of CHCHD2T61I with CSNK1E / D ( B ) , p - NEFL472 ( C , , and p - α - SYN129 ( D , Dashed lines indicate cell shapes .

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iPSCs were prepared from a healthy control and a PD patient harboring the CHCHD2T61I mutation. An isogenic control iPSC line was generated by the correction of the gene mutation Then, these iPSC cells were differentiated into dopaminergic (DA) neurons, and analyzed by immunofluorescence using anti-CHCHD2 (A-E), anti-ANT1/2 (A), anti-CSNK1E/D (B), anti-p-NEFL472 (C), and anti-p-α-SYN129 antibodies (D) Arrowheads indicate extra-mitochondrial CHCHD2T61I (A), puncta showing colocalization of CHCHD2T61I with CSNK1E/D (B), p-NEFL472 (C, , and p-α-SYN129 (D, Dashed lines indicate cell shapes.


CHCHD2T61I iPSC - derived DA neurons were treated with PF - 670462 ( 10 µM ) for 20 hr and analyzed by immunofluorescence using anti - CHCHD2 and anti - p - α - SYN129 antibodies ( G ) , and ProteoStat aggresome dye ( H ) . Arrowheads indicate puncta showing colocalization of CHCHD2T61I with p - α - SYN129 G ) or aggresomes H ) . Dashed lines indicate cell shapes .

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CHCHD2T61I iPSC-derived DA neurons were treated with PF-670462 (10 µM) for 20 hr and analyzed by immunofluorescence using anti-CHCHD2 and anti-p-α-SYN129 antibodies (G), and ProteoStat aggresome dye (H). Arrowheads indicate puncta showing colocalization of CHCHD2T61I with p-α-SYN129 G) or aggresomes H). Dashed lines indicate cell shapes.


A . MDCK cells were cultured on cell culture inserts for 4 days . On the last day , the culture medium was replaced with EV - depleted medium . EVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation . Cell lysates and EV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated . Note that the PEG pellets did not contain mitochondrial protein TOMM20 , suggesting that the PEG pellets were not contaminated by intracellular organelles .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O

A. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. EVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation. Cell lysates and EV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated. Note that the PEG pellets did not contain mitochondrial protein TOMM20, suggesting that the PEG pellets were not contaminated by intracellular organelles.


B . PEG pellets were subjected to OptiPrep flotation analysis .

B. PEG pellets were subjected to OptiPrep flotation analysis.


C . MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD63 antibody .

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C. MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD63 antibody.


D . sEVs were eluted from the beads with a glycine buffer and analyzed by nanoparticle tracking analysis ( NTA ) . Representative NTA traces were shown . E . Quantification of the NTA data obtained in five independent experiments .

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D. sEVs were eluted from the beads with a glycine buffer and analyzed by nanoparticle tracking analysis (NTA). Representative NTA traces were shown. E. Quantification of the NTA data obtained in five independent experiments.


F . MDCK cells were cultured sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody .

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F. MDCK cells were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody.


G . sEVs were eluted from the beads with a glycine buffer and analyzed by NTA . Representative NTA traces were shown . H . Quantification of the NTA data obtained in five independent experiments .

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G. sEVs were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown. H. Quantification of the NTA data obtained in five independent experiments.


A . MDCK cells were cultured on cell culture inserts for 4 days . On the last day , the culture medium was replaced with serum - free medium . sEVs released from the apical and basolateral sides of MDCK cells were purified by ultracentrifugation . Apical and basolateral sEVs ( P100 ) were analyzed by silver staining .

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A. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with serum-free medium. sEVs released from the apical and basolateral sides of MDCK cells were purified by ultracentrifugation. Apical and basolateral sEVs (P100) were analyzed by silver staining.


C . MDCK cells were cultured on cell culture inserts for 4 days . On the last day , the culture medium was replaced with EV - depleted medium . sEVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation . Cell lysates and sEV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated . GPRC5C was detected only in the apical sEV

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O

C. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation. Cell lysates and sEV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated. GPRC5C was detected only in the apical sEV samples


D . PEG pellets were subjected to OptiPrep flotation analysis . GPRC5C was detected only in the apical sEV

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D. PEG pellets were subjected to OptiPrep flotation analysis. GPRC5C was detected only in the apical sEV samples


E . MDCK cells were cultured sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody . GPRC5C was detected only in the apical sEV

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E. MDCK cells were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. GPRC5C was detected only in the apical sEV samples


F . MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD63 antibody .

F. MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD63 antibody.


A . MDCK cells were transfected with siControl or the siRNAs indicated , and the cells were transferred to cell culture inserts and cultured for 4 days . On the last day , the culture medium was replaced with EV - depleted medium . sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody . Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O

A. MDCK cells were transfected with siControl or the siRNAs indicated, and the cells were transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated.


B . The intensity of the bands was measured in three independent experiments .

B. The intensity of the bands was measured in three independent experiments.


C . sEVs were eluted from the beads with a glycine buffer and analyzed by NTA . Representative NTA traces were shown .

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C. sEVs were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown.


D . Quantification of the NTA data obtained in five independent experiments .

D. Quantification of the NTA data obtained in five independent experiments.


A . MDCK cells were transfected with siControl or siALIX , and the cells were transferred to cell culture inserts and cultured for 4 days . On the last day , the culture medium was replaced with EV - depleted medium with or without 10 nM GW4869 . sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody . Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated .

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A. MDCK cells were transfected with siControl or siALIX, and the cells were transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium with or without 10 nM GW4869. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated.


( A ) Hep3B hepatocytes were treated with nontargeting control siRNA ( siNT ) or siRNA targeting Dyn2 ( siDyn2 ) , followed by re - expression of either vector alone , or GFP - tagged versions of Dyn2 , loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0 . 1 % FBS . LDs were visualized by immunofluorescence using Oil Red O staining . Cell boundaries are outlined and those cells re - expressing GFP , GFP - wtDyn2 , or GFP - - K44A are denoted with asterisks . Bars , 20 µM . ( B ) Representative blot showing the efficiency of the Dyn2 knockdown in these cells . ( C and D ) Quantitation of the average LD number and area ( in pixels2 ) per cell from three independent experiments . The data are represented as mean ± SE . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 . NS , not significant .

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(A) Hep3B hepatocytes were treated with nontargeting control siRNA (siNT) or siRNA targeting Dyn2 (siDyn2), followed by re-expression of either vector alone, or GFP-tagged versions of Dyn2, loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS. LDs were visualized by immunofluorescence using Oil Red O staining. Cell boundaries are outlined and those cells re-expressing GFP, GFP-wtDyn2, or GFP--K44A are denoted with asterisks. Bars, 20 µM. (B) Representative blot showing the efficiency of the Dyn2 knockdown in these cells. (C and D) Quantitation of the average LD number and area (in pixels2) per cell from three independent experiments. The data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01. NS, not significant.