( A _ B ) Vero E6 - TMPRSS2 ( A ) or Vero E6 ( B ) were inoculated with a total of 126 clinical NP swab samples representing a range of viral , both total E ( Clinical Sample E CT detected by WHO - E primer / probe set ) subgenomic E ( CT detected by Mills - SgE primer set developed in this study ) . Culture supernatants were harvested when displayed CPE or at day 7 if no CPE was observed earlier , clarified , presence ( blue square ) or absence ( yellow circle ) of SARS - CoV - 2 determined by immuno - focus .

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

(A_B) Vero E6-TMPRSS2 (A) or Vero E6 (B) cells were inoculated with a total of 126 clinical NP swab samples representing a range of viral RNA levels, both total E RNA (Clinical Sample E CT detected by WHO-E primer/probe set) and subgenomic E RNA (CT detected by the Mills-SgE primer set developed in this study). Culture supernatants were harvested when cells displayed CPE or at day 7 if no CPE was observed earlier, clarified, and presence (blue square) or absence (yellow circle) of SARS-CoV-2 determined by immuno-focus assay.


( A - B ) Supernatants from panel of infected ( A ) Vero E6 - TMPRSS2 ( B ) Vero E6 were harvested when CPE was observed viral titer ( FFU / mL measured by focus forming ) was determined for final time point of each sample that successfully isolated . Samples are colored according to day CPE was observed sample was harvested . Total E CT of original clinical sample used for viral isolation is plotted on X axis . Data shown is from subset of samples that successfully isolated in each . Limit of detection for infectious titer is 400 FFU / mL .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-B) Supernatants from the panel of infected (A) Vero E6-TMPRSS2 and (B) Vero E6 cells were harvested when CPE was observed and the viral titer (FFU/mL measured by focus forming assay) was determined for the final time point of each sample that successfully isolated. Samples are colored according to the day CPE was observed and the sample was harvested. Total E RNA CT of the original clinical sample used for viral isolation is plotted on the X axis. Data shown is from the subset of samples that successfully isolated in each cell line. Limit of detection for infectious titer is 400 FFU/mL.


( A ) amount of subgenomic E in panel of clinical samples used to infect Vero E6 - TMPRSS2 was measured using a previously - published primer - probe set ( Wölfel - sgE )

O O O O O B-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

(A) The amount of subgenomic E RNA in the panel of clinical samples used to infect Vero E6-TMPRSS2 cells was measured using a previously-published primer-probe set (Wölfel-sgE)


( B ) For a subset of 85 NP swab samples for which sufficient material remained , SARS - CoV - 2 negative - strand E were determined by strand - specific RT - PCR with tagged primers . Data information : Culture supernatants were harvested when displayed CPE or at day 7 if no CPE was observed earlier , clarified , presence ( blue square ) or absence ( yellow circle ) of infectious SARS - CoV - 2 determined by immuno - focus .

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 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

(B) For a subset of 85 NP swab samples for which sufficient material remained, SARS-CoV-2 negative-strand E RNA levels were determined by strand-specific RT-PCR with tagged primers. Data information: Culture supernatants were harvested when cells displayed CPE or at day 7 if no CPE was observed earlier, clarified, and presence (blue square) or absence (yellow circle) of infectious SARS-CoV-2 determined by immuno-focus assay.


of housekeeping genes GAPDH 18S rRNA , negative - strand E ( negE ) , sgE relative to total of E were measured in infected , clarified supernatants concentrated through a sucrose cushion by ultracentrifugation . Data points represent four technical replicates from two independent experiments ( Supernatant ) or three replicates ( Concentrated ) are normalized to total of E for each sample , plotted as mean with error bars plotted as + / - .

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

Levels of the cellular housekeeping genes GAPDH and 18S rRNA, negative-strand E (negE), and sgE relative to total levels of E RNA were measured in infected cells, clarified supernatants and virus concentrated through a sucrose cushion by ultracentrifugation. Data points represent four technical replicates from two independent experiments (Cell and Supernatant) or three replicates (Concentrated Virus) and are normalized to the total levels of E for each sample, plotted as mean with error bars plotted as +/- SEM.


( A ) Immunoblot of BAG1 , BAG3 , Hsc / Hsp70 Hsp90 in young old I90 . For detection of BAG , an antibody directed against conserved BAG domain was used .

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

(A) Immunoblot analysis of BAG1, BAG3, Hsc/Hsp70 and Hsp90 in young and old I90 cells. For detection of BAG proteins, an antibody directed against the conserved BAG domain was used.


( B ) Real ‐ time PCR of human BAG family members Hsc70 Hsp90 in young old I90 . Depicted is expression ( log2 ) of target genes in old relative to young . * P0 . 05 * * P0 . 01 versus young , n = 3 .

O O O O O O O O O O O O B-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

(B) Real‐time PCR analysis of human BAG family members and Hsc70 and Hsp90 in young and old I90 cells. Depicted is the expression ratio (log2) of target genes in old cells relative to young cells. *P0.05 and **P0.01 versus young, n=3.


( C ) Co ‐ IP of Hsc / Hsp70 interaction with BAG1 BAG3 in young ( Y ) old ( O ) I90 . Upper panel shows relative amounts of in lysates used for Co ‐ IP ( Input ) . Middle panel shows of BAG1 BAG3 found in Hsc / Hsp70 immunoprecipitates . Lower panel shows of Hsc / Hsp70 co ‐ sedimented upon immunoprecipitation of BAG3 .

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

(C) Co‐IP analysis of Hsc/Hsp70 interaction with BAG1 and BAG3 in young (Y) and old (O) I90 cells. Upper panel shows relative amounts of proteins in cell lysates used for Co‐IP (Input). Middle panel shows levels of BAG1 and BAG3 found in Hsc/Hsp70 immunoprecipitates. Lower panel shows levels of Hsc/Hsp70 co‐sedimented upon immunoprecipitation of BAG3.


( D , E ) 293 were treated with indicated agents for 8 h followed by immunoblot of indicated .

(D, E) 293 cells were treated with the indicated agents for 8 h followed by immunoblot analysis of indicated proteins.


( A ) 293 were transfected with bag1 , bag3 or nonsense ( nons ) siRNAs , as indicated . After 48 h , were transfected with d2GFP expression plasmid together with half amounts of indicated siRNAs . After additional 24 h , of indicated were detected by immunoblot .

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

(A) 293 cells were transfected with bag1, bag3 or nonsense (nons) siRNAs, as indicated. After 48 h, cells were transfected with d2GFP expression plasmid together with half the amounts of the indicated siRNAs. After additional 24 h, levels of indicated proteins were detected by immunoblot analysis.


( B ) 293 were transfected with indicated siRNAs for 48 h followed by real ‐ time PCR of BAG1 BAG3 mRNA . Depicted is mean relative expression ( log2 ) ± s . e . m . * P0 . 05 * * * P0 . 001 versus nons , n = 3 .

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 O O O O O O O O O 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) 293 cells were transfected with indicated siRNAs for 48 h followed by real‐time PCR analysis of BAG1 and BAG3 mRNA levels. Depicted is the mean relative expression ratio (log2) ±s.e.m. *P0.05 and ***P0.001 versus nons, n=3.


( C ) BAG1 knockdown was performed in 293 for 48 h . Thereafter , were transfected with GFP or GFP ‐ UPS reporter genes as indicated . After additional 24 h , of GFP ‐ positive were detected by western ‐ blot using a GFP antibody .

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

(C) BAG1 knockdown was performed in 293 cells for 48 h. Thereafter, cells were transfected with GFP or GFP‐based UPS reporter genes as indicated. After additional 24 h, levels of GFP‐positive proteins were detected by western‐blot analysis using a GFP antibody.


( D , E ) Immunoblot of indicated from d2HEK treated with different concentrations of MG132 for 5 h ( D ) or transfected with indicated amounts of BAG1S plasmid for 24 h ( E ) .

O O O O 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

(D, E) Immunoblot analysis of indicated proteins from d2HEK cells treated with different concentrations of MG132 for 5 h (D) or transfected with indicated amounts of BAG1S plasmid for 24 h (E).


( A ) 293 were transfected with nonsense ( nons ) , bag1 or bag3 siRNAs for 48 h then treated for 2 h with lysosomal inhibitors pepstatinA E64 ( both 10 μg / ml ; Pep . A / E64 ) or DMSO as control , followed by immunoblot of indicated .

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

(A) 293 cells were transfected with nonsense (nons), bag1 or bag3 siRNAs for 48 h and then treated for 2 h with the lysosomal inhibitors pepstatinA and E64 (both 10 μg/ml; Pep.A/E64) or DMSO as control, followed by immunoblot analysis of the indicated proteins.


( B ) 293 were transfected for 48 h with indicated siRNAs BAG3 expression plasmid ( BAG3 ‐ N1 ) or vector control ( N1 ) followed by same as in ( A ) .

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

(B) 293 cells were transfected for 48 h with the indicated siRNAs and BAG3 expression plasmid (BAG3‐N1) or vector control (N1) followed by the same analysis as in (A).


( C ) Diagram shows autophagic flux of 293 with differently modulated BAG1 BAG3 as described in ( A ) ( B ) . Autophagic flux was determined by strength of LC3 ‐ II accumulation in a 2 ‐ h treatment period with Pep . A / E64 . Therefore , normalised LC3 ‐ II in absence of inhibitors were subtracted from corresponding obtained in presence of Pep . A / E64 . Values are expressed as mean ± s . e . m . * P0 . 05 versus control ‐ transfected , n = 3 .

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

(C) Diagram shows the autophagic flux of 293 cells with differently modulated BAG1 and BAG3 levels as described in (A) and (B). Autophagic flux was determined by the strength of LC3‐II accumulation in a 2‐h treatment period with Pep.A/E64. Therefore, normalised LC3‐II levels in the absence of inhibitors were subtracted from corresponding levels obtained in the presence of Pep.A/E64. Values are expressed as mean±s.e.m. *P0.05 versus control‐transfected cells, n=3.


( D ) I90 were transfected with GFP fused to LC3 ( pGFP . LC3 ) co ‐ transfected either with BAG3 ‐ N1 or N1 . After transfection for 48 h , were treated as in ( A ) of indicated were detected by immunoblot .

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

(D) I90 cells were transfected with GFP fused to LC3 (pGFP.LC3) and co‐transfected either with BAG3‐N1 or N1. After transfection for 48 h, cells were treated as in (A) and levels of indicated proteins were detected by immunoblot analysis.


( E ) I90 , transfected as in ( D ) for 24 h , were microscopically analysed for GFP ‐ LC3 . Representative pictures are shown . Bar : 20 μm . Values expressed in diagram are mean ± s . e . m . from three independent experiments ( 50 were counted per experiment ) . * P0 . 05 versus N1 , n = 3 .

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

(E) I90 cells, transfected as in (D) for 24 h, were microscopically analysed for GFP‐LC3 fluorescence. Representative pictures are shown. Bar: 20 μm. Values expressed in diagram are mean±s.e.m. from three independent experiments (50 cells were counted per experiment). *P0.05 versus N1, n=3.


( A ) I90 were transfected with BAG3 ( BAG3 ‐ N1 ) or vector control . After transfection for 48 h , of indicated were detected by western ‐ blot .

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

(A) I90 cells were transfected with BAG3 (BAG3‐N1) or vector control. After transfection for 48 h, levels of indicated proteins were detected by western‐blot analysis.


( B ) Transcript of SQSTM1 in I90 transfected as in ( A ) were compared by real ‐ time PCR . Depicted is fold ‐ increase of SQSTM1 mRNA in BAG3 ‐ N1 ‐ transfected relative to control ( N1 ) ‐ transfected . Values are expressed as mean ± s . e . m . * * P0 . 01 versus N1 , n = 3 .

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

(B) Transcript levels of SQSTM1 in I90 cells transfected as in (A) were compared by real‐time PCR analysis. Depicted is the fold‐increase of SQSTM1 mRNA levels in BAG3‐N1‐transfected cells relative to control (N1)‐transfected cells. Values are expressed as mean±s.e.m. **P0.01 versus N1 cells, n=3.


( C ) I90 were transfected with a BAG3 ‐ GFP fusion plasmid for 48 h followed by indirect immunofluorescence of endogenous SQSTM1 . ( a ) Direct of BAG3 ‐ GFP ( green ) , ( b ) indirect immunofluorescence of SQSTM1 ( red ) ( c ) stainings of ( a ) ( b ) overlapped . DAPI ( blue ) was used to stain . Representative pictures are shown . 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 B-MEASURED_VAR O O O O O O B-MEASURED_VAR 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

(C) I90 cells were transfected with a BAG3‐GFP fusion plasmid for 48 h followed by indirect immunofluorescence staining of endogenous SQSTM1. (a) Direct fluorescence of BAG3‐GFP (green), (b) indirect immunofluorescence of SQSTM1 (red) and (c) the stainings of (a) and (b) overlapped. DAPI (blue) was used to stain DNA. Representative pictures are shown. Bar: 10 μm.


( D ) Co ‐ IP studies testing interaction of SQSTM1 BAG3 . I90 were transfected as in ( A ) . After 24 h , SQSTM1 BAG3 were immunoprecipitated followed by of co ‐ sedimented , as indicated . As negative control purified rabbit ( rb ) mouse ( ms ) IgG was used . Left panel shows relative amounts of in lysates used for Co ‐ IP ( Input ) .

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

(D) Co‐IP studies testing the interaction of SQSTM1 and BAG3. I90 cells were transfected as in (A). After 24 h, SQSTM1 and BAG3 were immunoprecipitated followed by analysis of co‐sedimented proteins, as indicated. As negative control purified rabbit (rb) and mouse (ms) IgG was used. Left panel shows relative amounts of proteins in lysates used for Co‐IP (Input).


( A ) Proteasomal chymotrypsin cathepsin activity in lysates from young ( Y ) old ( O ) I90 was determined using specific probes , as described in Material methods section . Values are expressed are mean ± s . e . m . * P0 . 05 * * P0 . 01 versus young , n = 3 .

O O O O B-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

(A) Proteasomal chymotrypsin and cathepsin activity in lysates from young (Y) and old (O) I90 cells was determined using specific fluorescence probes, as described in the Material and methods section. Values are expressed are mean±s.e.m. *P0.05 and **P0.01 versus young, n=3.


( B ) Real ‐ time PCR of LC3 , WIPI1 SQSTM1 mRNA in young old I90 . Depicted is mean expression ( log2 ) ± s . e . m . of target genes in old relative to young . * * P0 . 01 * * * P0 . 001 versus young , n = 3 .

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

(B) Real‐time PCR analysis of LC3, WIPI1 and SQSTM1 mRNA levels in young and old I90 cells. Depicted is the mean expression ratio (log2) ±s.e.m. of target genes in old cells relative to young cells. **P0.01 and ***P0.001 versus young, n=3.


( C ) Immunoblot of LC3 SQSTM1 in young old I90 .

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

(C) Immunoblot analysis of LC3 and SQSTM1 in young and old I90 cells.


( D ) Young old I90 were treated for 2 h with bafilomycin A1 ( BafA1 , 2 μM ) or DMSO as control followed by immunoblot of 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

(D) Young and old I90 cells were treated for 2 h with bafilomycin A1 (BafA1, 2 μM) or DMSO as control followed by immunoblot analysis of indicated proteins.


( E ) Immunoblot of WIPI1 expression in young old I90 .

(E) Immunoblot analysis of WIPI1 expression in young and old I90 cells.


( F ) Indirect immunofluorescence of endogenous LC3 ( green ) , SQSTM1 ( red ) WIPI1 ( ) in I90 of young old . DAPI ( blue ) was used to stain . Representative pictures are shown . Bar : 20 μm . Diagrams show of with indicated characteristics counted as in Figure 3E .

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

(F) Indirect immunofluorescence staining of endogenous LC3 (green), SQSTM1 (red) and WIPI1 (white) in I90 cells of young and old age. DAPI (blue) was used to stain DNA. Representative pictures are shown. Bar: 20 μm. Diagrams show percentage of cells with indicated characteristics counted as in Figure 3E.


( G ) Old young I90 were treated for 1 h with DMSO as control ( C ) , lactacystin ( L , 2 μM ) or NH4Cl ( 20 mM ) plus leupeptin ( Leu , 5 μM ) ( N , NH4Cl / Leu ) . Western ‐ blot analyses were performed for detection of indicated . In diagram ( right panel ) , of polyUb ‐ SQSTM1 are depicted after normalisation to corresponding Tubulin . Values are expressed as mean ± s . e . m . * P0 . 05 versus old control , # P0 . 05 versus young control , n = 3 .

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

(G) Old and young I90 cells were treated for 1 h with DMSO as control (C), lactacystin (L, 2 μM) or NH4Cl (20 mM) plus leupeptin (Leu, 5 μM) (N, NH4Cl/Leu). Western‐blot analyses were performed for detection of indicated proteins. In the diagram (right panel), levels of polyUb‐proteins and SQSTM1 are depicted after normalisation to corresponding Tubulin levels. Values are expressed as mean±s.e.m. *P0.05 versus old control, #P0.05 versus young control, n=3.


( H ) I90 of old ( upper panel ) young ( lower panel ) were transfected with atg7 or nonsense ( nons ) siRNA . After transfection for 4 days , were treated with NH4Cl / Leu or DMSO for 1 h followed by fractioning of lysates in TritonX ‐ 100 ( TX ‐ 100 ) soluble insoluble material . Equal amounts of both fractions were directed to immunoblot for of indicated . Gapdh Histone H3 were used as loading controls of soluble insoluble fractions , respectively .

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

(H) I90 cells of old (upper panel) and young (lower panel) age were transfected with atg7 or nonsense (nons) siRNA. After transfection for 4 days , the cells were treated with NH4Cl/Leu or DMSO for 1 h followed by fractioning of cell lysates in TritonX‐100 (TX‐100) soluble and insoluble material. Equal protein amounts of both fractions were directed to immunoblot analysis for analysis of indicated proteins. Gapdh and Histone H3 were used as loading controls of soluble and insoluble fractions, respectively.


( I ) Transmission electron microscopic of young ( a ) old ( b ) I90 . Magnifications of marked areas in ( a ) ( b ) are shown in ( c ) ( d ) , respectively . An arrow indicates an autolysosome , arrow heads indicate autophagosomes . M , mitochondrion . N , nucleus .

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

(I) Transmission electron microscopic analysis of young (a) and old (b) I90 cells. Magnifications of marked areas in (a) and (b) are shown in (c) and (d), respectively. An arrow indicates an autolysosome, and arrow heads indicate autophagosomes. M, mitochondrion. N, nucleus.


( A ) Immunoblot of indicated of young ( Y ) old ( O ) I90 upon fractionation as described in Figure 5H .

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) Immunoblot analysis of indicated proteins of young (Y) and old (O) I90 cells upon fractionation as described in Figure 5H.


( B , C ) After transfection for 96 h of old I90 with bag3 or nonsense ( nons ) siRNA , BAG1 BAG3 mRNA were analysed by immunoblot ( B ) real ‐ time PCR ( C ) , respectively . Transcript in bag3 siRNA are depicted as mean log2 expression ± s . e . m . relative to nons siRNA . * P0 . 05 * * * P0 . 001 versus nons , n = 3 .

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

(B, C) After transfection for 96 h of old I90 cells with bag3 or nonsense (nons) siRNA, BAG1 and BAG3 protein and mRNA levels were analysed by immunoblot (B) and real‐time PCR (C) analysis, respectively. Transcript levels in bag3 siRNA cells are depicted as the mean log2 expression ratio ±s.e.m. relative to nons siRNA cells. *P0.05 and ***P0.001 versus nons, n=3.


( D ) Indirect immunofluorescence of endogenous LC3 ( green ) WIPI1 ( ) in old I90 transfected with nons ( a , b ) bag3 siRNA ( c , d ) for 96 h . DAPI ( blue ) was used as a marker . Representative pictures are shown . Bar : 20 μm . Diagrams show of counted as in Figure 3E .

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

(D) Indirect immunofluorescence analysis of endogenous LC3 (green) and WIPI1 (white) in old I90 cells transfected with nons (a, b) and bag3 siRNA (c, d) for 96 h. DAPI (blue) was used as a nuclear marker. Representative pictures are shown. Bar: 20 μm. Diagrams show percentage of cells counted as in Figure 3E.


( E ) Same as in Figure 5D but old transfected as in ( A ) were used . Values expressed in diagram are mean ± s . e . m . * P0 . 05 versus nons , n = 3 .

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

(E) Same analysis as in Figure 5D but old cells transfected as in (A) were used. Values expressed in the diagram are mean±s.e.m. *P0.05 versus nons, n=3.


( F ) Same as in Figure 5G but old I90 transfected as in ( A ) were used . C , control ; L , lactacystin , N , NH4Cl / Leu . Values are expressed as mean ± s . e . m . * P0 . 05 versus nons control , or as indicated , n = 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Same analysis as in Figure 5G but old I90 cells transfected as in (A) were used. C, control; L, lactacystin, N, NH4Cl/Leu. Values are expressed as mean±s.e.m. *P0.05 versus nons control, or as indicated, n=3.


( G , H ) Same as in ( F ) but young I90 48 h after transfection with a BAG3 expression plasmid ( BAG3 ‐ N1 ) or vector control ( N1 ) together with nons or sqstm1 siRNA ( G ) nons or atg7 siRNA ( H ) , as indicated , were used . * , # , ∼ + , P0 . 05 versus C N1 , C BAG3 ‐ N1 , C BAG3 ‐ N1 sqstm1 or atg7 C N1 sqstm1 or atg7 , n = 3 .

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

(G, H) Same analysis as in (F) but young I90 cells 48 h after transfection with a BAG3 expression plasmid (BAG3‐N1) or vector control (N1) together with nons or sqstm1 siRNA (G) and nons or atg7 siRNA (H), as indicated, were used. *, #, ∼ and +, P0.05 versus C N1, C BAG3‐N1, C BAG3‐N1 sqstm1 or atg7 and C N1 sqstm1 or atg7, n=3.


( A ) extracts from cerebellum ( CER ) of young ( Y , 3 months ) old ( O , 24 months ) mice were analysed for BAG1 BAG3 expression by immunoblot .

O O O O 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 O O O

(A) Protein extracts from cerebellum (CER) of young (Y, 3 months) and old (O, 24 months) mice were analysed for BAG1 and BAG3 expression by immunoblot analysis.


( B ) Real ‐ time PCR of indicated mRNA in cerebellum of young old mice . Depicted is log2 expression of target genes in old mice relative to young mice . Values are expressed as mean ± s . e . m . * P0 . 05 * * P0 . 01 versus young , n = 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Real‐time PCR analysis of indicated mRNA levels in cerebellum of young and old mice. Depicted is the log2 expression ratio of target genes in old mice relative to young mice. Values are expressed as mean±s.e.m. *P0.05 and **P0.01 versus young, n=3.


( C ) Immunoblot as in ( A ) but in cortex ( CTX ) , hippocampus ( HIP ) mid ‐ brain ( MB ) .

O O O 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) Immunoblot analysis as in (A) but in cortex (CTX), hippocampus (HIP) and mid‐brain (MB).


( D ) Western ‐ blot of indicated in different brain regions of young old mice .

(D) Western‐blot analysis of indicated proteins in different brain regions of young and old mice.


( E ) Total cathepsin specific cathepsin B activity in brain extracts from young old mice was determined using probes Z ‐ FR ‐ AMC Z ‐ RR ‐ AMC , respectively , as described in Material methods section . Values are expressed as mean ± s . e . m . * P0 . 05 versus young , n = 3 .

O O O O B-MEASURED_VAR O B-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

(E) Total cathepsin and specific cathepsin B activity in brain extracts from young and old mice was determined using fluorescence probes Z‐FR‐AMC and Z‐RR‐AMC, respectively, as described in the Material and methods section. Values are expressed as mean±s.e.m. *P0.05 versus young, n=3.


( F ) Expression of indicated in hippocampal astrocytic neuronal cultures from young ( 2 months ) old ( 24 months ) rats . Detection of GLT1 NeuN served as astrocyte neuron markers , respectively . Note NeuN signals in astrocytic cultures . We attributed these signals to presence of resting nuclei of dead neurons , which we observed under microscope .

O O 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 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

(F) Expression analysis of indicated proteins in primary hippocampal astrocytic and neuronal cell cultures from young (2 months) and old (24 months) rats. Detection of GLT1 and NeuN served as astrocyte and neuron markers, respectively. Note the NeuN signals in astrocytic cultures. We attributed these signals to the presence of resting nuclei of dead neurons, which we observed under the microscope.


Growth rate trajectories for duplicate experiments ( n = 2 evolution experiments per substrate condition ) ( green purple ) for example case of D - lyxose . Population growth rates are plotted against cumulative divisions . Clones were isolated for whole genome sequencing at notable growth - rate plateaus as indicated by arrows . Mutations gained at each plateau are highlighted beside arrows ( mutations arising earlier along trajectory persisted in later sequenced clones ) .

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

Growth rate trajectories for duplicate experiments (n=2 evolution experiments per substrate condition) (green and purple) for the example case of D-lyxose. Population growth rates are plotted against cumulative cell divisions. Clones were isolated for whole genome sequencing at notable growth-rate plateaus as indicated by the arrows. Mutations gained at each plateau are highlighted beside the arrows (mutations arising earlier along the trajectory persisted in later sequenced clones).


YihS V314L + R315S mutant enzyme activity on D - mannose D - lyxose . LC - MS was used to analyze YihS activity at saturating substrate concentrations to compare turnover rates on each substrate . Product formation was followed over time at a constant enzyme . Turnover rates were calculated using linear regression ( n = 3 replicates for each enzyme , Dataset EV4 ) . error bars represent standard deviation ( n = 3 ) of peak .

B-MEASURED_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 O

YihS V314L + R315S mutant enzyme activity on D-mannose and D-lyxose. LC-MS was used to analyze YihS activity at saturating substrate concentrations to compare turnover rates on each substrate. Product formation was followed over time at a constant enzyme concentration. Turnover rates were calculated using linear regression (n=3 replicates for each enzyme, Dataset EV4). The error bars represent standard deviation (n=3) of the peak area.


Turnover ratios of substrate conversion of D - lyxose / D - mannose are shown for wild type YihS mutant YihS enzymes . A < 1 indicates a higher turnover rate on D - mannose compared to D - lyxose . Error bars represent standard error ( n = 3 ) calculated from linear regression .

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

Turnover ratios of substrate conversion of D-lyxose / D-mannose are shown for the wild type YihS and mutant YihS enzymes. A ratio < 1 indicates a higher turnover rate on D-mannose compared to D-lyxose. Error bars represent standard error (n=3) calculated from the linear regression analysis.


Expression data ( RNAseq ) for significantly differentially expressed genes ( q - value < 0 . 05 , FDR - adjusted p - value , n = 2 biological replicates for each condition ) . Scatter plot shows log2 ( fold change ) of gene expression data comparing endpoint to initial populations for Exp . 1 Exp . 2 ( grey dots ) with location of gene in reference genome as x - axis . Those genes that are associated with AraC transcription units are highlighted ( red dots for Exp . 1 blue dots for Exp . 2 ) . Above plot , transcription units are labeled green if AraC activates expression ( in presence of arabinose ) or red if AraC represses expression of those genes .

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

Expression data (RNAseq) for significantly differentially expressed genes (q-value < 0.05, FDR-adjusted p-value, n=2 biological replicates for each condition). Scatter plot shows log2(fold change) of gene expression data comparing endpoint to initial populations for Exp. 1 and Exp. 2 (grey dots) with the location of the gene in the reference genome as the x-axis. Those genes that are associated with AraC transcription units are highlighted (red dots for Exp. 1 and blue dots for Exp. 2). Above the plot, the transcription units are labeled green if AraC activates expression (in the presence of arabinose) or red if AraC represses expression of those genes.


Growth rate of various weaned ( starting point of static phase ) optimized ( endpoint of static phase ) strains with or without fucK or araB genes knocked out . Strains were grown in triplicate ( n = 3 ) on M9 minimal media with D - arabinose as sole carbon source . colored bars represent calculated mean growth rate error bars represent standard deviation . p - values reported were calculated using a two - sided Welch ' s t - .

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

Growth rate analysis of various weaned (starting point of static phase) and optimized (endpoint of static phase) strains with or without fucK or araB genes knocked out. Strains were grown in triplicate (n=3) on M9 minimal media with D-arabinose as the sole carbon source. The colored bars represent the calculated mean growth rate and the error bars represent the standard deviation. The p-values reported were calculated using a two-sided Welch's t-test.


A : on phase contrast microscopy for parental fibroblast lines ( upper panel ) iPSCs ( lower panel ) from control Detroit 551 , WS5A CP2A POLG ( scale bars , 50 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 B-CONTROLLED_VAR O O O O O O O O

A: Morphology on phase contrast microscopy for parental fibroblast lines (upper panel) and iPSCs (lower panel) from control Detroit 551, WS5A and CP2A POLG patients (scale bars, 50 µm).


B : Immunofluorescence of markers POU5F1 ( green ) SSEA4 ( red ) : upper panel - Detroit 551 control iPSCs , middle panel - WS5A iPSCs lower panel - CP2A iPSCs ( Scale bar , 100 µm ) . Nuclei are stained with DAPI ( blue ) .

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

B: Immunofluorescence staining of stem cell markers POU5F1 (green) and SSEA4 (red): upper panel - Detroit 551 control iPSCs, middle panel - WS5A iPSCs and lower panel - CP2A iPSCs (Scale bar, 100 µm). Nuclei are stained with DAPI (blue).


C : RT - qPCR of gene expression for LIN28A , NANOG , POU5F1 for iPSCs from control Detroit 551 , WS5A CP2A POLG ( n = 7 , technical replicates per for ESCs ; n = 4 , technical replicates per clone for control , WS5A CP2A iPSCs ) . gene expression of clones is assessed with fold change using comparative ΔΔCt method by normalizing iPSCs to ESC1 .

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

C: RT-qPCR quantification of gene expression for LIN28A, NANOG, and POU5F1 for all iPSCs from control Detroit 551, WS5A and CP2A POLG patients (n=7, technical replicates per line for ESCs; n=4, technical replicates per clone for control, WS5A and CP2A iPSCs). The gene expression of the individual clones is assessed with fold change using the comparative ΔΔCt method by normalizing iPSCs to ESC1.


D , E : Flow cytometric of expression level of SSEA4 ( D , n = 9 , technical replicates per for ESCs ; n = 5 , technical replicates per clone for control iPSCs ; n = 3 , technical replicates per clone for WS5A iPSCs , n = 8 , technical replicates per clone for CP2A iPSCs ) POU5F1 ( E , n = 9 , technical replicates per for ESCs ; n = 5 , technical replicates per clone for control iPSCs ; n = 3 , technical replicates per clone for WS5A iPSCs , n = 8 , technical replicates per clone for CP2A iPSCs ) for ESCs iPSCs for both ESC control lines iPSCs generated from Detroit 551 control , WS5A CP2A fibroblasts .

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

D, E: Flow cytometric quantification of expression level of SSEA4 (D, n=9, technical replicates per line for ESCs; n=5, technical replicates per clone for control iPSCs; n=3, technical replicates per clone for WS5A iPSCs, n=8, technical replicates per clone for CP2A iPSCs) and POU5F1 (E, n=9, technical replicates per line for ESCs; n=5, technical replicates per clone for control iPSCs; n=3, technical replicates per clone for WS5A iPSCs, n=8, technical replicates per clone for CP2A iPSCs) for ESCs and iPSCs for both ESC control lines and iPSCs generated from Detroit 551 control, WS5A and CP2A fibroblasts.


A : Representative confocal of iPSC lineage specific differentiation into germ layers of endoderm derived hepatocytes with positive expression of ALBUMIN ( red ) HNF4A ( green ) ( a ) ( scale bar , 100 µm ) , mesodermal derived cardiomyocytes with positive expression of TNNT2 ( red ) ( b ) ( scale bar , 100 µm ) ectodermal derived dopaminergic neurons with positive expression of TH ( green ) MAP2 ( red ) ( c ) ( scale bar , 10 µm ) . Nuclei are stained with DAPI ( blue ) .

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 B-MEASURED_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 B-MEASURED_VAR 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

A: Representative confocal images of iPSC lineage specific differentiation into germ layers of endoderm derived hepatocytes with positive expression of ALBUMIN (red) and HNF4A (green) (a) (scale bar, 100 µm), mesodermal derived cardiomyocytes with positive expression of TNNT2 (red) (b) (scale bar, 100 µm) and ectodermal derived dopaminergic neurons with positive expression of TH (green) and MAP2 (red) (c) (scale bar, 10 µm). Nuclei are stained with DAPI (blue).


B : Confocal of mitochondrial for iPSC lines with co - of MTG ( upper panel ) TMRE ( lower panel ) ( scale bars , 25 µm ) . Nuclei are stained with DAPI ( blue ) .

O O O O O O O O O O O O O O O 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: Confocal images of mitochondrial morphology for iPSC lines with co-staining of MTG (upper panel) and TMRE (lower panel) (scale bars, 25 µm). Nuclei are stained with DAPI (blue).


Flow cytometric of iPSCs generated from Detroit 551 , WS5A CP2A fibroblasts for mitochondrial volume ( MTG ) ( C , n = 6 , technical replicates per clone for control CP2A ; n = 5 , technical replicates per clone for WS5A )

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

Flow cytometric analysis of iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts for mitochondrial volume (MTG) (C, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A)


Flow cytometric of iPSCs generated from Detroit 551 , WS5A CP2A fibroblasts for total MMP ( TMRE ) ( D , n = 6 , technical replicates per clone for control CP2A ; n = 5 , technical replicates per clone for WS5A ) specific MMP ( E , n = 6 , technical replicates per clone for control CP2A ; n = 5 , technical replicates per clone for WS5A ) calculated by dividing median ( MFI ) for total TMRE expression by MTG .

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

Flow cytometric analysis of iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts for total MMP (TMRE) (D, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A) and specific MMP (E, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A) calculated by dividing median fluorescence intensity (MFI) for total TMRE expression by MTG.


F : Intracellular ATP production in iPSCs generated from Detroit 551 , WS5A CP2A fibroblasts ( n = 3 , technical replicates per clone for control WS5A ; n = 4 , technical replicates per clone for CP2A ) .

O O O O O O O O O O O O O O O O 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: Intracellular ATP production in iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts (n=3, technical replicates per clone for control and WS5A; n=4, technical replicates per clone for CP2A).


G - H : Flow cytometric of TOMM20 expression level ( G , n = 4 , technical replicates per clone ) specific TFAM level ( total TFAM / TOMM20 ) ( H , n = 4 , technical replicates per clone ) .

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

G-H: Flow cytometric analysis of TOMM20 expression level (G, n=4, technical replicates per clone) and specific TFAM level (total TFAM/TOMM20) (H, n=4, technical replicates per clone).


I : Relative mtDNA copy number in Detroit 551 , WS5A CP2A iPSCs by RT - qPCR using ND1 APP ( n = 5 , technical replicates per clone for control ; n = 5 , technical replicates per clone for WS5A CP2A ) . Values are presented as Log2 of between expression values of ND1 in relation to APP .

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

I: Relative mtDNA copy number in Detroit 551, WS5A and CP2A iPSCs by RT-qPCR analysis using ND1 and APP (n=5, technical replicates per clone for control; n=5, technical replicates per clone for WS5A and CP2A). Values are presented as Log2 of the ratio between the expression values of ND1 in relation to APP.


B : Representative phase contrast ( upper panel ) immunostaining for specific stages during neural induction from iPSCs to NSCs . Upper panel displays in culture of different types during neural induction to NSCs from iPSCs including iPSCs ( a ) ; neuroepithelium with rosette - like structures ( b ) ; neurospheres with defined round shapes in suspension culture ( c ) NSCs in monolayers ( d ) ( scale bars , 50 µm ) . Lower panel demonstrates representative phase contrast for immunostaining corresponding to specific stages in upper panel : iPSCs with positive of SSEA4 ( red ) POU5F1 ( green ) ( e ) ( scale bar , 50 µm ) ; neuroepithelium with rosette - like structures with positive of PAX6 ( green ) NESTIN ( red ) ( f ) ( scale bar , 50 µm ) ; neurospheres with positive of NESTIN ( red ) ( g ) ( scale bar , 100 µm ) ; NSCs with positive of PAX6 ( green ) ( h ) ( scale bar , 50 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O O O O O O O O O O

B: Representative phase contrast images (upper panel) and immunostaining for specific stages during neural induction from iPSCs to NSCs. Upper panel displays the morphology in culture of different cell types during neural induction to NSCs from iPSCs including iPSCs (a); neuroepithelium with rosette-like structures (b); neurospheres with defined round shapes in suspension culture (c) and NSCs in monolayers (d) (scale bars, 50 µm). Lower panel demonstrates the representative phase contrast images for the immunostaining corresponding to the specific stages in the upper panel: iPSCs with positive staining of SSEA4 (red) and POU5F1 (green) (e) (scale bar, 50 µm); neuroepithelium with rosette-like structures with positive staining of PAX6 (green) and NESTIN (red) (f) (scale bar, 50 µm); neurospheres with positive staining of NESTIN (red) (g) (scale bar, 100 µm); NSCs with positive staining of PAX6 (green) (h) (scale bar, 50 µm).


C : Flow cytometric assessment of stemness marker POU5F1 NSC markers NESTIN PAX6 during neural induction from day 0 to day 5 ( n = 3 , technical replicates ) .

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

C: Flow cytometric assessment of stemness marker POU5F1 and NSC markers NESTIN and PAX6 during neural induction from day 0 to day 5 (n=3, technical replicates).


D : Representative of immunofluorescent labeling for NSC markers SOX2 ( green ) NESTIN ( red ) ( scale bar , 25 µm ) from Detroit 551 control iPSC - derived NSCs . Nuclei are stained with DAPI ( blue ) .

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

D: Representative images of the immunofluorescent labeling for NSC markers SOX2 (green) and NESTIN (red) (scale bar, 25 µm) from Detroit 551 control iPSC-derived NSCs. Nuclei are stained with DAPI (blue).


A : of gene expression for NSC markers PAX6 , NESTIN SOX2 for NSCs from RT - qPCR . expression of neural markers is assessed with fold change using comparative ΔΔCt method by normalizing NSCs to iPSCs ( n = 3 , technical replicates per ESC or iPSC clone ) .

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

A: Quantification of gene expression for NSC markers PAX6, NESTIN and SOX2 for all NSCs from RT-qPCR analysis. The expression of the neural stem cell markers is assessed with fold change using the comparative ΔΔCt method by normalizing NSCs to iPSCs (n=3, technical replicates per ESC line or iPSC clone).


B , C : of expression level for PAX6 ( B , n = 6 , technical replicates per for ESCs ; n = 7 , technical replicates per clone for control ; n = 8 , technical replicates per clone for WS5A ; n = 9 , technical replicates per clone for CP2A ) NESTIN ( C , n = 6 , technical replicates per for ESCs ; n = 7 , technical replicates per clone for control ; n = 8 , technical replicates per clone for WS5A ; n = 9 , technical replicates per clone for CP2A ) for iPSC - derived NSCs using flow cytometry .

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 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

B, C: Quantification of protein expression level for PAX6 (B, n=6, technical replicates per line for ESCs; n=7, technical replicates per clone for control; n=8, technical replicates per clone for WS5A; n=9, technical replicates per clone for CP2A) and NESTIN (C, n=6, technical replicates per line for ESCs; n=7, technical replicates per clone for control; n=8, technical replicates per clone for WS5A; n=9, technical replicates per clone for CP2A) for iPSC-derived NSCs using flow cytometry.


D : Sequencing chromatogram showing homozygous c . 2243G > C variation in POLG in WS5A iPSC - derived NSCs heterozygous c . 1399G > A c . 2243G > C variation in POLG in CP2A iPSC - derived NSCs .

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

D: Sequencing chromatogram showing the homozygous c.2243G>C variation in POLG in WS5A iPSC-derived NSCs and the heterozygous c.1399G>A and c.2243G>C variation in POLG in CP2A iPSC-derived NSCs.


E : Representative confocal showing glial neuronal lineages derived from NSCs . a , Immunostaining of NSC - derived astrocytes with GFAP ( red ) ( scale bar , 50 µm ) . b , Immunostaining of oligodendrocytes showing GALC ( red ) positive labeling ( scale bar , 50 µm ) . c , Dopaminergic neurons showing TH ( green ) TUJ1 ( red ) positive ( scale bar , 25 µm ) . Nuclei are stained with DAPI ( blue ) .

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

E: Representative confocal images showing glial and neuronal lineages derived from NSCs. a, Immunostaining of NSC-derived astrocytes with GFAP (red) staining (scale bar, 50 µm). b, Immunostaining of oligodendrocytes showing GALC (red) positive labeling (scale bar, 50 µm). c, Dopaminergic neurons showing TH (green) and TUJ1 (red) positive staining (scale bar, 25 µm). Nuclei are stained with DAPI (blue).


A : Representative transmission electron microscopy of mitochondrial structures in Detroit 551 control , WS5A CP2A NSCs ( scale bar , 400 nm ) .

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

A: Representative transmission electron microscopy images of mitochondrial structures in Detroit 551 control, WS5A and CP2A NSCs (scale bar, 400 nm).


Flow cytometric of mitochondrial volume ( MTG ) ( B , n = 5 , technical replicates per clone ) , total MMP ( TMRE ) ( C , n = 5 , technical replicates per clone ) calculated by dividing median ( MFI ) for total TMRE expression by MTG in NSCs generated from control lines , WS5A CP2A iPSCs .

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

Flow cytometric analysis of mitochondrial volume (MTG) (B, n=5, technical replicates per clone), total MMP (TMRE) (C, n=5, technical replicates per clone) calculated by dividing median fluorescence intensity (MFI) for total TMRE expression by MTG in NSCs generated from control lines, WS5A and CP2A iPSCs.


Flow cytometric of specific MMP ( D , n = 5 , technical replicates per clone ) , calculated by dividing median ( MFI ) in NSCs generated from control lines , WS5A CP2A iPSCs .

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

Flow cytometric analysis of specific MMP (D, n=5, technical replicates per clone), calculated by dividing median fluorescence intensity (MFI) in NSCs generated from control lines, WS5A and CP2A iPSCs.


E : Intracellular ATP production in NSCs ( n = 3 , technical replicates per clone ) .

E: Intracellular ATP production in NSCs (n=3, technical replicates per clone).


Flow cytometric of mitochondrial volume ( MTG ) ( F , n = 6 , technical replicates for control ; n = 5 , technical replicates for WS5A CP2A ) , total MMP ( TMRE ) ( G , n = 5 , technical replicates for control ; n = 4 , technical replicates for WS5A CP2A ) in Detroit 551 , WS5A CP2A fibroblasts .

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

Flow cytometric analysis of mitochondrial volume (MTG) (F, n=6, technical replicates for control; n=5, technical replicates for WS5A and CP2A), total MMP (TMRE) (G, n=5, technical replicates for control; n=4, technical replicates for WS5A and CP2A) in Detroit 551, WS5A and CP2A fibroblasts.


Flow cytometric of specific MMP ( TMRE / MTG ) ( H , n = 5 , technical replicates for control ; n = 4 , technical replicates for WS5A CP2A ) in Detroit 551 , WS5A CP2A fibroblasts .

Flow cytometric analysis of specific MMP (TMRE/MTG) (H, n=5, technical replicates for control; n=4, technical replicates for WS5A and CP2A) in Detroit 551, WS5A and CP2A fibroblasts.


I : Intracellular ATP production in Detroit 551 , WS5A CP2A fibroblasts ( n = 6 , technical replicates for control ; n = 3 , technical replicates for WS5A CP2A ) .

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

I: Intracellular ATP production in Detroit 551, WS5A and CP2A fibroblasts (n=6, technical replicates for control; n=3, technical replicates for WS5A and CP2A).


A , B : Flow cytometric of TOMM20 ( A , n = 3 , technical replicates per clone for control ; n = 4 , technical replicates per clone for WS5A CP2A ) TFAM expression ( B , n = 3 , technical replicates per clone for control CP2A ; n = 5 , technical replicates per clone for WS5A ) in NSCs . Expressed as specific TFAM level ( total TFAM / TOMM20 ) .

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

A, B: Flow cytometric analysis of TOMM20 (A, n=3, technical replicates per clone for control; n=4, technical replicates per clone for WS5A and CP2A) and TFAM protein expression (B, n=3, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A) in NSCs. Expressed as specific TFAM level (total TFAM/TOMM20).


C : Relative mtDNA copy number analyzed by RT - qPCR using primers fluorogenic probes for regions of ND1 gene APP in NSCs ( n = 4 , technical replicates per clone for control ; n = 5 , technical replicates per clone for WS5A ; n = 7 , technical replicates per clone for CP2A ) .

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 O O O O O O O O O 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: Relative mtDNA copy number analyzed by RT-qPCR using primers and fluorogenic probes for regions of ND1 and nuclear gene APP in all NSCs (n=4, technical replicates per clone for control; n=5, technical replicates per clone for WS5A; n=7, technical replicates per clone for CP2A).


D , E : Flow cytometric of TOMM20 expression level ( D , n = 4 , technical replicates per clone ) TFAM expression ( E , n = 4 , technical replicates for control WS5A ; n = 5 , technical replicates for CP2A ) in fibroblasts . Expressed as specific TFAM level ( total TFAM / TOMM20 ) .

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

D, E: Flow cytometric analysis of TOMM20 expression level (D, n=4, technical replicates per clone) and TFAM protein expression (E, n=4, technical replicates for control and WS5A; n=5, technical replicates for CP2A) in fibroblasts. Expressed as specific TFAM level (total TFAM/TOMM20).


F : Relative mtDNA copy number analyzed by RT - qPCR for regions of ND1 gene APP in fibroblasts ( n = 3 , technical replicates for control ; n = 4 , technical replicates for WS5A CP2A ) .

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

F: Relative mtDNA copy number analyzed by RT-qPCR for regions of ND1 and nuclear gene APP in fibroblasts (n=3, technical replicates for control; n=4, technical replicates for WS5A and CP2A).


G : Long - PCR for detection of mtDNA deletions in NSCs their parental fibroblasts from WS5A CP2A .

G: Long-PCR for detection of mtDNA deletions in NSCs and their parental fibroblasts from WS5A and CP2A patients.


H : Representative of confocal microscopy with immunofluorescence labeling of DA neuron - specific marker TH ( green ) neuron - specific marker MAP2 ( red ) for iPSC - derived DA neurons ( scale bars , 25 µm ) . Nuclei are stained with DAPI ( blue ) .

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 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

H: Representative images of confocal microscopy with immunofluorescence labeling of DA neuron-specific marker TH (green) and neuron-specific marker MAP2 (red) for iPSC-derived DA neurons (scale bars, 25 µm). Nuclei are stained with DAPI (blue).


I : Histogram of positive population stained with DA marker TH in iPSC - derived DA neurons using flow cytometric .

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

I: Histogram of the positive cell population stained with DA marker TH in iPSC-derived DA neurons using flow cytometric analysis.


J : Flow cytometric measurement of specific TFAM level ( total TFAM / TOMM20 ) in iPSC - derived DA neurons from Detroit 551 control , WS5A CP2A POLG ( n = 8 , technical replicates per clone for control ; n = 5 , technical replicates per clone for WS5A ; n = 4 , technical replicates per clone for CP2A ) .

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

J: Flow cytometric measurement of specific TFAM level (total TFAM/TOMM20) in iPSC-derived DA neurons from Detroit 551 control, WS5A and CP2A POLG patients (n=8, technical replicates per clone for control; n=5, technical replicates per clone for WS5A; n=4, technical replicates per clone for CP2A).


K : Relative mtDNA copy number analyzed by RT - qPCR for regions of ND1 gene APP in iPSC - derived DA neurons ( n = 3 , technical replicates per clone ) .

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

K: Relative mtDNA copy number analyzed by RT-qPCR for regions of ND1 and nuclear gene APP in all iPSC-derived DA neurons (n=3, technical replicates per clone).


A : I immunohistochemistry in occipital cortex of a neurologically healthy control ( a ) two with POLG - disease ( b c ) ( Scale bar , 20 µm ) . have numerous - I negative neurons ( examples marked by arrows ) . ( d ) mtDNA relative in microdissected neurons from occipital cortex of with POLG diseases ( n = 5 ) neurologically healthy controls ( n = 5 ) . Each point represents mean value of three technical replicates from a pooled sample of 10 neurons . For purposes of comparison , a control sample has been arbitrarily set to one . medians of two groups are compared by Mann - Whitney U . Data are presented as mean ( horizontal bars ) ± ( vertical bars ) .

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

A: Complex I immunohistochemistry in the occipital cortex of a neurologically healthy control (a) and two patients with POLG-disease (b and c) (Scale bar, 20 µm). Patients have numerous complex-I negative neurons (examples marked by arrows). (d) mtDNA relative quantification in microdissected neurons from the occipital cortex of patients with POLG diseases (n=5) and neurologically healthy controls (n=5). Each point represents the mean value of three technical replicates from a pooled sample of 10 neurons. For the purposes of comparison, a control sample has been arbitrarily set to one. The medians of the two groups are compared by Mann-Whitney U test. Data are presented as mean (horizontal bars) ± SEM (vertical bars).


B : Representative confocal of immunostaining for mitocondrial I subunit NDUFB10 ( green ) TOMM20 ( red ) in control , WS5A CP2A NSCs ( scale bars , 50 µm ) . Nuclei are stained with DAPI ( blue ) .

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

B: Representative confocal images of immunostaining for mitocondrial complex I subunit NDUFB10 (green) and TOMM20 (red) in control, WS5A and CP2A NSCs (scale bars, 50 µm). Nuclei are stained with DAPI (blue).


C - E : Flow cytometric measurements of mitochondrial I ( C , n = 4 , technical replicates per clone ) , II ( D , n = 3 , technical replicates per clone for control ; n = 4 , technical replicates per clone for WS5A CP2A ) IV ( E , n = 3 , technical replicates per clone for control CP2A ; n = 4 , technical replicates per clone for WS5A ) level in iPSC - derived NSCs . Expressed as specific I , II IV level ( total I , II , IV level / TOMM20 ) .

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

C-E: Flow cytometric measurements of mitochondrial complex I (C, n=4, technical replicates per clone), II (D, n=3, technical replicates per clone for control; n=4, technical replicates per clone for WS5A and CP2A) and IV (E, n=3, technical replicates per clone for control and CP2A; n=4, technical replicates per clone for WS5A) protein level in iPSC-derived NSCs. Expressed as specific complex I, II and IV level (total complex I, II, IV level/TOMM20).


F - H : Flow cytometric measurements of mitochondrial I ( F , n = 4 , technical replicates per clone ) , II ( G , n = 4 , technical replicates per clone ) IV ( H , n = 4 , technical replicates per clone ) level in iPSCs . Expressed as specific I , II IV level .

F-H: Flow cytometric measurements of mitochondrial complex I (F, n=4, technical replicates per clone), II (G, n=4, technical replicates per clone) and IV (H, n=4, technical replicates per clone) protein level in iPSCs. Expressed as specific complex I, II and IV level.


I - K : Flow cytometric measurements of mitochondrial I ( I , n = 4 , technical replicates ) , II ( J , n = 4 , technical replicates ) IV ( K , n = 4 , technical replicates ) level in parental fibroblasts expressed as specific I , II IV level .

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

I-K: Flow cytometric measurements of mitochondrial complex I (I, n=4, technical replicates), II (J, n=4, technical replicates) and IV (K, n=4, technical replicates) protein level in parental fibroblasts expressed as specific complex I, II and IV level.


A - C : LCMS - metabolomics for quantitative measurements of NAD + / NADH ( A , n = 3 , technical replicates per clone ) , NAD + ( B , n = 3 , technical replicates per clone ) NADH ( C , n = 3 , technical replicates per clone ) level in NSCs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: LCMS-based metabolomics for quantitative measurements of NAD+/NADH ratio (A, n=3, technical replicates per clone), NAD+ (B, n=3, technical replicates per clone) and NADH (C, n=3, technical replicates per clone) level in NSCs.


D - F : LCMS - metabolomics for quantitative measurement of NAD + / NADH ( D , n = 3 , technical replicates per clone ) , NAD + ( E , n = 3 , technical replicates per clone ) NADH ( F , n = 3 , technical replicates per clone ) level in iPSCs .

D-F: LCMS-based metabolomics for quantitative measurement of NAD+/NADH ratio (D, n=3, technical replicates per clone), NAD+ (E, n=3, technical replicates per clone) and NADH (F, n=3, technical replicates per clone) level in iPSCs.


G : Intracellular ROS production measurements of specific ROS level calculated by total ROS / MTDR in control , WS5A CP2A iPSC - derived NSCs using DCFDA MTDR ( n = 6 , technical replicates per clone for control ; n = 5 , technical replicates per clone for WS5A ; n = 3 , technical replicates per clone for CP2A ) .

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

G: Intracellular ROS production measurements of the specific ROS level calculated by total ROS/MTDR in control, WS5A and CP2A iPSC-derived NSCs using DCFDA and MTDR (n=6, technical replicates per clone for control; n=5, technical replicates per clone for WS5A; n=3, technical replicates per clone for CP2A).


H : Flow cytometric measurements of mitochondrial ROS production at specific ROS level in Detroit 551 , WS5A CP2A iPSC - derived NSCs calculated by total ROS ( MitoSox Red ) / MTG ( n = 4 , technical replicates per clone for control CP2A ; n = 3 , technical replicates per clone for WS5A ) .

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

H: Flow cytometric measurements of mitochondrial ROS production at the specific ROS level in Detroit 551, WS5A and CP2A iPSC-derived NSCs calculated by total ROS (MitoSox Red)/MTG (n=4, technical replicates per clone for control and CP2A; n=3, technical replicates per clone for WS5A).


I - J : Representative of senescence β - galactosidase ( scale bars , 20 µm ) ( I ) by calculating of positively stained by division of negative from I ( J , n = 3 , technical replicates per clone ) .

O 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

I-J: Representative images of senescence β-galactosidase staining (scale bars, 20 µm) (I) and quantification by calculating the percentage of positively stained cells by division of the negative cells from I (J, n=3, technical replicates per clone).


K - L : Representative ( K ) ( L ) for western blotting with Phospho - SirT1 ( Ser47 ) , LC3B , UCP2 , PINK1 , Parkin , BNIP3 β - ACTIN . Three independent experiments are included .

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

K-L: Representative images (K) and quantification (L) for western blotting with Phospho-SirT1 (Ser47), LC3B, UCP2, PINK1, Parkin, BNIP3 and β-ACTIN. Three independent experiments are included.


a , microscopyHeLa infected with Shigella Listeria visualized with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) ( pseudo - coloured red ) , Salmonella carrying mCherry - expressing vector ( red ) . cis - Golgi ( green ) was detected by α - GM130 antibodies . 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 O O O O 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, Fluorescence microscopyHeLa cells infected with Shigella and Listeria visualized with 4′,6-diamidino-2-phenylindole (DAPI) (pseudo-coloured red), and Salmonella carrying mCherry-expressing vector (red). The cis-Golgi (green) was detected by α-GM130 antibodies. Scale bar, 10 µm.


b , microscopy of HeLa transfected with either IpaJ or VirA . cis - Golgi ( GM130 , green ) F - actin ( red ) are shown . Scale bar , 10 µm .

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

b, Fluorescence microscopy of HeLa cells transfected with either IpaJ or VirA. The cis-Golgi (GM130, green) and F-actin (red) are shown. Scale bar, 10 µm.


c , microscopy of HeLa infected with indicated Shigella mutant strains . Scale bar , 10 µm .

c, Fluorescence microscopy of HeLa cells infected with indicated Shigella mutant strains. Scale bar, 10 µm.


d , of HeLa with disrupted Golgi in 100 infected with either wild - type Shigella ( M90T ) or indicated mutants . Error bars , means ± s . e . m . calculated from three independent experiments .

O O O O O O O O O O O O O O O O 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, Percentage of HeLa cells with disrupted Golgi morphology in 100 cells infected with either wild-type Shigella (M90T) or the indicated mutants. Error bars, means ± s.e.m. calculated from three independent experiments.


e , Number of recoverable c . f . u . per mouselung24 h after intranasal inoculation with 1 × 106 of wild - type M90T or each mutant Shigella as indicated . Limit of detection ( LOD ) for this is indicated by dotted . Error bars , geometrical means ± s . e . m . * * P < 0 . 001 .

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

e, Number of recoverable c.f.u. per mouselung24 h after intranasal inoculation with 1 × 106 of wild-type M90T or each mutant Shigella strain as indicated. Limit of detection (LOD) for this assay is indicated by the dotted line. Error bars, geometrical means ± s.e.m. **P<0.001.


b , microscopy of HeLa transfected with indicated IpaJ mutants . cis - Golgi ( GM130 , green ) F - actin ( red ) are shown . Scale bar , 10 µm .

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

b, Fluorescence microscopy of HeLa cells transfected with the indicated IpaJ mutants. The cis-Golgi (GM130, green) and F-actin (red) are shown. Scale bar, 10 µm.


c , IpaJ or its catalytic mutants were expressed from a galactose - inducible promoter ( pGal413 vector ) assayed for a growth arrest phenotype on galactose or glucose ( control ) carbon source .

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

c, IpaJ or its catalytic mutants were expressed from a galactose-inducible promoter (pGal413 vector) and assayed for a growth arrest phenotype on galactose or glucose (control) carbon source.


e , Yeast harbouring a galactose - inducible IpaJ gene were transformed with a multi - copy vector containing indicated genes . Yeast were assayed for a growth arrest phenotype on galactose or glucose ( control ) carbon source .

e, Yeast strain harbouring a galactose-inducible IpaJ gene were transformed with a multi-copy vector containing the indicated genes. Yeast were assayed for a growth arrest phenotype on galactose or glucose (control) carbon source.


a - c , Mass spectra of purified ARF1 - strep in untreated ( a ) , IpaJ - treated ( b ) or IpaJ C64A - treated ( c ) , with observed monoisotopic mass reported in daltons . * Observed 1 Da shift in molecular mass is accounted for in MS / MS data ( Supplementary Fig . 7 ) .

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

a-c, Mass spectra of purified ARF1-strep in untreated (a), IpaJ-treated (b) or IpaJ C64A-treated (c) cells, with the observed monoisotopic mass reported in daltons. *Observed 1 Da shift in molecular mass is accounted for in MS/MS data (Supplementary Fig. 7).


f , In - gel ( top panel ) visualizing Alexa Fluor 647 - labelled myristoylated ARF1 - His isolated from either not expressing ( lane 1 ) or expressing ( lane 2 ) NMTp . lysates treated with recombinant IpaJ or IpaJ C64A as indicated . expression of ARF1 - His were determined by Coomassie blue stain .

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

f, In-gel fluorescence assay (top panel) visualizing Alexa Fluor 647-labelled myristoylated ARF1-His isolated from bacteria either not expressing (lane 1) or expressing (lane 2) NMTp. Bacterial cell lysates treated with recombinant IpaJ or IpaJ C64A as indicated. The expression levels of ARF1-His were determined by Coomassie blue stain.


g , In - gel ( top panel ) visualizing Alexa Fluor 647 - labelled myristoylated ARF1 - strep purified from HeLa lysates left untreated or incubated with IpaJ or its catalytic mutant as indicated . expressed amounts of ARF1 - strep are shown ( bottom panel ) .

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

g, In-gel fluorescence assay (top panel) visualizing Alexa Fluor 647-labelled myristoylated ARF1-strep purified from HeLa cell lysates left untreated or incubated with IpaJ or its catalytic mutant as indicated. The expressed amounts of ARF1-strep are shown (bottom panel).


h , In - gel visualizing extracts isolated from HeLa incubated with azide myristic acid , azide palmitic acid or geranylgeranyl alcohol azide subsequently labelled with Alexa Fluor 647 alkyne by click chemistry . Arrows indicate that are proteolytically demyristoylated by IpaJ .

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

h, In-gel fluorescence assay visualizing protein extracts isolated from HeLa cells incubated with azide myristic acid, azide palmitic acid or geranylgeranyl alcohol azide and subsequently labelled with Alexa Fluor 647 alkyne by click chemistry. Arrows indicate proteins that are proteolytically demyristoylated by IpaJ.


b , In - gel showing myristoylated peptides of indicated in expressing IpaJ or catalytic mutant . peptide - eGFP IpaJ inputs are indicated . * Demyristoylation of GRASP65 hVPS15 resulted in slower mobility of resulting peptide , potentially owing to reduced mobility in SDS - polyacrylamide gel electrophoresis caused by proteolytic reaction .

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

b, In-gel fluorescence assay showing myristoylated peptides of the indicated proteins in cells expressing IpaJ or catalytic mutant. The peptide-eGFP and IpaJ inputs are indicated. *Demyristoylation of GRASP65 and hVPS15 resulted in slower mobility of the resulting peptide, potentially owing to reduced mobility in SDS-polyacrylamide gel electrophoresis caused by proteolytic reaction.


c , In - gel showing myristoylated ARF1 T31N mutant ( a GDP - locked mutant ) or ARF1 Q71L ( a GTP - locked mutant ) after in co - expressing IpaJ or catalytic mutants as indicated . Equal loading of ARF1 ( middle panel ) IpaJ ( lower panel ) was determined by western blot .

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

c, In-gel fluorescence assay showing myristoylated ARF1 T31N mutant (a GDP-locked mutant) or ARF1 Q71L (a GTP-locked mutant) after in cells co-expressing IpaJ or catalytic mutants as indicated. Equal loading of ARF1 (middle panel) and IpaJ (lower panel) was determined by western blot analysis.


d , microscopy showing ARF1 - mCherry Golgi apparatus ( GM130 ) after microinjection of recombinant IpaJ or IpaJ C64A , over indicated period . Scale bar , 10 µm .

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

d, Fluorescence microscopy showing ARF1-mCherry and the Golgi apparatus (GM130) after cellular microinjection of recombinant IpaJ or IpaJ C64A, over the indicated period. Scale bar, 10 µm.


e , microscopy showing ARF1 - eGFP ( green ) Golgi membranes ( blue , GM130 ) after infection with Shigella M90T indicated mutants ( red ; mCherry - expressing ) . Scale bar , 10µm .

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

e, Fluorescence microscopy showing ARF1-eGFP (green) and Golgi membranes (blue, GM130) after infection with Shigella M90T and the indicated mutants (red; mCherry-expressing bacteria). Scale bar, 10µm.


( a ) PC12 stably expressing A53T mutant α - synuclein were induced for 24 h , after which expression was switched off by removing doxycycline from medium for next 24 h . Transgene product clearance can be inferred by assessing at various times after switching off expression after an initial induction period , as expression decay when synthesis is reduced or stopped . were treated with EHNA or AMP - PNP or left untreated during switch - off period . Blots were probed with antibody to HA to detect transgene antibody to actin as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PC12 cells stably expressing A53T mutant α-synuclein were induced for 24 h, after which expression was switched off by removing doxycycline from the medium for the next 24 h. Transgene product clearance can be inferred by assessing levels at various times after switching off expression after an initial induction period, as expression levels decay when synthesis is reduced or stopped. Cells were treated with EHNA or AMP-PNP or left untreated during the switch-off period. Blots were probed with antibody to HA to detect the transgene and antibody to actin as a loading control.


( b ) PC12 expressing Q74 - GFP were induced for 15 h then treated with inhibitors for 48 h after switch - off . Band ( three independent experiments ) is 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

(b) PC12 cells expressing Q74-GFP were induced for 15 h and then treated with inhibitors for 48 h after switch-off. Band intensity quantification (three independent experiments) is shown.


( c ) Immunofluorescence of expressing Q74 - GFP after 48 h of switch - off after treatment with ( EHNA ) or without ( Control ) EHNA .

O O O O O 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) Immunofluorescence images of cells expressing Q74-GFP after 48 h of switch-off after treatment with (EHNA) or without (Control) EHNA.


( d ) of in COS - 7 transiently transfected with Q74 - GFP either with ( EHNA ) or without ( Control ) EHNA ( added during last 33 h before fixation ) .

O O O O O O O O O O O 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) Quantification of aggregates and cell death in COS-7 cells transiently transfected with Q74-GFP either with (EHNA) or without (Control) EHNA (added during the last 33 h before fixation).


( e ) Western - blot of HeLa stably expressing GFP - UbG76V treated with lactacystin ( Lac ) , EHNA or AMP - PNP or left untreated ( Control ) using antibody to GFP . * * * P 0 . 0001 ; * * P 0 . 001 .

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

(e) Western-blot analysis of HeLa cells stably expressing GFP-UbG76V treated with lactacystin (Lac), EHNA or AMP-PNP or left untreated (Control) using antibody to GFP. ***P 0.0001; **P 0.001.


( a ) Stable PC12 expressing A53T α - synuclein were induced for 24 h , after which expression was switched off were simultaneously transfected with p50 or dynamitin ( p50 ) , CC1 , CC2 , motor domain - deleted kinesin heavy chain ( ΔM - KHC ) or tail region of kinesin heavy chain ( DN - KHC ) for next 24 h . Dominant - negative constructs along with pEGFP - C1 ( 3 : 1 of dominant - negative construct : pEGFP - C1 ) were used to transfect , FACS was used to select transfected , which were analyzed by western blotting as in Figure 1a . of band from multiple experiments ( three independent experiments for dynein mutants two for kinesin mutants ) is shown . * * * P 0 . 0001 ; * P 0 . 01 ; NS , not significant .

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

(a) Stable PC12 cells expressing A53T α-synuclein were induced for 24 h, after which expression was switched off and cells were simultaneously transfected with p50 or dynamitin (p50), CC1, CC2, motor domain-deleted kinesin heavy chain (ΔM-KHC) or the tail region of kinesin heavy chain (DN-KHC) for the next 24 h. Dominant-negative constructs along with pEGFP-C1 (3:1 ratio of dominant-negative construct:pEGFP-C1) were used to transfect cells, and FACS was used to select transfected cells, which were analyzed by western blotting as in Figure 1a. Quantification of the band intensity from multiple experiments (three independent experiments for dynein mutants and two for kinesin mutants) is shown. ***P 0.0001; *P 0.01; NS, not significant.


( b ) COS - 7 were cotransfected with HA - Q74 or GFP - Q74 with empty vector ( pEGFP or pcDNA3 . 1 ) , p50 or dynamitin ( p50 ) , p62 subunit of dynactin ( p62 ) , tail region of kinesin heavy chain ( DN - KHC ) , CC1 or CC2 for 48 h , after which were fixed for immunofluorescence immunocytochemistry ( using to antibody to HA ) . of GFP - positive with is shown . * * * P 0 . 0001 ; * * P 0 . 001 ; NS , not significant .

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

(b) COS-7 cells were cotransfected with HA-Q74 or GFP-Q74 with empty vector (pEGFP or pcDNA3.1), p50 or dynamitin (p50), p62 subunit of dynactin complex (p62), the tail region of kinesin heavy chain (DN-KHC), CC1 or CC2 for 48 h, after which the cells were fixed for immunofluorescence and immunocytochemistry (using to antibody to HA). The percentage of GFP-positive cells with aggregates and cell death is shown. ***P 0.0001; **P 0.001; NS, not significant.


( c ) Immunocytochemistry ( using antibody to HA ) of COS - 7 cotransfected for 48 h with HA - Q74 ( red ) pEGFP or p50 ( green ) . Panels on left are magnifications of boxed areas in second panels . boxes are same . * * * P 0 . 0001 ; * * P 0 . 001 . Examples of fragmented nuclei in overexpressing p50 nuclei in overexpressing p50 but treated with Z - VAD - fmk ( broad - spectrum caspase inhibitor ) are shown in Supplementary Figure 4 online .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunocytochemistry analysis (using antibody to HA) of COS-7 cells cotransfected for 48 h with HA-Q74 (red) and pEGFP or p50 (green). Panels on left are magnifications of boxed areas in second panels. The boxes are the same size. ***P 0.0001; **P 0.001. Examples of fragmented nuclei in cells overexpressing p50 and nuclei in cells overexpressing p50 but treated with Z-VAD-fmk (broad-spectrum caspase inhibitor) are shown in Supplementary Figure 4 online.


( a ) Immunofluorescence of COS - 7 cotransfected for 15 h with either empty vector ( control ) or CC1 GFP - LC3 ( green ) , in a 3 : 1 . Panels on right are magnifications of boxed areas .

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

(a) Immunofluorescence analysis of COS-7 cells cotransfected for 15 h with either empty vector (control) or CC1 and GFP-LC3 (green), in a 3:1 ratio. Panels on right are magnifications of boxed areas.


( b ) Western - blot of COS - 7 treated with EHNA or left untreated using antibodies to LC3 to actin . of band from two independent experiments is shown .

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

(b) Western-blot analysis of COS-7 cells treated with EHNA or left untreated using antibodies to LC3 and to actin. Quantification of the band intensity from two independent experiments is shown.


( c ) NRK transiently transfected for 15 h with either empty vector ( control ) or CC1 GFP - LC3 ( green ) , in a 3 : 1 were immunostained with antibody to lgp120 , numbers of GFP - LC3 lgp120 doubly stained vesicles in were scored .

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

(c) NRK cells transiently transfected for 15 h with either empty vector (control) or CC1 and GFP-LC3 (green), in a 3:1 ratio were immunostained with antibody to lgp120, and the numbers of GFP-LC3 and lgp120 doubly stained vesicles in individual cells were scored.


Frequencies of ommatidia with different numbers of rhabdomeres are shown at 3 days after eclosion in progeny of flies expressing mutant huntingtin exon 1 ( gmrQ120 ) crossed to either a control stock ( w1118 ; W ) or to Dhc64C ( dynein heavy chain mutant ; Dhc ; a ) or Roblk ( dynein chain mutant ; Robl ; b ) flies . P ≤ 0 . 0001 ( Mann - Whitney U ) .

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

Frequencies of ommatidia with different numbers of rhabdomeres are shown at 3 days after eclosion in progeny of flies expressing mutant huntingtin exon 1 (gmrQ120) crossed to either a control stock (w1118; W) or to Dhc64C (dynein heavy chain mutant; Dhc; a) or Roblk (dynein light chain mutant; Robl; b) flies. P ≤ 0.0001 (Mann-Whitney U test).


( c ) Ommatidia from progeny of flies expressing mutant huntingtin exon 1 ( Q120 ) crossed with either control stock ( w1118 ; W ) or with Dhc64C ( Dhc ) flies . fly experiments were repeated three times independently with similar significance . One representative experiment is 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

(c) Ommatidia from the progeny of flies expressing mutant huntingtin exon 1 (Q120) crossed with either control stock (w1118; W) or with Dhc64C (Dhc) flies. All fly experiments were repeated three times independently with similar significance levels. One representative experiment is shown.


( a ) Tremor onset severity in a mouse model of Huntington disease are enhanced by Loa mutation . At 7 : HdhHD / + Dnchc1Loa / + n = 9 , HdhHD / + Dnchc1 + / + n = 9 , P = 0 . 14 . At 9 : HdhHD / + Dnchc1Loa / + n = 8 , HdhHD / + Dnchc1 + / + n = 9 , P = 0 . 027 . At 11 : HdhHD / + Dnchc1Loa / + n = 6 , HdhHD / + Dnchc1 + / + n = 9 , P = 0 . 001 . At 13 : HdhHD / + Dnchc1Loa / + n = 5 , HdhHD / + Dnchc1 + / + n = 8 , P = 0 . 021 . At 15 : HdhHD / + Dnchc1Loa / + n = 3 , HdhHD / + Dnchc1 + / + n = 5 , P = 0 . 021 .

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

(a) Tremor onset and severity in a mouse model of Huntington disease are enhanced by the Loa mutation. At 7 weeks: HdhHD/+ Dnchc1Loa/+ n = 9, HdhHD/+ Dnchc1+/+ n = 9, P = 0.14. At 9 weeks: HdhHD/+ Dnchc1Loa/+ n = 8, HdhHD/+ Dnchc1+/+ n = 9, P = 0.027. At 11 weeks: HdhHD/+ Dnchc1Loa/+ n = 6, HdhHD/+ Dnchc1+/+ n = 9, P = 0.001. At 13 weeks: HdhHD/+ Dnchc1Loa/+ n = 5, HdhHD/+ Dnchc1+/+ n = 8, P = 0.021. At 15 weeks: HdhHD/+ Dnchc1Loa/+ n = 3, HdhHD/+ Dnchc1+/+ n = 5, P = 0.021.


( b , c ) Dynein mutation reduces motor coordination muscle function in a mouse model of Huntington disease . HdhHD / + Dnchc1Loa / + , HdhHD / + Dnchc1 + / + Hdh + / + Dnchc1Loa / + mice are shown . An average of six mice were used at every time point , with at least three mice per group after 13 . ( b ) Grip strength . Overall effect for time points between three genotypes : P = 0 . 0024 . Overall pair comparison between HdhHD / + Dnchc1Loa / + HdhHD / + Dnchc1 + / + : P = 0 . 001 . Overall pair comparison between HdhHD / + Dnchc1Loa / + Hdh + / + Dnchc1Loa / + : P = 0 . 0157 . Effect at each time point between three genotypes : 7 , P = 0 . 023 ; 9 , P = 0 . 001 ; 11 , P 0 . 0001 ; 13 , P = 0 . 004 ; 15 , P = 0 . 336 ( most of double mutants were dead by 15 ) . ( c ) Accelerating rotarod . Overall effect for time points between three genotypes : P = 0 . 0367 . Overall pair comparison between HdhHD / + Dnchc1Loa / + HdhHD / + Dnchc1 + / + : P = 0 . 0245 . Overall pair comparison between HdhHD / + Dnchc1Loa / + Hdh + / + Dnchc1Loa / + : P = 0 . 0553 . Effect at each time point between three genotypes : 8 , P 0 . 001 ; 10 , P = 0 . 005 ; 12 , P = 0 . 001 ; 14 , P = 0 . 009 ; 16 , P = 0 . 039 .

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

(b,c) Dynein mutation reduces motor coordination and muscle function in a mouse model of Huntington disease. HdhHD/+ Dnchc1Loa/+, HdhHD/+ Dnchc1+/+ and Hdh+/+ Dnchc1Loa/+ mice are shown. An average of six mice were used at every time point, with at least three mice per group after 13 weeks. (b) Grip strength. Overall effect for all time points between the three genotypes: P = 0.0024. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and HdhHD/+ Dnchc1+/+: P = 0.001. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and Hdh+/+ Dnchc1Loa/+: P = 0.0157. Effect at each time point between the three genotypes: 7 weeks, P = 0.023; 9 weeks, P = 0.001; 11 weeks, P 0.0001; 13 weeks, P = 0.004; 15 weeks, P = 0.336 (most of the double mutants were dead by 15 weeks). (c) Accelerating rotarod. Overall effect for all time points between the three genotypes: P = 0.0367. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and HdhHD/+ Dnchc1+/+: P = 0.0245. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and Hdh+/+ Dnchc1Loa/+: P = 0.0553. Effect at each time point between the three genotypes: 8 weeks, P 0.001; 10 weeks, P = 0.005; 12 weeks, P = 0.001; 14 weeks, P = 0.009; 16 weeks, P = 0.039.


( d ) Reduced lifespan of HdhHD / + Dnchc1Loa / + mice . HdhHD / + Dnchc1Loa / + ( red ) n = 13 , HdhHD / + Dnchc1 + / + ( green ) n = 9 , P = 0 . 038 . Mice were subjected to humane end points , so survival curve reflects onset of end points rather than complete lifespan . Asterisks ( * ) represent mice that were found dead without reaching humane end points . No mortality of Hdh + / + Dnchc1Loa / + mice was observed .

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

(d) Reduced lifespan of HdhHD/+ Dnchc1Loa/+ mice. HdhHD/+ Dnchc1Loa/+ (red line) n = 13, HdhHD/+ Dnchc1+/+ (green line) n = 9, P = 0.038. Mice were subjected to humane end points, and so the survival curve reflects the onset of the end points rather than the complete lifespan. Asterisks (*) represent mice that were found dead without reaching humane end points. No mortality of Hdh+/+ Dnchc1Loa/+ mice was observed.


( a ) Brain sections from HdhHD / + Dnchc1 + / + or HdhHD / + Dnchc1Loa / + mice were stained with antibody to huntingtin ( EM48 ; green ) . No EM48 - stained were detected in brains of Hdh + / + Dnchc1Loa / + mice . Arrows indicate with .

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

(a) Brain sections from HdhHD/+ Dnchc1+/+ or HdhHD/+ Dnchc1Loa/+ mice were stained with antibody to huntingtin (EM48; green). No EM48-stained aggregates were detected in brains of Hdh+/+ Dnchc1Loa/+ mice. Arrows indicate cells with aggregates.


( b ) Western - blot of mouse brain lysates from wild - type ( WT ) or Loa mutant mice using antibodies to LC3 to actin .

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

(b) Western-blot analysis of mouse brain lysates from wild-type (WT) or Loa mutant mice using antibodies to LC3 and to actin.


( A ) Growth curves of S . pombe cultures in minimal medium without ( EMM ) or with antimycin A ( EMM + antA ) . Cultures were supplemented with either complete - acid mix ( AA mix , black ) , without any ( no AA , red ) , with arginine only ( R , blue ) , or with other than arginine ( AAs , grey ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Growth curves of S. pombe cultures in minimal medium without (EMM) or with antimycin A (EMM + antA). Cultures were supplemented with either the complete amino-acid mix (all AA mix, black), without any amino acids (no AA, red), with arginine only (R, blue), or with individual amino acids other than arginine (individual AAs, grey).


( B ) Growth curves as in ( A ) for cultures supplemented with complete - acid mix ( black ) , without any ( red ) , or - acid mix without arginine ( green ) .

O O O O O O O O O O O O O O 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) Growth curves as in (A) for cultures supplemented with complete amino-acid mix (black), without any amino acids (red), or amino-acid mix without arginine (green).


( C ) Scatter plot of normalised colony sizes of grown on EMM against normalised colony sizes of grown on EMM supplemented with arginine ( EMM + R ) ( Dataset file 1 ) . Mutants that passed our filtering ( Methods ) whose colony - ratios were 30 % bigger when supplemented with arginine are shown in red ; mutants encoding known genes of arginine biosynthesis pathway are shown with blue circles .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Scatter plot of normalised colony sizes of cells grown on EMM against normalised colony sizes of cells grown on EMM supplemented with arginine (EMM+R) (Dataset file 1). Mutants that passed our filtering (Methods) and whose colony-size ratios were 30% bigger when supplemented with arginine are shown in red; mutants encoding known genes of the arginine biosynthesis pathway are shown with blue circles.


( E ) Box plot of mean colony ratios of mutants grown with vs without arginine for mutants ( grey ) respiratory deficient mutants ( orange , on previous screens ( Malecki & Bähler , 2016 ) ( Welch ' s t - pval * * < 0 . 002 ) . same was performed for mutants grown on EMM ( left ; 4 biological repeats , Dataset file 1 ) or on EMM supplemented with antimycin A ( right ; 2 biological repeats , Dataset file 2 ) . Boxplots were created using R boxplot command with default settings . central band indicates median , upper lower limits of box indicate first third quartiles , whiskers show most extreme data , limited to 1 . 5 - time interquartile range .

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

(E) Box plot of mean colony size ratios of mutants grown with vs without arginine for all mutants (grey) and respiratory deficient mutants (orange, based on previous screens (Malecki & Bähler, 2016) (Welch's t-test pval** < 0.002). The same analysis was performed for mutants grown on EMM (left; 4 biological repeats, Dataset file 1) or on EMM supplemented with antimycin A (right; 2 biological repeats, Dataset file 2). Boxplots were created using the R boxplot command with default settings. The central band indicates the median, the upper and lower limits of the box indicate first and third quartiles, and the whiskers show the most extreme data, limited to 1.5-time interquartile range.


( A ) Normalised values of 16 in grown in EMM without ( grey ) or with ( blue ) antimycin A . For each condition , four independent samples were analysed . Concentrations are presented relative to mean value for given acid in EMM medium . * Ornithine ( Orn ) is abbreviated as O . Boxplots were created using R boxplot command with default settings . central band indicates median , upper lower limits of box indicate first third quartiles , whiskers show most extreme data , limited to 1 . 5 - time interquartile range .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Normalised concentration values of 16 amino acids in cells grown in EMM without (grey) or with (blue) antimycin A. For each condition, four independent samples were analysed. Concentrations are presented relative to the mean value for the given amino acid in EMM medium. *Ornithine (Orn) is abbreviated as O. Boxplots were created using R boxplot command with default settings. The central band indicates the median, the upper and lower limits of the box indicate first and third quartiles, and the whiskers show the most extreme data, limited to 1.5-time interquartile range.


( B ) - acid concentrations before after addition of antimycin A to exponentially growing in EMM . Left : heat map showing changes ( log2 fold change ) of each measured acid during time course relative to time point 0 ( before antimycin A addition ) . Right : simplified scheme of - acid biosynthetic pathways . for which any biosynthetic enzyme has an annotated mitochondrial location are highlighted in yellow . * Ornithine ( Orn ) is abbreviated as O .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Amino-acid concentrations before and after addition of antimycin A to cells exponentially growing in EMM. Left: heat map showing concentration changes (log2 fold change) of each measured amino acid during the time course relative to time point 0 (before antimycin A addition). Right: simplified scheme of amino-acid biosynthetic pathways. Amino acids for which any biosynthetic enzyme has an annotated mitochondrial location are highlighted in yellow. *Ornithine (Orn) is abbreviated as O.