F . Western blot analyses of cortical hippocampal lysates from APP / PS1mice for APP sAPP expressions .

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

F. Western blot analyses of cortical and hippocampal lysates from APP/PS1mice for APP and sAPP expressions.


G . ThT assays for inhibition of Aβ aggregation . was normalized to Aβaggregates ( 100 % , day 5 ) .

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

G. ThT assays for the inhibition of Aβ aggregation. Fluorescence intensity was normalized to Aβaggregates (100%, day 5).


H . SDS - PAGE of PICUP cross - linked Aβaggregates visualized by silver .

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

H. SDS-PAGE analysis of PICUP cross-linked Aβaggregates visualized by silver staining.


A . Western blot of phosphorylation of tau expression of indicated in cortical hippocampal regions of brain in APP / PS1mice after administration of Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) . membranes analysed in ( A ) are identical to those in Fig 5F .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-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

A. Western blot analysis of phosphorylation levels of tau and expression levels of indicated proteins in the cortical and hippocampal regions of the brain in APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS). The membranes analysed in (A) are identical to those in Fig 5F.


B . Immunohistochemical of cortical hippocampal regions of brains in wild - type ( Wt ) APP / PS1mice after administration of Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) for phosphorylated tau expression . Anti - tau ( phospho Ser199 ) antibody ( red colour ) was applied for tau phosphorylation on Serine - 199 Hoechst 33342 ( blue colour ) for counterstaining in brain sections . Scale bars = 200 μM .

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

B. Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild-type (Wt) and APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS) for phosphorylated tau expression levels. Anti-tau (phospho Ser199) antibody (red colour) was applied for staining tau phosphorylation on Serine-199 and Hoechst 33342 (blue colour) for nuclear counterstaining in the brain sections. Scale bars = 200 μM.


C . Western blot analyses of phosphorylated RIPK3 expressions in cortex hippocampus of APP / PS1mice injected with Nec - 1 ( upper ) . membranes analysed in ( C ) are identical to those in Fig 1G 5F .

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

C. Western blot analyses of phosphorylated RIPK3 expressions in cortex and hippocampus of APP/PS1mice injected with Nec-1 (upper). The membranes analysed in (C) are identical to those in Fig 1G and 5F.


C . Western blot analyses of phosphorylated RIPK3 expressions in cultured astrocytes treated with Nec - 1 ( lower ) . membranes analysed in ( C ) are identical to those in Fig 1G 5F .

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

C. Western blot analyses of phosphorylated RIPK3 expressions in primary cultured astrocytes treated with Nec-1 (lower). The membranes analysed in (C) are identical to those in Fig 1G and 5F.


D , E . ThT assays for inhibition of tau aggregation ( D ) for disaggregation of tau ( E ) . was normalized to tauaggregates ( 100 % , day 5 ) .

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 B-MEASURED_VAR O O O O O O O O

D, E. ThT assays for the inhibition of tau aggregation (D) and for disaggregation of tau (E). Fluorescence intensity was normalized to tauaggregates (100%, day 5).


F . Surface plasmon resonance sensorgrams of Nec - 1 targeting tau ( left ) , corresponding dissociation fitting curve from saturated region ( right ) under various concentrations of Nec - 1 .

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

F. Surface plasmon resonance sensorgrams of Nec-1 targeting tau (left), and the corresponding dissociation fitting curve from the saturated region (right) under various concentrations of Nec-1.


MEFs expressing vector , TRPML - 1 , or GRP1 - DD were treated with BSA or palmitate ( 250 μM ) for 3 h stained as in ( A ) . aspect ( O ) , branch ( P ) , roundness ( Q ) of mitochondria in in ( N ) .

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

MEFs expressing vector, TRPML-1, or GRP1-DD were treated with BSA or palmitate (250 μM) for 3 h and stained as in (A). aspect ratio (O), branch length (P), and roundness (Q) of mitochondria in the cells in (N).


TMRE ( 100 nM ) MitoTracker green ( 200 nM ) in MEFs treated with BSA or palmitate ( 250 μM ) for 12 h after a 3 h pre - treatment with vehicle or SH - BC - 893 ( 893 , 5 μM ) . TMRE in ( A ) on a per basis . MitoSOX ( 5 μM ) MitoTracker Green ( 200 nM ) in MEFs treated with vehicle ( DMSO ) or C2 - ceramide ( 50 μM ) for 12 h after a 3 h pre - treatment with vehicle or SH - BC - 893 ( 893 , 5 μM ) . MitoSOX for ( C ) on a per basis .

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

TMRE (100 nM) and MitoTracker green (200 nM) staining in MEFs treated with BSA or palmitate (250 μM) for 12 h after a 3 h pre-treatment with vehicle or SH-BC-893 (893, 5 μM). TMRE intensity in (A) on a per cell basis. MitoSOX (5 μM) and MitoTracker Green (200 nM) staining in MEFs treated with vehicle (DMSO) or C2-ceramide (50 μM) for 12 h after a 3 h pre-treatment with vehicle or SH-BC-893 (893, 5 μM). MitoSOX intensity for (C) on a per cell basis.


NAD ( P ) H autofluorescence evaluated by confocal microscopy in freshly resected livers from mice in ( A ) 4 - 8 h after treatment with vehicle ( n = 5 ) or 120 mg / kg SH - BC - 893 ( n = 4 ) by gavage at ZT8 . 5 . Mice were sacrificed imaged in pairs ( SD HFD ) .

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

NAD(P)H autofluorescence evaluated by confocal microscopy in freshly resected livers from mice in (A) 4-8 h after treatment with vehicle (n=5) or 120 mg/kg SH-BC-893 (n=4) by gavage at ZT8.5. Mice were sacrificed and imaged in pairs (SD and HFD).


Citrate synthase in arcuate nucleus of mice in ( E ) 4 h after treatment with vehicle or 120 mg / kg SH - BC - 893 by gavage at ZT8 . 5 ; n = 3 .

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

Citrate synthase staining in the arcuate nucleus of mice in (E) 4 h after treatment with vehicle or 120 mg/kg SH-BC-893 by gavage at ZT8.5; n=3.


imaging was performed in the areas of the cortex


NAD ( P ) H autofluorescence was evaluated in epididymal fat ( eWAT ) freshly resected from mice in ( A ) 4 h after treatment with vehicle or 120 mg / kg SH - BC - 893 by gavage at ZT8 . 5 ; n = 5 . NAD ( P ) H from ( B ) calculated on a per basis ; 16 - 25 from each of 5 mice per group were quantified ( 87 - 109 total per treatment group ) .

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

NAD(P)H autofluorescence was evaluated in epididymal fat (eWAT) freshly resected from the mice in (A) 4 h after treatment with vehicle or 120 mg/kg SH-BC-893 by gavage at ZT8.5; n=5. NAD(P)H intensity from (B) calculated on a per cell basis; 16 - 25 cells from each of the 5 mice per group were quantified (87 - 109 total cells per treatment group).


TMRE ( 100 nM ) MitoTracker Green ( 200 nM ) in eWAT resected from mice in ( D ) treated as in ( B , C ) ; n = 5 . TMRE ( F ) or MitoTracker ( G ) for ( E ) calculated on a per basis ; 15 - 24 from each of 5 mice in each group were quantified ( 99 - 104 total per treatment group ) .

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

TMRE (100 nM) and MitoTracker Green staining (200 nM) in eWAT resected from mice in (D) treated as in (B,C); n=5. TMRE (F) or MitoTracker (G) intensity for (E) calculated on a per cell basis; 15 - 24 cells from each of the 5 mice in each group were quantified (99 - 104 total cells per treatment group).


MitoSOX ( 5 μM ) MitoTracker Green in eWAT resected from mice in ( D ) treated as in ( B E ) ; n = 5 . MitoSOX ( I ) or MitoTracker ( J ) calculated as in ( F G ) ; 10 - 13 from each of 5 mice in each group were quantified ( 57 - 58 total per treatment group ) .

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

MitoSOX (5 μM) and MitoTracker Green staining in eWAT resected from the mice in (D) treated as in (B and E); n=5. MitoSOX (I) or MitoTracker (J) intensity calculated as in (F and G); 10 - 13 cells from each of the 5 mice in each group were quantified (57 - 58 total cells per treatment group).


Chop , Xbp1s , Atf6 , Grp78 mRNA measured in eWAT resected from mice maintained on a HFD for 13 gavaged with vehicle or 120 mg / kg SH - BC - 893 on Monday , Wednesday , Friday for final 3 ; n = 7 - 9 .

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

Chop, Xbp1s, Atf6, and Grp78 mRNA measured in eWAT resected from mice maintained on a HFD for 13 weeks and gavaged with vehicle or 120 mg/kg SH-BC-893 on Monday, Wednesday, and Friday for the final 3 weeks; n=7-9.


Plasma adiponectin ( A ) or leptin ( B ) in mice maintained on a SD or HFD for 8 - 9 then gavaged with vehicle or 120 mg / kg SH - BC - 893 4 h prior to blood collection . Pair - fed mice were provided with amount of food consumed by SH - BC - 893 - treated mice in 4 h but left untreated ; n = 8 - 10 .

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

Plasma adiponectin (A) or leptin (B) in mice maintained on a SD or HFD for 8-9 weeks then gavaged with vehicle or 120 mg/kg SH-BC-893 4 h prior to blood collection. Pair-fed mice were provided with the amount of food consumed by SH-BC-893-treated mice in 4 h but left untreated; n=8-10.


Average daily run by HFD - fed mice treated with vehicle ( n = 8 ) or SH - BC - 893 ( 120 mg / kg , n = 6 ) on Mondays , Wednesdays , Fridays over 4 when housed with a running wheel .

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

Average daily distance run by HFD-fed mice treated with vehicle (n=8) or SH-BC-893 (120 mg/kg, n=6) on Mondays, Wednesdays, and Fridays over 4 weeks when housed with a running wheel.


change in mice in ( O , P ) over 24 h after first treatment ( Q ) or after 3 week course of treatment ( R ) ; n = 9 .

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

Body weight change in the mice in (O,P) over the 24 h after the first treatment (Q) or after the 3 week course of treatment (R); n=9.


Representative H & E stained liver sections from mice shown in Fig . 6F after 4 of treatment with vehicle or 120 mg / kg SH - BC - 893 . 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O

Representative H&E stained liver sections from the mice shown in Fig. 6F after 4 weeks of treatment with vehicle or 120 mg/kg SH-BC-893. Scale bar, 10 μm.


Blood glucose ( F ) or under curve ( AUC , ( G ) ) during an oral glucose tolerance performed in same mice as in ( E ) .

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

Blood glucose levels (F) or area under the curve (AUC, (G)) during an oral glucose tolerance test performed in the same mice as in (E).


Insulin in plasma collected 4 h after indicated treatment with vehicle or SH - BC - 893 from mice in Fig . 5D , E or in 9 week old SD - fed mice treated with vehicle ( same mice as in Fig . 5J , K , 0 on HFD ) ; n = 8 . Insulin in plasma collected 4 h after treatment with vehicle or SH - BC - 893 from same mice as in Fig . 5A , B ; n = 8 .

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

Insulin levels in plasma collected 4 h after the indicated treatment with vehicle or SH-BC-893 from the mice in Fig. 5D,E or in 9 week old SD-fed mice treated with vehicle (same mice as in Fig. 5J,K, 0 weeks on HFD); n=8. Insulin levels in the plasma collected 4 h after treatment with vehicle or SH-BC-893 from the same mice as in Fig. 5A,B; n=8.


C TIMM50S in immortalized skin fibroblast from control ( C1 - 4 ) mutant TIMM50 ( Pt ) . SDHA ETHE1 are shown as loading controls .

O B-MEASURED_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

C TIMM50S levels in primary and immortalized skin fibroblast from control (C1-4) and mutant TIMM50 (Pt). SDHA and ETHE1 are shown as loading controls.


D qPCR of TIMM50 transcript in control TIMM50 mutant fibroblasts . Data are shown as mean ± SD , n = 4 .

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

D qPCR quantification of TIMM50 transcript levels in control and TIMM50 mutant fibroblasts. Data are shown as mean ± SD, n = 4.


A Western - blot of whole extracts for different components of import machinery in control TIMM50 mutant fibroblasts grown in either glucose or galactose containing medium .

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

A Western-blot of whole cell extracts for different components of the protein import machinery in control and TIMM50 mutant fibroblasts grown in either glucose or galactose containing medium.


B Western - blot of whole extracts for different components of import machinery in control TIMM50 mutant fibroblasts , transduced with empty vector ( EV ) or with wild - type TIMM50 grown in either glucose or galactose containing medium .

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

B Western-blot of whole cell extracts for different components of the protein import machinery in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in either glucose or galactose containing medium.


C Mitochondrial potential in fibroblasts grown in glucose or galactose using JC - 1 in untreated or FCCP treated conditions . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 .

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

C Mitochondrial membrane potential in fibroblasts grown in glucose or galactose using JC-1 staining in untreated or FCCP treated conditions. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.


D TMRM live of control TIMM50 mutant fibroblasts , transduced with empty vector ( EV ) or with wild - type TIMM50 grown in either glucose or galactose containing medium . Scale bar corresponds to 10 μm .

O O O O O B-CONTROLLED_VAR 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

D TMRM live staining of control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in either glucose or galactose containing medium. Scale bar corresponds to 10 μm.


A In organello import of precursors of TFAM AAC1 into isolated mitochondria from control TIMM50 mutant fibroblasts in absence or presence of uncoupler FCCP . radiolabeled precursors were incubated with isolated mitochondria for indicated times followed by trypsin treatment only in case of AAC1 . Coomassie Blue ( CB ) was used as loading control radiolabeled ( TNT ) as reference for . position of precursor ( empty arrowheads ) mature ( filled arrowhead ) are also shown . Western blot for TIMM50 is shown as control . B of TFAM AAC1 import from control TIMM50 mutant fibroblasts expressed as fraction of input radiolabeled precursor converted into mature normalized to Coomassie Blue signal . Data are shown as mean ± SD , n = 3 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 grouping both controls together .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR 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-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 B-MEASURED_VAR I-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 O O O O O O O O O O O O O O O O O O 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 In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts in the absence or presence of the uncoupler FCCP. The radiolabeled precursors were incubated with isolated mitochondria for the indicated times followed by trypsin treatment only in the case of AAC1. Coomassie Blue (CB) staining was used as loading control and the radiolabeled protein (TNT) as reference for quantification. The position of the precursor (empty arrowheads) and the mature protein (filled arrowhead) are also shown. Western blot for TIMM50 is shown as cell line control. B Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together.


C In organello import of precursors of TFAM AAC1 into isolated mitochondria from control TIMM50 mutant fibroblasts transduced with empty vector ( EV ) or with wild - type TIMM50 in absence or presence of uncoupler FCCP position of precursor ( empty arrowheads ) mature ( filled arrowhead ) are also shown . D of TFAM AAC1 import from control TIMM50 mutant fibroblasts transduced with empty vector ( EV ) or with wild - type TIMM50 expressed as fraction of input radiolabeled precursor converted into mature normalized to Coomassie Blue signal . Data are shown as mean ± SD , n = 3 biological replicates ; ∗ p < 0 . 05 .

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

C In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts transduced with the empty vector (EV) or with wild-type TIMM50 in the absence or presence of the uncoupler FCCP The position of the precursor (empty arrowheads) and the mature protein (filled arrowhead) are also shown. D Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts transduced with the empty vector (EV) or with wild-type TIMM50 expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05.


Western blot for representatives of five respiratory complexes in control TIMM50 mutant fibroblasts grown in glucose or galactose . Two biological replicates are shown for each condition . Significant changes in in TIMM50 mutant fibroblasts compared to controls have been boxed in red .

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

Western blot for representatives of all five respiratory complexes in control and TIMM50 mutant fibroblasts grown in glucose or galactose. Two biological replicates are shown for each cell line and condition. Significant changes in protein levels in TIMM50 mutant fibroblasts compared to controls have been boxed in red.


B Western blot for representatives of five respiratory complexes in control TIMM50 mutant fibroblasts , transduced with empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . Two biological replicates were obtained for each condition . Significant changes in in TIMM50 mutant fibroblasts complemented with TIMM50 compared to EV have been boxed in red .

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

B Western blot for representatives of all five respiratory complexes in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Two biological replicates were obtained for each cell line and condition. Significant changes in protein levels in TIMM50 mutant fibroblasts complemented with TIMM50 compared to EV have been boxed in red.


Oxygen consumption measurements in control TIMM50 mutant fibroblasts grown in glucose or galactose . Values of basal maximal respiration along with ATP - production dependent , proton leak respiration spare capacity are shown . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 grouping both controls together .

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

Oxygen consumption measurements in control and TIMM50 mutant fibroblasts grown in glucose or galactose. Values of basal and maximal respiration along with ATP-production dependent, proton leak respiration and spare capacity are shown. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together.


D Oxygen consumption measurements in control TIMM50 mutant fibroblasts , transduced with empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . Values of basal maximal respiration along with ATP - production dependent , proton leak respiration spare capacity are shown . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 .

O O O O O O B-CONTROLLED_VAR 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

D Oxygen consumption measurements in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Values of basal and maximal respiration along with ATP-production dependent, proton leak respiration and spare capacity are shown. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.


A ROS production as measured by intracellular oxidation of - permeant probe DCF - DA in control TIMM50 mutant fibroblasts grown in glucose or galactose . Measurements were made in untreated after treatment with 250μM hydrogen peroxide for 30 min . Data are shown as mean ± SD , n = 3 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 grouping both controls together .

O B-MEASURED_VAR 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 B-CONTROLLED_VAR I-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

A ROS production as measured by intracellular oxidation of the cell-permeant probe DCF-DA in control and TIMM50 mutant fibroblasts grown in glucose or galactose. Measurements were made in untreated cells and after treatment with 250μM hydrogen peroxide for 30 min. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together.


B Steady state of ROS related SOD2 ACO2 in control TIMM50 mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control . C of steady state of SOD2 ACO2 in control TIMM50 mutant fibroblasts growing in either glucose or galactose . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 grouping both controls together .

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

B Steady state levels of ROS related proteins SOD2 and ACO2 in control and TIMM50 mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control. C Quantification of the steady state levels of SOD2 and ACO2 in control and TIMM50 mutant fibroblasts growing in either glucose or galactose. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01 grouping both controls together.


D Steady state of mitophagy related VDAC1 , p62 LC3 - II in control TIMM50 mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control . E of steady state of VDAC1 , p62 LC3 - II in control TIMM50 mutant fibroblasts growing in either glucose or galactose . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 grouping both controls together .

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

D Steady state levels of mitophagy related proteins VDAC1, p62 and LC3-II in control and TIMM50 mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control. E Quantification of the steady state levels of VDAC1, p62 and LC3-II in control and TIMM50 mutant fibroblasts growing in either glucose or galactose. Data are shown as mean ± SD, n = 4 biological replicates; ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together.


A in control TIMM50 mutant fibroblasts , transduced with empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . Data are shown as mean ± SD , n = 5 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 .

O O O B-CONTROLLED_VAR 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

A Cell size in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Data are shown as mean ± SD, n = 5 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.


B Growth curves of control TIMM50 mutant fibroblasts , transduced with empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . growth was monitored continuously by Incucyte live imager ( Essen Bioscience ) . One of three independent experiments carried out is presented . Data are shown as mean of three technical replicates ± SD .

O O O O O B-CONTROLLED_VAR 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

B Growth curves of control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Cell growth was monitored continuously by the Incucyte live cell imager (Essen Bioscience). One of the three independent experiments carried out is presented. Data are shown as mean of three technical replicates ± SD.


C Plot of Annexin V against propidium iodine signal in control TIMM50 mutant fibroblasts , transduced with empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . in late stage of apoptosis are boxed in top right corner .

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

C Plot of Annexin V intensity against propidium iodine signal in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Cells in the late stage of apoptosis are boxed in the top right corner.


D of number of late apoptotic in control TIMM50 mutant fibroblasts , transduced with empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . Data are shown as mean ± SD , n = 5 or 4 biological replicates in case of transduced ; ∗ * ∗ p < 0 . 001 .

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 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

D Quantification of the number of late apoptotic cells in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Data are shown as mean ± SD, n = 5 or 4 biological replicates in the case of transduced cells; ∗*∗p < 0.001.


A Western - blot of whole extracts for TIMM50 GAPDH in control TIMM50 , RNASEH1 ISCU mutant fibroblasts grown in either glucose or galactose containing medium .

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

A Western-blot of whole cell extracts for TIMM50 and GAPDH in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in either glucose or galactose containing medium.


B Mitochondrial potential in control TIMM50 , RNASEH1 ISCU mutant fibroblasts grown in glucose or galactose using JC - 1 in untreated or FCCP treated conditions . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 .

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

B Mitochondrial membrane potential in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose using JC-1 staining in untreated or FCCP treated conditions. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.


C Western blot for representatives of five respiratory complexes in control TIMM50 , RNASEH1 ISCU mutant fibroblasts grown in glucose or galactose . GAPDH is from same blot as panel A . on two biological replicates for RNASEH1 ISCU mutant fibroblasts Significant changes in in RNASEH1 ISCU mutant fibroblasts compared to controls have been boxed in red .

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

C Western blot for representatives of all five respiratory complexes in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. GAPDH is from the same blot as panel A. Quantification based on two biological replicates for RNASEH1 and ISCU mutant fibroblasts Significant changes in protein levels in RNASEH1 and ISCU mutant fibroblasts compared to controls have been boxed in red.


D Oxygen consumption measurements in control TIMM50 , RNASEH1 ISCU mutant fibroblasts grown in glucose or galactose . Values of basal maximal respiration along with ATP - production dependent , proton leak respiration spare capacity are shown . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 .

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

D Oxygen consumption measurements in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. Values of basal and maximal respiration along with ATP-production dependent, proton leak respiration and spare capacity are shown. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.


E Steady state of ROS related SOD2 ACO2 mitophagy related VDAC1 , p62 LC3 in control TIMM50 , RNASEH1 ISCU mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control . GAPDH is from same blot as panel A .

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

E Steady state levels of ROS related proteins SOD2 and ACO2 and mitophagy related proteins VDAC1, p62 and LC3 in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control. GAPDH is from the same blot as panel A.


F of number of late apoptotic in control TIMM50 , RNASEH1 ISCU mutant fibroblasts grown in glucose or galactose . Data are shown as mean ± SD , n = 4 or more biological replicates ; ∗ * ∗ p < 0 . 001 .

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

F Quantification of the number of late apoptotic cells in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. Data are shown as mean ± SD, n = 4 or more biological replicates; ∗*∗p < 0.001.


Huh7 were infected with HCV ( Jc1 ) using 30 TCID50 / 48 h later were fixed processed for microscopy . In case of samples shown in panels B - D , were first transfected with expression constructs specified in left of each panel 24 h later were infected as described above . Samples were analyzed with a Nikon TE2000 - E inverted confocal microscope at 60 × magnification . ( A ) - ( D ) Colocalization of HCV specified in top of each panel with disulphide isomerase ( PDI ; an ER marker ) , GFP - Rab21 ( marker for early endosomes ) , GFP - Rab7 ( marker for late endosomes ) or βCOP - YFP ( marker for COP I vesicles ) . upper panels represent a low magnification overview ; boxed areas are shown as enlargement in corresponding panel below . nucleus was stained with DAPI ( blue ) . Scale bars represent 10 µm ( top panels ) 2 µm ( lower panels ) . of degree of colocalization ( Pearson ' s correlation coefficient ) is given at top of enlarged pictures .

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

Huh7 cells were infected with HCV (strain Jc1) using 30 TCID50/cell and 48 h later cells were fixed and processed for fluorescence microscopy. In case of samples shown in panels B-D, cells were first transfected with expression constructs specified in the left of each panel and 24 h later cells were infected as described above. Samples were analyzed with a Nikon TE2000-E inverted confocal microscope at 60× magnification. (A)-(D) Colocalization of HCV proteins specified in the top of each panel with protein disulphide isomerase (PDI; an ER marker), GFP-Rab21 (marker for early endosomes), GFP-Rab7 (marker for late endosomes) or βCOP-YFP (marker for COP I vesicles). The upper panels represent a low magnification overview; boxed areas are shown as enlargement in the corresponding panel below. The nucleus was stained with DAPI (blue). Scale bars represent 10 µm (top panels) and 2 µm (lower panels). The quantification of the degree of colocalization (Pearson's correlation coefficient) is given at the top of the enlarged pictures.


( A ) Epifluorescence microscopy of live containing a subgenomic replicon with a GFP - tagged NS5A . Huh7 - Lunet were transfected with replicon seeded onto carbon - patterned sapphire discs . Twenty - four hours later were analyzed by microscopy immediately processed for EM . ( a ) image ; ( b ) enlarged image of of interest ; ( c ) merge of bright field . Coordinates etched onto surface of sapphire disc were used to record position of selected . squares in a c enclose shown in b . ( B ) EM micrograph of boxed in panel Ab overlapped with image . Areas marked with a green dotted indicate regions of intense . Note that do not match perfectly because image corresponds to complete whereas EM image represents one 60 nm ultrathin section of same . ( C ) Higher magnification of two different regions , labeled 1 2 in panel B , corresponding to regions with intense ( 1 ) or a region corresponding to intersection of high to low ( 2 ) . Region 1 ( top panel ) corresponds to a DMV - containing residing in close proximity of ER ; region 2 ( bottom panel ) corresponds to a LD - enriched containing DMVs in very close proximity . Areas marked with squares in left are magnified in corresponding right panels . LD , lipid droplet ; ER , endoplasmic reticulum ; DMV , double vesicle ; m , mitochondrium ; if , intermediate filaments .

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

(A) Epifluorescence microscopy of live cells containing a subgenomic replicon with a GFP-tagged NS5A. Huh7-Lunet cells were transfected with replicon RNA and seeded onto carbon-patterned sapphire discs. Twenty-four hours later cells were analyzed by fluorescence microscopy and immediately processed for EM. (a) Fluorescence image; (b) enlarged fluorescence image of the cell of interest; (c) merge of bright field and fluorescence images. Coordinates etched onto the surface of the sapphire disc were used to record the position of the selected cells. White squares in a and c enclose the cell shown in b. (B) EM micrograph of the cell boxed in panel Ab overlapped with the fluorescence image. Areas marked with a green dotted line indicate regions of intense fluorescence. Note that the images do not match perfectly because the fluorescence image corresponds to the complete cell whereas the EM image represents one 60 nm ultrathin section of the same cell. (C) Higher magnification images of the two different regions, labeled 1 and 2 in panel B, corresponding to regions with intense fluorescence (1) or a region corresponding to the intersection of high to low fluorescence (2). Region 1 (top panel) corresponds to a DMV-containing area residing in close proximity of the ER; region 2 (bottom panel) corresponds to a LD-enriched area containing DMVs in very close proximity. Areas marked with white squares in the left images are magnified in the corresponding right panels. LD, lipid droplet; ER, endoplasmic reticulum; DMV, double membrane vesicle; m, mitochondrium; if, intermediate filaments.


( A ) of mock - infected high - pressure - frozen freeze - substituted Huh7 . 5 . ( B - E ) of high pressure frozen freeze - substituted Huh7 . 5 harvested 16 , 24 , 36 48 h after infection with Jc1 ( 100 TCID50 / ) . Yellow squares indicate areas that are shown at higher magnification on right of each subpanel . Note time - dependent increase of complexity of HCV - induced alterations . ER , endoplasmic reticulum ; m , mitochondria ; MVB , multi - vesicular bodies ; LD , lipid droplet ; DMV , double vesicle ; MMV , multi vesicle ; DMT , double tubule ( labeled with a black arrow ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Images of mock-infected high-pressure-frozen and freeze-substituted Huh7.5 cells. (B-E) Micrographs of high pressure frozen freeze-substituted Huh7.5 cells harvested 16, 24, 36 and 48 h after infection with Jc1 (100 TCID50/cell). Yellow squares indicate the areas that are shown at higher magnification on the right of each subpanel. Note the time-dependent increase of complexity of HCV-induced membrane alterations. ER, endoplasmic reticulum; m, mitochondria; MVB, multi-vesicular bodies; LD, lipid droplet; DMV, double membrane vesicle; MMV, multi membrane vesicle; DMT, double membrane tubule (labeled with a black arrow).


Immuno - EM localization of NS5A NS3 in HCV - infected . Huh7 . 5 were infected with 100 TCID50 / of Jc1 fixed 16 h post - infection . ( A ) shown in panels b , c e to g were post - fixed with 0 . 1 % OsO4 providing best preservation , especially in case of DMVs ( f g ) LDs ( e ) . Both NS5A NS3 localize predominantly to ER ( b d ) 50 - 70 nm diameter single - vesicles ( SMVs ; subpanel a , c d ) . NS5A also localizes to lipid droplets ( e ) .

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

Immuno-EM localization of NS5A and NS3 in HCV-infected cells.Huh7.5 cells were infected with 100 TCID50/cell of Jc1 and fixed 16 h post-infection. (A) Cells shown in panels b, c and e to g were post-fixed with 0.1% OsO4 providing best membrane preservation, especially in case of DMVs (f and g) and LDs (e). Both NS5A and NS3 localize predominantly to the ER (b and d) and 50-70 nm diameter single-membrane vesicles (SMVs; subpanel a, c and d). NS5A also localizes to lipid droplets (e).


( B ) Amount of gold particles per µm2 in Jc1 - infected versus mock - infected after immunolabeling with NS3 - NS5A - specific antibodies . Note higher immunolabeling with samples prepared without OsO4 , but also lower preservation under this condition .

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

(B) Amount of gold particles per µm2 in Jc1-infected versus mock-infected cells after immunolabeling with NS3- and NS5A-specific primary antibodies. Note the higher immunolabeling with samples prepared without OsO4, but also the lower membrane preservation under this condition.


( C ) Relative labeling distribution of NS3 NS5A . Thawed cryosections of post - fixed or not with OsO4 were labelled with NS3 - or NS5A - specific antibodies by using two different blocks 3 different labeling experiments . Per labeling experiment two grids were considered , counting ∼ 100 - 200 gold particles per grid attributing particles to indicated structures . In case of uninfected only background labeling in cytoplasm , on mitochondria undefined structures was seen . Numbers refer to of total gold particles counted per sample . ER , endoplasmic reticulum ; SV , small vesicles ; Cyto , cytosol ; Mito , mitochondria ; n . d . , non - defined structures ; NE , envelope ; EE / LE , early / late endosomes ; PM , plasma ; DMV , double vesicle ; LD , lipid droplet .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 labeling distribution of NS3 and NS5A. Thawed cryosections of cells post-fixed or not with OsO4 were labelled with NS3- or NS5A-specific antibodies by using two different blocks and 3 different labeling experiments. Per labeling experiment two grids were considered, counting ∼100-200 gold particles per grid and attributing the particles to the indicated structures. In the case of uninfected cells only background labeling in the cytoplasm, on mitochondria and undefined structures was seen. Numbers refer to the percent of total gold particles counted per sample. ER, endoplasmic reticulum; SV, small vesicles; Cyto, cytosol; Mito, mitochondria; n.d., non-defined structures; NE, nuclear envelope; EE/LE, early/late endosomes; PM, plasma membrane; DMV, double membrane vesicle; LD, lipid droplet.


Correlation between HCV replication appearance of double vesicles . ( A ) Time course of spread of HCV infection in Huh7 . 5 infected with 100 TCID50 / of Jc1 . Infected were detected by immunofluorescence using an NS5A - specific antiserum ( upper panels ) . graph shown below represents result of counting ∼ 200 for each time point to determine of infected . Scale bars represent 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 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

Correlation between HCV RNA replication and appearance of double membrane vesicles.(A) Time course of spread of HCV infection in Huh7.5 cells infected with 100 TCID50/cell of Jc1. Infected cells were detected by immunofluorescence using an NS5A-specific antiserum (upper panels). The graph shown below represents the result of counting ∼200 cells for each time point to determine the percentage of infected cells. Scale bars represent 50 µm.


( B ) Time course of accumulation of intracellular HCV in infected Huh7 . 5 . graph shows result of two independent experiments ( 3 replicas each ) . Whiskers indicate minimum maximum values .

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

(B) Time course of accumulation of intracellular HCV RNA in infected Huh7.5 cells. The graph shows the result of two independent experiments (3 replicas each). Whiskers indicate the minimum and maximum values.


( C ) Colocalization of dsRNA NS5A in infected with Jc1 ( 10 TCID50 / ) . were fixed at time points specified in left of each panel row NS5A dsRNA were detected by indirect immunofluorescence microscopy . was stained with DAPI ( blue ) . Boxed areas in left panels indicate areas that are shown as enlargements in corresponding right panels . of degree of colocalization ( Pearson ' s correlation coefficient ) is given in enlarged pictures . Scale bars represent 10 µm 2 µm ( left right panels , respectively ) .

O O O O O B-MEASURED_VAR 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 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

(C) Colocalization of dsRNA and NS5A in cells infected with Jc1 (10 TCID50/cell). Cells were fixed at time points specified in the left of each panel row and NS5A and dsRNA were detected by indirect immunofluorescence microscopy. DNA was stained with DAPI (blue). Boxed areas in the left panels indicate areas that are shown as enlargements in the corresponding right panels. The quantification of the degree of colocalization (Pearson's correlation coefficient) is given in the enlarged pictures. Scale bars represent 10 µm and 2 µm (left and right panels, respectively).


( D ) Time course of accumulation of DMVs MMVs . For each time point , 10 profiles were counted . Mann - Whitney ( non - parametric ) was applied to determine statistical significance . Error bars refer to standard deviation . Note striking correlation between increase of intracellular HCV DMV number between 16 24 hpi .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Time course of accumulation of DMVs and MMVs. For each time point, 10 cellular profiles were counted. The Mann-Whitney (non-parametric) test was applied to determine statistical significance. Error bars refer to the standard deviation. Note the striking correlation between the increase of intracellular HCV RNA and DMV number between 16 and 24 hpi.


( A ) Huh7 . 5 were infected with 100 TCID50 / of Jc1 , fixed 16 h later after HPF - FS processed for ET as described in materials methods . Left : slice of a dual axis tomogram showing various alterations . Right : 3D reconstruction of complete tomogram . Note high number of DMVs . Panels B , C D are part of tomogram displayed in panel A their position in panel A is highlighted by either a yellow dashed square ( B C ) or by a star ( D ) . In 3D models shown on right , ER is depicted in brown , inner of DMVs DMTs in yellowish brown their outer in semi - transparent brown . Single vesicles are colored in pink , intermediate filaments in blue Golgi apparatus in green . ( B ) Left : serial single slices through same tomogram shown in panel ( A ) displaying a connection between outer of a DMV ER ( black arrows ) . Right : 3D surface model showing connection . ( C ) Left : serial single slices through same tomogram illustrating a lasso - like structure of a DMV that after rendering reveals a pore - like opening that connects interior of DMV with cytosol . position of this opening in 2D slice is marked with a black arrow . Right : 3D view of this DMV showing ' pore ' . ( D ) Left : serial single slices through same tomogram showing a DMV with a large inter - space between its inner outer membranes . Right : 3D view of this DMV . Scale bars represent 100 nm . This tomogram is shown in movie S1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Huh7.5 cells were infected with 100 TCID50/cell of Jc1, fixed 16 h later and after HPF-FS processed for ET as described in materials and methods. Left: slice of a dual axis tomogram showing the various membrane alterations. Right: 3D reconstruction of the complete tomogram. Note the high number of DMVs. Panels B, C and D are part of the tomogram displayed in panel A and their position in panel A is highlighted by either a yellow dashed square (B and C) or by a star (D). In the 3D models shown on the right, the ER is depicted in dark brown, the inner membrane of DMVs and DMTs in yellowish brown and their outer membrane in semi-transparent light brown. Single membrane vesicles are colored in pink, intermediate filaments in dark blue and the Golgi apparatus in green. (B) Left: serial single slices through the same tomogram shown in panel (A) displaying a connection between the outer membrane of a DMV and the ER membrane (black arrows). Right: 3D surface model showing the membrane connection. (C) Left: serial single slices through the same tomogram illustrating a lasso-like structure of a DMV that after rendering reveals a pore-like opening that connects the interior of the DMV with the cytosol. The position of this opening in the 2D slice is marked with a black arrow. Right: 3D view of this DMV showing the 'pore'. (D) Left: serial single slices through the same tomogram showing a DMV with a large inter-membrane space between its inner and outer membranes. Right: 3D view of this DMV. Scale bars represent 100 nm. This tomogram is shown in movie S1.


( A ) Left : slice of a dual axis tomogram taken from Huh7 . 5 36 h after infection with 100 TCID50 / of Jc1 . Right : 3D reconstruction of tomogram . Note extensive reorganization appearance of MMVs predominating at late time points after infection . Black arrows show invaginations of DMVs . star indicates a large MMV ; due to its complexity only its middle part could be rendered . ( B ) Left : slices through same tomogram highlighting a DMT enwrapping a DMV presumably leading to formation of a MMV . Right : 3D surface rendering of this structure . ( C ) Left : slices through same tomogram highlighting a ' self - invagination ' event of a DMV , also leading to formation of a MMV . Right : 3D surface rendering of this late structure , revealing an opening of this MMV towards cytosol as a result of self - invagination . Panels B C are part of tomogram displayed in panel A their positions are highlighted by yellow dashed squares in panel A . Scale bars represent 100 nm . For further details see legend to Figure 6 . This tomogram is shown in movie S2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left: slice of a dual axis tomogram taken from Huh7.5 cells 36 h after infection with 100 TCID50/cell of Jc1. Right: 3D reconstruction of the tomogram. Note the extensive membrane reorganization and the appearance of MMVs predominating at late time points after infection. Black arrows show invaginations of DMVs. The white star indicates a large MMV; due to its complexity only its middle part could be rendered. (B) Left: slices through the same tomogram highlighting a DMT enwrapping a DMV and presumably leading to the formation of a MMV. Right: 3D surface rendering of this structure. (C) Left: slices through the same tomogram highlighting a 'self-invagination' event of a DMV, also leading to the formation of a MMV. Right: 3D surface rendering of this late structure, revealing an opening of this MMV towards the cytosol as a result of the self-invagination. Panels B and C are part of the tomogram displayed in panel A and their positions are highlighted by yellow dashed squares in panel A. Scale bars represent 100 nm. For further details see legend to Figure 6. This tomogram is shown in movie S2.


alterations induced by expression of HCV . Huh7 . 5 transfected with replicon RNAs ( A , B ) or Huh7 - Lunet - T7 transfected with pTM - expression constructs ( C - H ) specified in left of each panel , respectively , were high pressure frozen 24 h after transfection , freeze substituted , embedded into epon resin sections were analyzed by transmission EM . Representative showing HCV - induced alterations are shown .

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

Membrane alterations induced by expression of individual HCV proteins.Huh7.5 cells transfected with replicon RNAs (A, B) or Huh7-Lunet-T7 cells transfected with pTM-based expression constructs (C-H) specified in the left of each panel, respectively, were high pressure frozen 24 h after transfection, freeze substituted, embedded into epon resin and sections were analyzed by transmission EM. Representative images showing HCV-induced membrane alterations are shown.


( I ) Average diameter of specific vesicular structures , either DMVs or SMVs , detected in that had been transfected with constructs specified in upper panels . Whiskers represent minimum maximum values . ( J ) Number of vesicular structures detected in profiles of 10 transfected . transfected with pTM expression vector without HCV insert were used as reference . number of vesicular structures per µm2 is given ; whiskers represent minimum maximum values . ( K ) Relative abundance of membranous structures . Note that only upon expression of NS3 - 5A polyprotein NS5A two different structures were observed . In other cases , only one membranous structure was detected .

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

(I) Average diameter of specific vesicular structures, either DMVs or SMVs, detected in cells that had been transfected with constructs specified in the upper panels. Whiskers represent minimum and maximum values. (J) Number of vesicular structures detected in profiles of 10 transfected cells. Cells transfected with the pTM expression vector without HCV insert were used as reference. The number of vesicular structures per µm2 is given; whiskers represent minimum and maximum values. (K) Relative abundance of membranous structures. Note that only upon expression of the NS3-5A polyprotein and NS5A two different structures were observed. In all other cases, only one membranous structure was detected.


B . Coomassie - stained SDS - PAGE ( from left to right ) of Hchim , V1ΔH , VoND , C .

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

B. Coomassie-stained SDS-PAGE (from left to right) of Hchim, V1ΔH, VoND, and C.


C . Representative ATPase activity ( ATP regenerating system ) . At indicated time , 200 nM ConA is added to .

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

C. Representative ATPase activity assay (ATP regenerating system). At the indicated time, 200 nM ConA is added to the assay.


C . Growth of wild type c ″ - mutant yeast strains probed on ( 1 ) rich media buffered to pH 5 ( YPD pH 5 ) ; ( 2 ) synthetic dropout ( SD ) plus ( SD + a . a . ) ; ( 3 ) SD minus uracil ( SD - ura ) ; ( 4 , 5 ) YPD buffered to pH 7 plus 4 mM Zn2 + or 60 mM Ca2 + , respectively ; ( 6 ) YPD buffered to pH 7 ; ( 7 ) YPD plus 60 mM Ca2 + .

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

C. Growth phenotypes of wild type and c″-mutant yeast strains probed on (1) rich media buffered to pH 5 (YPD pH 5); (2) synthetic dropout (SD) plus all amino acids (SD + all a.a.); (3) SD minus uracil (SD - ura); (4,5) YPD buffered to pH 7 plus 4 mM Zn2+ or 60 mM Ca2+, respectively; (6) YPD buffered to pH 7; (7) YPD plus 60 mM Ca2+.


B . Mass spectrometry of V1Hchim . IDs are plotted as a function of overall peptide spectrum matches ( PSM ) posterior error probability ( PEP ) scores .

O O O O O 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. Mass spectrometry analysis of V1Hchim. Protein IDs are plotted as a function of overall peptide spectrum matches (PSM) and the posterior error probability (PEP) scores.


A . exclusion chromatography elution profile ( 1 . 6 cm × 50 cm Superdex S75 ) SDS - PAGE of full - N - terminally 7 × His tagged yeast Oxr1 . B . Inhibition of V1ΔH V1Hchim ' s MgATPase activity by Oxr1 in absence presence of subunit C . data from one representative of two experiments using two different preparations are shown . data points ( n = 2 ) mean from one representative of two biological replicates are shown with error bars representing S . E . M . . C . Oxr1 binds wild type V1 . Samples of V1 , Oxr1 , mixtures of V1 with a 2 - fold molar of Oxr1 in absence or presence of a 3 - fold molar of subunit C were subjected to exclusion chromatography on a 1 cm × 30 cm Superose 6 Increase column . SDS - PAGE is shown for four samples immunoblot of column fractions using a penta - His antibody is shown for samples containing Oxr1 Oxr1 plus C . Note that incubation of V1 with Oxr1 in presence of excess subunit C leads to partial release of subunit H ( highlighted by rectangular box in V1 + Oxr1 + C gel ) . Also , some degradation of Oxr1 ' s unstructured N - terminus is observed as indicated by asterisk ( only full - is detected by αHis - tag antibody ) . data from one representative of two experiments using two different preparations are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR 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 O O O O 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 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 B-CONTROLLED_VAR I-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 I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR 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 O O O O O O 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. Size exclusion chromatography elution profile (1.6 cm × 50 cm Superdex S75) and SDS-PAGE of full-length N-terminally 7×His tagged yeast Oxr1. B. Inhibition of V1ΔH and V1Hchim's MgATPase activity by Oxr1 in absence and presence of subunit C. The data from one representative of two experiments using two different preparations are shown. Individual data points (n=2) and mean from one representative of two biological replicates are shown with error bars representing S.E.M. . C. Oxr1 binds wild type V1. Samples of V1, Oxr1, and mixtures of V1 with a 2-fold molar ratio of Oxr1 in absence or presence of a 3-fold molar ratio of subunit C were subjected to size exclusion chromatography on a 1 cm × 30 cm Superose 6 Increase column. SDS-PAGE is shown for all four samples and immunoblot analysis of column fractions using a penta-His antibody is shown for samples containing Oxr1 and Oxr1 plus C. Note that incubation of V1 with Oxr1 in presence of excess subunit C leads to partial release of subunit H (highlighted by the rectangular box in the V1 + Oxr1 + C gel). Also, some degradation of Oxr1's unstructured N-terminus is observed as indicated by the asterisk (only the full-length protein is detected by the αHis-tag antibody). The data from one representative of two experiments using two different preparations are shown.


D . Dissociation kinetics of Hwt Hchim from V1ΔH in presence of Oxr1 subunit C as probed by BLI . Recombinant wild type ( left ) chimeric subunit H ( right ) with an N - terminal fusion to maltose binding ( MBP ) was immobilized on anti - mouse Fc ( AMC ) BLI sensors via an αMBP monoclonal antibody . sensors were then dipped into wells with V1ΔH followed by wells with buffer in absence or presence of Oxr1 C to monitor kinetics of V1ΔH dissociation . Only dissociation phase is shown . data from one representative of three experiments using two different preparations are shown .

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

D. Dissociation kinetics of Hwt and Hchim from V1ΔH in presence of Oxr1 and subunit C as probed by BLI. Recombinant wild type (left) and chimeric subunit H (right) with an N-terminal fusion to maltose binding protein (MBP) was immobilized on anti-mouse Fc (AMC) BLI sensors via an αMBP monoclonal antibody. The sensors were then dipped into wells with V1ΔH followed by wells with buffer in absence or presence of Oxr1 and C to monitor the kinetics of V1ΔH dissociation. Only the dissociation phase is shown. The data from one representative of three experiments using two different preparations are shown.


E . Oxr1 mediated inhibition of assembly of holo V - ATPase from V1Hchim , subunit C VoND . data from one representative of two experiments using two different preparations are shown . data points ( n = 2 ) mean from one representative of two biological replicates are shown with error bars representing S . E . M . .

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

E. Oxr1 mediated inhibition of assembly of holo V-ATPase from V1Hchim, subunit C and VoND. The data from one representative of two experiments using two different preparations are shown. Individual data points (n=2) and mean from one representative of two biological replicates are shown with error bars representing S.E.M. .


F . Oxr1 prevents binding of V1Hchim to VoND as probed by BLI . Streptavidin coated BLI sensors were loaded with Vo in lipid nanodiscs containing biotinylated MSP , sensors were then dipped into wells containing recombinant C V1Hchim in absence presence of Oxr1 . Only V1Hchim association dissociation phase from one representative of two experiments using two different preparations is shown .

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 B-MEASURED_VAR I-MEASURED_VAR 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

F. Oxr1 prevents binding of V1Hchim to VoND as probed by BLI. Streptavidin coated BLI sensors were loaded with Vo in lipid nanodiscs containing biotinylated MSP, and sensors were then dipped into wells containing recombinant C and V1Hchim in absence and presence of Oxr1. Only the V1Hchim association and dissociation phase from one representative of two experiments using two different preparations is shown.


G . Oxr1 mediated dissociation of holo V - ATPase as analyzed by negative stain electron microscopy . V1HchimVoND was incubated without with a 3 - fold molar of Oxr1 for 16 h . Samples were then spotted on carbon coated copper grids , stained with 1 % uranyl acetate observed by transmission electron microscopy . Number of assembled V1Vo disassembled V1 Vo particles were counted on a total of 20 each . horizontal dotted lines indicate mean . A few holoenzyme free V1 VoND particles are highlighted by circles boxes , respectively . Bar = 50 nm . data from one representative of two experiments using two different preparations are shown .

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-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

G. Oxr1 mediated dissociation of holo V-ATPase as analyzed by negative stain electron microscopy. V1HchimVoND was incubated without and with a 3-fold molar ratio of Oxr1 for 16 h. Samples were then spotted on carbon coated copper grids, stained with 1% uranyl acetate and observed by transmission electron microscopy. Number of assembled V1Vo and disassembled V1 and Vo particles were counted on a total of 20 micrographs each. The horizontal dotted lines indicate the mean. A few holoenzyme and free V1 and VoND particles are highlighted by white circles and boxes, respectively. Bar = 50 nm. The data from one representative of two experiments using two different preparations are shown.


A . Immunostaining of TUJ1 , DESMIN GATA6 after six days of mESC differentiation towards ectoderm , mesoderm endoderm . Bar : 50 μm .

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

A. Immunostaining of TUJ1, DESMIN and GATA6 after six days of mESC differentiation towards ectoderm, mesoderm and endoderm. Bar: 50 μm.


C . Venn diagram of genes with differential expression during mESC differentiation towards three germ layers .

C. Venn diagram of genes with differential expression during mESC differentiation towards the three germ layers.


D - F . Hierarchical clustering of mRNA expression of mESCs differentiated towards ectoderm ( E ) , mesoderm ( F ) endoderm ( G ) .

O O O 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-F. Hierarchical clustering of mRNA expression of mESCs differentiated towards ectoderm (E), mesoderm (F) and endoderm (G).


A . Hierarchical clustering of gene expression profiles of mESCs differentiated towards three germ layers differentiated mouse tissues .

A. Hierarchical clustering of gene expression profiles of mESCs differentiated towards the three germ layers and differentiated mouse tissues.


C . Hierarchical clustering of mRNA expression during differentiation of mESCs towards endoderm ( yellow ) or EpiSCs ( green ) .

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

C. Hierarchical clustering of mRNA expression during differentiation of mESCs towards endoderm (yellow) or EpiSCs (green).


B . Ectoderm differentiation of 3KI mESC ( marked by expression of Sox1 : : GFP ) . C . Mesendoderm differentiation of 3KI mESC ( marked by expression of T : : 3xTagBFP ) .

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. Ectoderm differentiation of the 3KI mESC line (marked by the expression of Sox1::GFP). C. Mesendoderm differentiation of the 3KI mESC line (marked by the expression of T::3xTagBFP).


D . Endoderm differentiation of 3KI mESC ( marked by expression of Eomes : : mCherry ) .

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

D. Endoderm differentiation of the 3KI mESC line (marked by the expression of Eomes::mCherry).


E , F . Spontaneous differentiation of 3KI mESC leading to coexistence of belonging to three germ layers . Bar : 100 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

E, F. Spontaneous differentiation of the 3KI mESC line leading to the coexistence of cells belonging to the three germ layers. Bar: 100 μm.


B . Differentiation of Atf1 - / - , Fos - / - Jun - / - , Foxj2 - / - , Meis3 - / - , Nr5a2 - / - , Sp1 + / - Zfp354c - / - mESCs in 3KI background to ectoderm . C . Differentiation of Atf1 - / - , Fos - / - Jun - / - , Foxj2 - / - , Meis3 - / - , Nr5a2 - / - , Sp1 + / - Zfp354c - / - mESCs in 3KI background to mesendoderm . D . Differentiation of Atf1 - / - , Fos - / - Jun - / - , Foxj2 - / - , Meis3 - / - , Nr5a2 - / - , Sp1 + / - Zff354c - / - mESCs in 3KI background to definitive endoderm .

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

B. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zfp354c-/- mESCs in the 3KI background to ectoderm. C. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zfp354c-/- mESCs in the 3KI background to mesendoderm. D. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zff354c-/- mESCs in the 3KI background to definitive endoderm.


F . Differentiation of wild type ( WT ) , Sp1 + / - , Nr5a2 - / - Fos - / - Jun - / - mESCs in 3KI background to definitive endoderm .

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

F. Differentiation of wild type (WT), Sp1+/-, Nr5a2-/- and Fos-/-Jun-/- mESCs in the 3KI background to definitive endoderm.


( B ) microscopy of LGCs , FBGCs osteoclasts stained with Giemsa ( upper panel ) . Representative Hoechst dye acquired by ImageStream ( lower panel ) of sorted showing mononuclear ( mono ) multinucleated ( multi ) populations for each type .

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

(B) Light microscopy images of LGCs, FBGCs and osteoclasts stained with Giemsa staining (upper panel). Representative Hoechst dye images acquired by ImageStream (lower panel) of sorted cells showing mononuclear (mono) and multinucleated (multi) populations for each cell type.


( C ) - seq volcano plots highlighting top 15 differentially expressed genes between mononuclear multinucleated populations for each type .

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

(C) RNA-seq volcano plots highlighting the top 15 differentially expressed genes between mononuclear and multinucleated populations for each cell type.


( C ) MRC1 surface marker expression ( red ) acquired by ImageStream in mononuclear ( mono ) multinucleated ( multi ) LGCs , FBGCs osteoclasts stained for Hoechst . Bar graphs ( lower panel ) represent normalized MRC1 mean ( MFI ) , measured by ImageStream ; n = 4 donors .

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

(C) MRC1 surface marker expression (red) acquired by ImageStream in mononuclear (mono) and multinucleated (multi) LGCs, FBGCs and osteoclasts stained for Hoechst. Bar graphs (lower panel) represent normalized MRC1 mean fluorescence intensity (MFI), measured by ImageStream; n=4 donors.


( E ) CSF1R , MRC1 , TLR2 FOS relative expression measured by qRT - PCR for LGCs ( upper ) , FBGCs ( middle ) osteoclasts ( lower ) , following fusion regulator DC - STAMP knockdown . si - Ctrl , scrambled siRNA ; si - DC - STAMP , DC - STAMP siRNA ; n = 7 donors .

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

(E) CSF1R, MRC1, TLR2 and FOS relative expression measured by qRT-PCR for LGCs (upper), FBGCs (middle) and osteoclasts (lower), following the cell membrane fusion regulator DC-STAMP knockdown. si-Ctrl, scrambled siRNA; si-DC-STAMP, DC-STAMP siRNA; n=7 donors.


( E ) effect of lysosomotropic agents ( hydroxychloroquine , hydroxy ; ammonium chloride , NH4Cl ) v - ATPase inhibitors ( Bafilomycin A1 , Baf A1 ; Concanamycin A , Con A ) on fusion multinucleation in LGCs ( upper panel ) , FBGCs ( middle panel ) osteoclasts ( lower panel ) . To effect of lysosome dysfunction on iron , FeCl3 was supplemented . Error bars are median with interquartile range ; significance tested by one - way Anova followed by Sídák ' s multiple comparisons on log transformed data ; n = 4 donors ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 ; * * * * , P < 0 . 0001 .

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

(E) The effect of lysosomotropic agents (hydroxychloroquine, hydroxy; ammonium chloride, NH4Cl) and v-ATPase inhibitors (Bafilomycin A1, Baf A1; Concanamycin A, Con A) on fusion and multinucleation in LGCs (upper panel), FBGCs (middle panel) and osteoclasts (lower panel). To test the effect of lysosome dysfunction on cellular iron, FeCl3 was supplemented. Error bars are median with interquartile range; significance tested by one-way Anova followed by Sídák's multiple comparisons on log transformed data; n=4 donors; *, P < 0.05; **, P < 0.01; *** P < 0.001; ****, P < 0.0001.


( E ) B7 - H3 surface marker expression ( red ) acquired by ImageStream in mononuclear multinucleated LGCs , FBGCs osteoclasts stained for Hoechst . Bar graphs ( lower panel ) show normalized B7 - H3 mean ( MFI ) , measured by ImageStream ; n = 4 donors . Error bars are mean ± SD ; significance tested by paired t - ; * * , P < 0 . 01 ; ns , non - significant .

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

(E) B7-H3 surface marker expression (red) acquired by ImageStream in mononuclear and multinucleated LGCs, FBGCs and osteoclasts stained for Hoechst. Bar graphs (lower panel) show normalized B7-H3 mean fluorescence intensity (MFI), measured by ImageStream; n=4 donors. Error bars are mean ± SD; significance tested by paired t-test; **, P < 0.01; ns, non-significant.


( C ) Phagocytosis quantified by ImageStream in LGCs , FBGCs osteoclasts . Representative showing uptake of S . Aureus bioparticles ( yellow ) in mononuclear ( mono ) multinucleated ( multi ) . Bar graphs ( lower panel ) show normalized S . Aureus bioparticles mean ( MFI ) measured by ImageStream ; n = 5 donors . Error bars are mean ± SD ; significance tested by paired t - ; * * , P < 0 . 01 ; ns , non - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Phagocytosis quantified by ImageStream analysis in LGCs, FBGCs and osteoclasts. Representative images showing the uptake of S. Aureus bioparticles (yellow) in mononuclear (mono) and multinucleated (multi) cells. Bar graphs (lower panel) show normalized S. Aureus bioparticles mean fluorescence intensity (MFI) measured by ImageStream; n=5 donors. Error bars are mean ± SD; significance tested by paired t-test; **, P < 0.01; ns, non-significant.


( C ) TRAP immunohistochemistry ( upper panel ) hydroxyapatite resorption ( lower panel ) in LGCs , FBGCs osteoclasts . Note presence of TRAP + MGCs in types hydroxyapatite resorption only in osteoclasts .

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

(C) TRAP immunohistochemistry (upper panel) and hydroxyapatite resorption (lower panel) in LGCs, FBGCs and osteoclasts. Note the presence of TRAP+ MGCs in all cell types and hydroxyapatite resorption only in osteoclasts.


( D ) Oxygen consumption rate ( OCR , upper panel ) , its related readout ( middle panel ) extracellular acidification rate ( ECAR , lower panel ) measured by extracellular flux in sorted mononuclear multinucleated osteoclasts ; n = 4 donors . ( E ) Extracellular acidification rate ( ECAR , left ) glycolysis ( right ) in mononuclear multinucleated osteoclasts ; n = 4 donors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Oxygen consumption rate (OCR, upper panel), its related readout (middle panel) and extracellular acidification rate (ECAR, lower panel) measured by extracellular flux analysis in sorted mononuclear and multinucleated osteoclasts; n=4 donors. (E) Extracellular acidification rate (ECAR, left) and glycolysis (right) in mononuclear and multinucleated osteoclasts; n=4 donors.


( F ) Mitochondria copy number measured by qPCR in mononuclear multinucleated osteoclasts ; n = 3 donors .

(F) Mitochondria copy number measured by qPCR in mononuclear and multinucleated osteoclasts; n=3 donors.


( G ) Relative expression of mitochondrial transcripts in sorted mononuclear multinucleated osteoclasts after normalisation by mitochondrial copy number ; n = 6 donors .

O 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) Relative expression of mitochondrial transcripts in sorted mononuclear and multinucleated osteoclasts after normalisation by mitochondrial copy number; n=6 donors.


A Western blot of GFUS . Expression of GFUS was analyzed by Western blotting on a 15 % SDS PAGE with fractions derived from control fibroblasts . revealed a significant decrease of GFUS . Data were obtained from fibroblasts ; n = 4 ; experiment was independently repeated 4 times , for statistics an unpaired t was performed . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Bars error bars represent mean ± SD .

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 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

A Western blot analysis and quantification of GFUS. Expression of GFUS was analyzed by Western blotting on a 15% SDS PAGE with cytosolic fractions derived from control and patient fibroblasts. Quantification revealed a significant decrease of GFUS protein. Data were obtained from fibroblasts; n = 4; experiment was independently repeated 4 times, for statistics an unpaired t test was performed. Data information: *p < 0.05; **p < 0.01; ***p<0.001. Bars and error bars represent mean ± SD.


B mRNA expression studies . qRT - PCR studies showed a significant decrease in mRNA level of GFUS by 40 . 0 % ( ± 9 . 3 % , * * p = 0 . 0010 ) normalized to a control . Also , expression of SLC35C1 SLC35C2 were significantly decreased in - derived fibroblasts . Data were obtained from fibroblasts ; n = 10 ; experiment was independently repeated 3 times , for statistics an one - way ANOVA was performed . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Bars error bars represent mean ± SD .

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

B mRNA expression studies. qRT-PCR studies showed a significant decrease in mRNA level of GFUS by 40.0% (± 9.3%, **p = 0.0010) normalized to a control. Also, expression of SLC35C1 and SLC35C2 were significantly decreased in patient-derived fibroblasts. Data were obtained from fibroblasts; n = 10; experiment was independently repeated 3 times, for statistics an one-way ANOVA was performed. Data information: *p < 0.05; **p < 0.01; ***p<0.001. Bars and error bars represent mean ± SD.


C Conversion of GDP - D - mannose into GDP - L - fucose . conversion GDP - D - [ 14C ] mannose to GDP - L - [ 14C ] fucose was analyzed in an in - vitro . After 1 h nearly half of GDP - D - mannose was transformed to GDP - fucose in control , whereas ´ s fibroblasts were nearly unable to catalyze reaction . Data were obtained from fibroblasts ; n = 1 .

C Conversion of GDP-D-mannose into GDP-L-fucose. The conversion GDP-D-[14C]mannose to GDP-L-[14C]fucose was analyzed in an in-vitro assay. After 1 h nearly half of GDP-D-mannose was transformed to GDP-fucose in the control cell line, whereas the patient´s fibroblasts were nearly unable to catalyze the reaction. Data were obtained from fibroblasts; n = 1.


D Lectin binding studies in fibroblasts . Aleuria aurantia lectin ( AAL ) was used to address fucosylation level in fibroblasts . AAL was performed before after treatment of with 100 µM L - fucose . Before sugar supplementation a significant loss of AAL signal strength was measured , whereas after treatment a significant re - fucosylation was detected . Data were obtained from fibroblasts ; n = 4 ; experiment was independently repeated 4 times , for statistics an unpaired t was performed . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Bars error bars represent mean ± SD .

O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Lectin binding studies and quantification in fibroblasts. Aleuria aurantia lectin (AAL) was used to address the fucosylation level in fibroblasts. AAL staining was performed before and after treatment of the patient cell line with 100 µM L-fucose. Before sugar supplementation a significant loss of AAL signal strength was measured, whereas after treatment a significant re-fucosylation was detected. Data were obtained from fibroblasts; n = 4; experiment was independently repeated 4 times, for statistics an unpaired t test was performed. Data information: *p < 0.05; **p < 0.01; ***p<0.001. Bars and error bars represent mean ± SD.


E Complementation study with - derived fibroblasts . Viral infection was used to introduce an empty cloning vector wild - type GFUS cDNA in , respectively . For lysates of a control - , a control - supplemented with 100 µM fucose , , supplemented with 100 µM fucose , transfected with an empty vector transfected with wild type GFUS were analyzed by AAL clearly indicating disease - causing influence of defective GFUS in . Western blot against GFUS further showed expression of wildtype - GFUS in infected derived fibroblasts . Data were obtained from fibroblasts ; n = 4 ; experiment was independently repeated 2 times , for statistics an one - way ANOVA was performed . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Bars error bars represent mean ± SD .

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 B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Complementation study with patient-derived fibroblasts. Viral infection was used to introduce an empty cloning vector and the wild-type GFUS cDNA in patient cells, respectively. For analysis lysates of a control cell-line, a control cell-line supplemented with 100 µM fucose, patient cells, patient cells supplemented with 100 µM fucose, patient cells transfected with an empty vector and patient cells transfected with wild type GFUS were analyzed by AAL staining clearly indicating the disease-causing influence of the defective GFUS in the patient. Western blot analysis against GFUS further showed expression of wildtype-GFUS protein in the infected patient derived fibroblasts. Data were obtained from fibroblasts; n = 4; experiment was independently repeated 2 times, for statistics an one-way ANOVA was performed. Data information: *p < 0.05; **p < 0.01; ***p<0.001. Bars and error bars represent mean ± SD.


A Aleuria aurantia lectin ( AAL ) was used to address fucosylation level in sera of controls ( left ) which revealed significant loss of fucose residues in case of . Data were obtained from serum ; n = 6 ; experiment was independently repeated 3 times , for statistics an unpaired t was performed . Data information : * * p < 0 . 01 . Bars error bars represent mean ± SD . Exact p ‑ values are reported in results part .

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 O O O O O O O O O O O O O O O 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 Aleuria aurantia lectin (AAL) was used to address the fucosylation level in sera of controls and the patient (left) which revealed significant loss of fucose residues in case of the patient. Data were obtained from serum; n = 6; experiment was independently repeated 3 times, for statistics an unpaired t test was performed. Data information: **p < 0.01. Bars and error bars represent mean ± SD. Exact p‑values are reported in the results part.


B FACS of PBMCs . Results of flow cytometry of ( red ) control ( green ) PBMCs stained with 1 µg / ml FAA lectin without ( ) or with 200 mM L - fucose ( ) before start of fucose therapy . C FACS of PBMCs . Results of flow cytometry of ( red ) control ( green ) PBMCs stained with 1 µg / ml FAA lectin without ( ) or with 200 mM L - fucose ( ) 8 after therapy start

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

B FACS analysis of PBMCs. Results of flow cytometry analysis of patient (red) and control (green) PBMCs stained with 1 µg/ml FAA lectin without (dark) or with 200 mM L-fucose (light) before the start of fucose therapy. C FACS analysis of PBMCs. Results of flow cytometry analysis of patient (red) and control (green) PBMCs stained with 1 µg/ml FAA lectin without (dark) or with 200 mM L-fucose (light) 8 weeks after therapy start


C . SDS - PAGE showing a representative fraction of purified spMis18 N - terminal Yippee - like globular domain ( acid residues 1 - 120 ; spMis181 - 120 ) .

O O O O O O O O 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. SDS-PAGE showing a representative fraction of the purified spMis18 N-terminal Yippee-like globular domain (amino acid residues 1-120; spMis181-120).


D . Cartoon representation of crystal structure of spMis181 - 120 in two different orientations , highlighting key residues within putative substrate - binding pocket .

O O 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. Cartoon representation of the crystal structure of spMis181-120 in two different orientations, highlighting key residues within the putative substrate-binding pocket.


E . Substrate - binding pocket mutations Y90A Y74A / Y90A / T105A / S107K affect ability of ectopically expressed spMis18fl to rescue sensitivity of mis18 - 262 to varying degrees . Five - fold serial dilutions of mis18 - 262 transformed with plasmids harbouring indicated spMis18fl constructs , spotted on PMG - uracil + Phloxine B media supplemented with ( repressed ) or without ( expressed ) thiamine , incubated at indicated temperatures ; dead stain pink .

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

E. Substrate-binding pocket mutations Y90A and Y74A/Y90A/T105A/S107K affect the ability of ectopically expressed spMis18fl to rescue the temperature sensitivity of mis18-262 cells to varying degrees. Five-fold serial dilutions of mis18-262 cells transformed with plasmids harbouring the indicated spMis18fl constructs, spotted on PMG - uracil + Phloxine B media supplemented with (repressed) or without (expressed) thiamine, and incubated at the indicated temperatures; dead cells stain dark pink.


A . Cartoon representation of assembly of spMis18 Yippee - like domains ( spMis181 - 120 ) as observed in crystal asymmetric unit . Close up views highlight acid composition interactions stabilizing dimeric arrangements . Interface - II is composed of conserved residues an extensive binding surface as compared to Interface - I ( see text for details ) , suggesting Dimer - II to be a physiologically relevant dimer . Residues mutated in this study are highlighted with 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 O O O O O O O

A. Cartoon representation of the assembly of spMis18 Yippee-like domains (spMis181-120) as observed in the crystal asymmetric unit. Close up views highlight the amino acid composition and interactions stabilizing the dimeric arrangements. Interface-II is composed of conserved residues and an extensive binding surface as compared to Interface-I (see text for details), suggesting Dimer-II to be a physiologically relevant dimer. Residues mutated in this study are highlighted with circles.


B . Exclusion Chromatography ( SEC ) SEC - MALS ( SEC combined with Multi Angle scattering ) analyses of recombinant wild - type ( wt ) dimer disrupting mutant ( I31A ) of spMis181 - 120 . Measured molecular weights from SEC - MALS confirm that spMis181 - 120 wt is a dimer in solution spMis181 - 120 I31A is a monomer .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Size Exclusion Chromatography (SEC) and SEC-MALS (SEC combined with Multi Angle Light scattering) analyses of the recombinant wild-type (wt) and dimer disrupting mutant (I31A) of spMis181-120. Measured molecular weights from SEC-MALS confirm that spMis181-120 wt is a dimer in solution and spMis181-120 I31A is a monomer.


C . Evaluation of ability of Yippee - like domains of human Mis18 ( hsMis18α77 - 187 hsMis18β56 - 183 ) to form a heterodimer . SEC SEC - MALS analyses demonstrate that while hsMis18α77 - 187 elutes at 15 . 5 ml as a dimer ( 31 . 3 kDa ) , hsMis18β56 - 183 elutes at 17 . 3 ml as a monomer ( 15 kDa ) . Purified hsMis18α77 - 187 hsMis18β56 - 183 when mixed together elute at 15 . 4 ml as a heterodimer ( 27 . 5 kDa ) .

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

C. Evaluation of the ability of Yippee-like domains of human Mis18 proteins (hsMis18α77-187 and hsMis18β56-183) to form a heterodimer. SEC and SEC-MALS analyses demonstrate that while hsMis18α77-187 elutes at 15.5 ml as a dimer (31.3 kDa), hsMis18β56-183 elutes at 17.3 ml as a monomer (15 kDa). Purified hsMis18α77-187 and hsMis18β56-183 when mixed together elute at 15.4 ml as a heterodimer (27.5 kDa).


D . Cartoon representation of structure of homology - modelled human Mis18α77 - 187 - Mis18β56 - 183 heterodimer using crystal structure of spMis181 - 120 reported here as a template . Residues mutated in this study are highlighted with circles . Modelling was carried out using Phyre2 web server ( www . sbg . bio . ic . ac . uk / phyre2 / ) .

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 O O O O 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. Cartoon representation of the structure of the homology-modelled human Mis18α77-187 - Mis18β56-183 heterodimer using the crystal structure of spMis181-120 reported here as a template. Residues mutated in this study are highlighted with circles. Modelling was carried out using Phyre2 web server (www.sbg.bio.ic.ac.uk/phyre2/).


E . SDS - PAGE of GST - pull down where wt dimer disrupting mutants of hsMis18α77 - 187 hsMis18β56 - 183 were co - expressed as His - GST - tagged , respectively in E . coli . While wt GST - hsMis18β56 - 183 showed interaction with wt His - hsMis18α77 - 187 , hsMis18 harbouring dimer - disrupting mutations , GST - hsMis18β56 - 183 V77E / Y172D His - hsMis18α77 - 187 V82E / Y176D did not show noticeable interaction . corresponding Ni - NTA pull downs showing expression of His - tagged are shown in Fig EV2D .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. SDS-PAGE analysis of the GST-pull down assay where wt and dimer disrupting mutants of hsMis18α77-187 and hsMis18β56-183 were co-expressed as His- and GST-tagged proteins, respectively in E. coli. While wt GST-hsMis18β56-183 showed interaction with wt His-hsMis18α77-187, hsMis18 proteins harbouring dimer-disrupting mutations, GST-hsMis18β56-183 V77E/Y172D and His-hsMis18α77-187 V82E/Y176D did not show noticeable interaction. The corresponding Ni-NTA pull downs showing the expression of His-tagged proteins are shown in Fig EV2D.


A B . SEC - MALS of His - GFP - spMis18fl His - spMis18ΔC , respectively . Refractive index ( RI , left Y - axis ) molecular mass ( kDa , right Y - axis ) profiles show that both His - GFP - spMis18fl ( predicted MW of a monomer is 51 . 97 kDa ) His - spMis18ΔC ( Predicted MW of a monomer 21 . 8 kDa ) predominantly exist as tetramers ( 187 . 6 kDa 78 . 6 kDa , respectively ) with a small population of dimer ( 128 . 5 kDa 45 . 8 kDa , respectively ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. SEC-MALS analysis of His-GFP-spMis18fl and His-spMis18ΔC, respectively. Refractive index (RI, left Y-axis) and molecular mass (kDa, right Y-axis) profiles show that both His-GFP-spMis18fl (predicted MW of a monomer is 51.97 kDa) and His-spMis18ΔC (Predicted MW of a monomer 21.8 kDa) predominantly exist as tetramers (187.6 kDa and 78.6 kDa, respectively) with a small population of dimer (128.5 kDa and 45.8 kDa, respectively).