( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

(B) 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 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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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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 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 .

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(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 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 ) .

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(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 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 .

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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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(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 , and C .

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


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

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


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 + .

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

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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 . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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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 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 . .

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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 , 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 .

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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 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 .

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

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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 the 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 ) and endoderm ( G ) .

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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 the three germ layers and differentiated mouse tissues .

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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 ) .

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


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 ) .

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

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


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

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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 + / - 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 .

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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 - / - and Fos - / - Jun - / - mESCs in the 3KI background to definitive endoderm .

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


( 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 .

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

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(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 ) 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 .

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(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 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 .

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(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 ) 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 .

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(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 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 .

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(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 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 .

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(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 ) 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 .

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(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 ) 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 .

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(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 and 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 and multinucleated osteoclasts after normalisation by mitochondrial copy number ; n = 6 donors .

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


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 .

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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 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 .

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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 . 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 .

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 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 .

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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 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 .

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

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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 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

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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 the purified spMis18 N - terminal Yippee - like globular domain ( amino acid residues 1 - 120 ; spMis181 - 120 ) .

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

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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 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 .

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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 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 .

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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 . 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 .

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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 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 ) .

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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 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 / ) .

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 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 .

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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 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 ) .

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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).


C . SEC profile of a sample containing an equimolar mixture of purified His - GFP - spMis18C - term - α and His - spMis18MeDiY ( upper panel ) and SDS - PAGE analysis of SEC - fractions ( bottom panel ) . His - GFP - spMis18C - term - α and His - spMis18MeDiY eluted separately at 12 . 3 and 15 . 3 ml , respectively , demonstrating the inability of these domains to interact with each other .

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C. SEC profile of a sample containing an equimolar mixture of purified His-GFP-spMis18C-term-α and His-spMis18MeDiY (upper panel) and SDS-PAGE analysis of SEC-fractions (bottom panel). His-GFP-spMis18C-term-α and His-spMis18MeDiY eluted separately at 12.3 and 15.3 ml, respectively, demonstrating the inability of these domains to interact with each other.


D . SEC - MALS analysis of His - GFP - spMis18C - term - α shows that it exists as a homotrimer with a measured molecular weight of 119 . 8 kDa . ( Theoretically calculated MW of a monomer is 38 . 3 kDa ) .

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D. SEC-MALS analysis of His-GFP-spMis18C-term-α shows that it exists as a homotrimer with a measured molecular weight of 119.8 kDa. (Theoretically calculated MW of a monomer is 38.3 kDa).


A . spMis18fl - Myc co - immunoprecipitates with both GFP - tagged spMis18fl and spMis18MeDiY , but shows reduced association when MeDiY - mediated dimerization is disrupted in spMis18flI31A . The asterisk ( * ) in the top panel denotes the IgG heavy chain .

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A. spMis18fl-Myc co-immunoprecipitates with both GFP-tagged spMis18fl and spMis18MeDiY, but shows reduced association when MeDiY-mediated dimerization is disrupted in spMis18flI31A. The asterisk (*) in the top panel denotes the IgG heavy chain.


B C . Dimer - II interface mutations I31A and Y114A affect the ability of ectopically expressed spMis18fl to rescue the temperature sensitivity of mis18 - 818 and mis18 - 262 cells , while expression of spMis18MeDiY alone confers a dominant negative effect on growth in a MeDiY dimerization - dependent manner . Five - fold serial dilutions of cells expressing the indicated spMis18 constructs integrated at the leu1 locus in the genome , spotted on complete PMG + Phloxine B media supplemented with ( repressed ) or without ( expressed ) thiamine , and incubated at the indicated temperatures ; dead cells stain dark pink .

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B C. Dimer-II interface mutations I31A and Y114A affect the ability of ectopically expressed spMis18fl to rescue the temperature sensitivity of mis18-818 and mis18-262 cells, while expression of spMis18MeDiY alone confers a dominant negative effect on growth in a MeDiY dimerization-dependent manner. Five-fold serial dilutions of cells expressing the indicated spMis18 constructs integrated at the leu1 locus in the genome, spotted on complete PMG + Phloxine B media supplemented with (repressed) or without (expressed) thiamine, and incubated at the indicated temperatures; dead cells stain dark pink.


D . Mutations that disrupt MeDiY dimerization lead to reduced levels of spMis18fl association with centromeres . qChIP analyses of spMis18fl - GFP association with centromere 2 ( cc2 ) in the indicated strains when grown in complete PMG media supplemented with ( repressed ) or without ( expressed ) thiamine . Error bars represent standard deviation between at least three biological replicates .

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D. Mutations that disrupt MeDiY dimerization lead to reduced levels of spMis18fl association with centromeres. qChIP analyses of spMis18fl-GFP association with centromere 2 (cc2) in the indicated strains when grown in complete PMG media supplemented with (repressed) or without (expressed) thiamine. Error bars represent standard deviation between at least three biological replicates.


A - B HT ‑ 29 cells were transfected with non - silencing control siRNA ( sictr ) or siRNAs against USP22 ( siUSP22 ) for 48 h at 20 nM . After transfection , cells were treated with 20 µM zVAD . fmk , 0 . 5 µM BV6 and 1 ng / ml TNFα either for 6 h and analyzed by Western blotting ( A ) or for 18 h , and the percentage of PI - positive cells was assessed by fluorescence - based PI staining ( B ) . Vinculin served as a loading control .

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A-B HT‑29 cells were transfected with non-silencing control siRNA (sictr) or siRNAs against USP22 (siUSP22) for 48 h at 20 nM. After transfection, cells were treated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα either for 6 h and analyzed by Western blotting (A) or for 18 h, and the percentage of PI-positive cells was assessed by fluorescence-based PI staining (B). Vinculin served as a loading control.


C HT ‑ 29 CRISPR / Cas9 control ( ctr ) and USP22 KO cells were analyzed by Western blotting for USP22 expression . GAPDH served as loading control .

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C HT‑29 CRISPR/Cas9 control (ctr) and USP22 KO cells were analyzed by Western blotting for USP22 expression. GAPDH served as loading control.


D HT ‑ 29 control and USP22 KO cells were treated with 20 µM zVAD . fmk , 0 . 5 µM BV6 and 1 ng / ml TNFα after pre - incubation with 30 µM Nec1 - s and 20 µM GSK ' 872 for 1 h and incubated for 18 h before fluorescence - based quantification of PI - positive cells .

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D HT‑29 control and USP22 KO cells were treated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα after pre-incubation with 30 µM Nec1-s and 20 µM GSK'872 for 1 h and incubated for 18 h before fluorescence-based quantification of PI-positive cells.


E HT ‑ 29 control and USP22 KO cells , generated with 2 guide RNAs ( 2g ) , expressing empty vector ( EV ) or PAM mutated 3xFLAG - HA - USP22 WT ( USP22 PAM ) , C185S ( USP22 PAM C185S ) or C185A ( USP22 PAM C185A ) were analyzed by Western blotting for USP22 expression levels . β - Actin was used as a loading control .

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E HT‑29 control and USP22 KO cells, generated with 2 guide RNAs (2g), expressing empty vector (EV) or PAM mutated 3xFLAG-HA-USP22 WT (USP22 PAM), C185S (USP22 PAM C185S) or C185A (USP22 PAM C185A) were analyzed by Western blotting for USP22 expression levels. β-Actin was used as a loading control.


F HT - 29 control and USP22 KO cells , generated with 2 guide RNAs ( 2g ) , expressing empty vector ( EV ) or PAM mutated 3xFLAG - HA - USP22 WT ( USP22 PAM ) , C185S ( USP22 PAM C185S ) or C185A ( USP22 PAM C185A ) were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 , 1 ng / ml TNFα for 18 h . The percentage of PI - positive cells was assessed by fluorescence - based PI staining .

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F HT-29 control and USP22 KO cells, generated with 2 guide RNAs (2g), expressing empty vector (EV) or PAM mutated 3xFLAG-HA-USP22 WT (USP22 PAM), C185S (USP22 PAM C185S) or C185A (USP22 PAM C185A) were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, 1 ng/ml TNFα for 18 h. The percentage of PI-positive cells was assessed by fluorescence-based PI staining.


A HT ‑ 29 control and USP22 KO cells were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 and 1 ng / ml TNFα for the indicated time points . Detection of indicated proteins was carried out by Western blotting . GAPDH served as a loading control .

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A HT‑29 control and USP22 KO cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for the indicated time points. Detection of indicated proteins was carried out by Western blotting. GAPDH served as a loading control.


B HT ‑ 29 control and USP22 KO cells were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 and 1 ng / ml TNFα for 4 h . Detection of indicated proteins was carried out by Western blotting . β - Actin served as a loading control .

B HT‑29 control and USP22 KO cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 4 h. Detection of indicated proteins was carried out by Western blotting. β-Actin served as a loading control.


C HT ‑ 29 control and USP22 KO cells were incubated with 30 µM Nec1 - s or 20 µM Dab for 18 h , as indicated . Cell were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 and 1 ng / ml TNFα for 5 h . 100 μg of each lysate were incubated with 400 U / μl λ - phosphatase for 30 min at 30 ° C . Protein expression of RIPK3 was monitored by Western blotting . β - Actin was used as loading control . High molecular weight RIPK3 ' smears ' were quantified after λ - phosphatase treatment and normalized to total RIPK3 and β - Actin levels .

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C HT‑29 control and USP22 KO cells were incubated with 30 µM Nec1-s or 20 µM Dab for 18 h, as indicated. Cell were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 5 h. 100 μg of each lysate were incubated with 400 U/μl λ-phosphatase for 30 min at 30 °C. Protein expression of RIPK3 was monitored by Western blotting. β-Actin was used as loading control. High molecular weight RIPK3 'smears' were quantified after λ-phosphatase treatment and normalized to total RIPK3 and β-Actin levels.


A HeLa TRex RIPK3 CRISPR / Cas9 control ( ctr ) and USP22 KO cells were treated with 1 µg / ml Dox overnight . Protein expression of induced Strep - RIPK3 was analyzed by Western blotting . GAPDH served as loading control . The asterisk marks an unspecific band .

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A HeLa TRex RIPK3 CRISPR/Cas9 control (ctr) and USP22 KO cells were treated with 1 µg/ml Dox overnight. Protein expression of induced Strep-RIPK3 was analyzed by Western blotting. GAPDH served as loading control. The asterisk marks an unspecific band.


B HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg / ml Dox for 18 h before pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα was added and cell death was measured after 4 and 5 h by analysis of PI - positive nuclei .

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B HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg/ml Dox for 18 h before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα was added and cell death was measured after 4 and 5 h by analysis of PI-positive nuclei.


C HeLa TRex RIPK3 control and USP22 KO cells were pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 1 , 2 , 3 , 4 and 5 h . Protein expression of phosphorylated RIPK1 , total RIPK1 , total RIPK3 , phosphorylated MLKL , total MLKL and USP22 , without ( left ) or with ( right ) 1 µg / ml Dox treatment overnight , was monitored by Western blotting . GAPDH was used as a loading control .

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C HeLa TRex RIPK3 control and USP22 KO cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1, 2, 3, 4 and 5 h. Protein expression of phosphorylated RIPK1, total RIPK1, total RIPK3, phosphorylated MLKL, total MLKL and USP22, without (left) or with (right) 1 µg/ml Dox treatment overnight, was monitored by Western blotting. GAPDH was used as a loading control.


D HT - 29 control cells and RIPK3 KO cells re - expressing PAM - mutated Dox - inducible RIPK3 WT were incubated overnight with 1 µg / ml Dox . Cells were pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 2 h , as indicated . Strep - RIPK3 was immunoprecipitated using anti ‑ Strep - beads and the indicated co - immunoprecipitated proteins were analyzed by Western blotting . β - Actin served as a loading control .

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D HT-29 control cells and RIPK3 KO cells re-expressing PAM-mutated Dox-inducible RIPK3 WT were incubated overnight with 1 µg/ml Dox. Cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h, as indicated. Strep-RIPK3 was immunoprecipitated using anti‑Strep-beads and the indicated co-immunoprecipitated proteins were analyzed by Western blotting. β-Actin served as a loading control.


E USP22 KO HT - 29 cells and USP22 KO cells re - expressing PAM - mutated 3xFLAG - HA - USP22 were pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα was added for 2 h , as indicated . 3xFLAG - HA - USP22 was immunoprecipitated using anti - HA - beads and the indicated co - immunoprecipitated proteins were analyzed by Western blotting . β - Actin served as a loading control .

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E USP22 KO HT-29 cells and USP22 KO cells re-expressing PAM-mutated 3xFLAG-HA-USP22 were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα was added for 2 h, as indicated. 3xFLAG-HA-USP22 was immunoprecipitated using anti-HA-beads and the indicated co-immunoprecipitated proteins were analyzed by Western blotting. β-Actin served as a loading control.


A HT - 29 control , USP22 KO and USP22 KO cells re - expressing PAM - mutated 3xFLAG - HA - USP22 WT or C185S were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 and 1 ng / ml TNFα for 4 h . Poly - ubiquitinated proteins were enriched by GST - TUBE pull - down , followed by incubation with the catalytic domain of USP2 , as indicated . RIPK3 and USP22 expression and levels of ubiquitinated RIPK3 were monitored using Western blotting with the indicated antibodies . β - Actin served as loading control . Ponceau - staining was used to confirm equal loading of GST - TUBE .

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A HT-29 control, USP22 KO and USP22 KO cells re-expressing PAM-mutated 3xFLAG-HA-USP22 WT or C185S were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 4 h. Poly-ubiquitinated proteins were enriched by GST-TUBE pull-down, followed by incubation with the catalytic domain of USP2, as indicated. RIPK3 and USP22 expression and levels of ubiquitinated RIPK3 were monitored using Western blotting with the indicated antibodies. β-Actin served as loading control. Ponceau-staining was used to confirm equal loading of GST-TUBE.


B HT - 29 control and USP22 KO cells were transfected with His - ubiquitin for 24 h , as indicated . Cells were pre ‑ stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 for 1 h . Following pre - treatment , 1 ng / ml TNFα was added for 4 h . His - ubiquitin was immunoprecipitated using Ni - NTA beads and detection of indicated proteins was performed by Western blotting . β - Actin served as loading control for the input , whereas His - ubiquitin levels served as loading control for immunoprecipitated ubiquitin .

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

B HT-29 control and USP22 KO cells were transfected with His-ubiquitin for 24 h, as indicated. Cells were pre‑stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 for 1 h. Following pre-treatment, 1 ng/ml TNFα was added for 4 h. His-ubiquitin was immunoprecipitated using Ni-NTA beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas His-ubiquitin levels served as loading control for immunoprecipitated ubiquitin.


C HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg / ml Dox and transfected with HA - ubiquitin for 24 h , as indicated . Cells were pre - stimulated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . Following pre - treatment , 10 ng / ml TNFα were added for 3 h . HA - ubiquitin was immunoprecipitated using anti - HA - beads and detection of indicated proteins was performed by Western blotting . β - Actin served as loading control for the input , whereas HA - levels served as loading control for immunoprecipitated ubiquitin .

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C HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg/ml Dox and transfected with HA-ubiquitin for 24 h, as indicated. Cells were pre-stimulated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. Following pre-treatment, 10 ng/ml TNFα were added for 3 h. HA-ubiquitin was immunoprecipitated using anti-HA-beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas HA-levels served as loading control for immunoprecipitated ubiquitin.


B Bar graphs demonstrating the number of up ‐ , non ‐ , and downregulated ubiquitination sites in USP22 KO HT - 29 cells versus control under TBZ - treatment .

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B Bar graphs demonstrating the number of up‐, non‐, and downregulated ubiquitination sites in USP22 KO HT-29 cells versus control under TBZ-treatment.


D HeLa cells expressing Dox - inducible RIPK3 WT , D160N , K42R , K351 , K518R , 2xKR or 3xKR were pre ‑ incubated with 1 µg / ml Dox , 20 µM Dab , or 10 µM NSA overnight , followed by pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 4 h . Cell death was measured by quantification of PI - positive nuclei .

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D HeLa cells expressing Dox-inducible RIPK3 WT, D160N, K42R, K351, K518R, 2xKR or 3xKR were pre‑incubated with 1 µg/ml Dox, 20 µM Dab, or 10 µM NSA overnight, followed by pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 4 h. Cell death was measured by quantification of PI-positive nuclei.


E HeLa cells expressing Dox - inducible RIPK3 mutants were treated with 1 µg / ml Dox overnight . Protein levels of inducible RIPK3 expression were analyzed by Western blotting . GAPDH served as loading control .

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E HeLa cells expressing Dox-inducible RIPK3 mutants were treated with 1 µg/ml Dox overnight. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. GAPDH served as loading control.


A HeLa cells expressing Dox - inducible RIPK3 WT , K518R or 3xKR were treated with 1 µg / ml Dox and / or 10 µM NSA and 20 µM Dab overnight . Protein levels of inducible RIPK3 expression and phosphorylated MLKL were examined by Western blotting . β - Actin was used as a loading control .

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

A HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were treated with 1 µg/ml Dox and/or 10 µM NSA and 20 µM Dab overnight. Protein levels of inducible RIPK3 expression and phosphorylated MLKL were examined by Western blotting. β-Actin was used as a loading control.


B HeLa cells expressing Dox - inducible RIPK3 WT or 3xKR were treated with 1 µg / ml Dox overnight before pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 1 , 2 , 3 and 4 h . Protein levels of inducible RIPK3 expression and phosphorylated MLKL were analyzed by Western blotting . β - Actin served as loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells expressing Dox-inducible RIPK3 WT or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1, 2, 3 and 4 h. Protein levels of inducible RIPK3 expression and phosphorylated MLKL were analyzed by Western blotting. β-Actin served as loading control.


C HeLa cells expressing Dox - inducible RIPK3 WT , 2xKR or 3xKR were treated with 1 µg / ml Dox overnight before pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 2 h . Protein levels of inducible RIPK3 expression were analyzed by Western blotting . β - Actin served as loading control . D HeLa cells expressing Dox - inducible RIPK3 WT , K518R or 3xKR were treated with 1 µg / ml Dox overnight before pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 2 h . Whole cell lysates were generated using RIPA lysis buffer containing 2 % SDS . Protein levels of inducible RIPK3 expression were analyzed by Western blotting . β - Actin served as loading control .

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

C HeLa cells expressing Dox-inducible RIPK3 WT, 2xKR or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. β-Actin served as loading control. D HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. Whole cell lysates were generated using RIPA lysis buffer containing 2% SDS. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. β-Actin served as loading control.


E HeLa cells expressing Dox - inducible RIPK3 WT , D160N , K518R or 3xKR were incubated overnight with 1 µg / ml Dox and pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 1 and 2 h . Strep - RIPK3 was immunoprecipitated by using anti - Strep - beads . Co - immunoprecipitated phosphorylated MLKL and RIPK1 , as well as protein expression of indicated proteins were analyzed by Western blotting . β - Actin served as a loading control .

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E HeLa cells expressing Dox-inducible RIPK3 WT, D160N, K518R or 3xKR were incubated overnight with 1 µg/ml Dox and pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1 and 2 h. Strep-RIPK3 was immunoprecipitated by using anti-Strep-beads. Co-immunoprecipitated phosphorylated MLKL and RIPK1, as well as protein expression of indicated proteins were analyzed by Western blotting. β-Actin served as a loading control.


F HeLa cells expressing Dox - inducible RIPK3 WT , K518R or 3xKR were incubated with 1 µg / ml Dox and transfected with HA - ubiquitin for 24 h , as indicated . Cells were pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 2 h . HA - ubiquitin was immunoprecipitated using anti - HA - beads and detection of indicated proteins was performed by Western blotting . β - Actin served as loading control for the input , whereas HA - ubiquitin levels served as loading control for immunoprecipitated ubiquitin .

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F HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were incubated with 1 µg/ml Dox and transfected with HA-ubiquitin for 24 h, as indicated. Cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. HA-ubiquitin was immunoprecipitated using anti-HA-beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas HA-ubiquitin levels served as loading control for immunoprecipitated ubiquitin.


A HT ‑ 29 RIPK3 KO cells re - expressing Dox - inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg / ml Dox overnight . Cells were treated with 20 µM zVAD . fmk , 5 µM BV6 and 10 ng / ml TNFα for 4 h . Cell death was measured by analysis of PI - positive nuclei .

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A HT‑29 RIPK3 KO cells re-expressing Dox-inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg/ml Dox overnight. Cells were treated with 20 µM zVAD.fmk, 5 µM BV6 and 10 ng/ml TNFα for 4 h. Cell death was measured by analysis of PI-positive nuclei.


B HT ‑ 29 RIPK3 KO cells re - expressing Dox - inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg / ml Dox overnight , as indicated . Cells were treated with 20 µM zVAD . fmk , 5 µM BV6 and 10 ng / ml TNFα for 2 h and analyzed by Western blotting for RIPK3 and phosphorylated MLKL expression levels . β - Actin served as a loading control .

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B HT‑29 RIPK3 KO cells re-expressing Dox-inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg/ml Dox overnight, as indicated. Cells were treated with 20 µM zVAD.fmk, 5 µM BV6 and 10 ng/ml TNFα for 2 h and analyzed by Western blotting for RIPK3 and phosphorylated MLKL expression levels. β-Actin served as a loading control.


The presence of GFP - PSF - 1 on mitotic chromatin ( indicated by white arrows ) was monitored by spinning disk confocal microscopy ( see Materials and Methods ) , in GFP - psf - 1 mCherry - Histone H2B worms that were fed on bacteria containing a single plasmid expressing the indicated RNAi treatments ( ' Control ' = empty vector ) . NEB = nuclear envelope breakdown . The scalebars correspond to 5 µm .

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The presence of GFP-PSF-1 on mitotic chromatin (indicated by white arrows) was monitored by spinning disk confocal microscopy (see Materials and Methods), in GFP-psf-1 mCherry-Histone H2B worms that were fed on bacteria containing a single plasmid expressing the indicated RNAi treatments ('Control' = empty vector). NEB = nuclear envelope breakdown. The scalebars correspond to 5 µm.


Ubiquitylation of CMG - MCM - 7 was reconstituted with the indicated factors as described in Materials and Methods . ' Neddylation ' indicates addition of the C . elegans ULA - 1 _ RFL - 1 E1 enzyme , the UBC - 12 E2 enzyme , the DCN - 1 E3 enzyme and NED - 8 .

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

Ubiquitylation of CMG-MCM-7 was reconstituted with the indicated factors as described in Materials and Methods. 'Neddylation' indicates addition of the C. elegans ULA-1_RFL-1 E1 enzyme, the UBC-12 E2 enzyme, the DCN-1 E3 enzyme and NED-8.


Reactions containing CMG and either CUL - 2LRR - 1 or CUL - 2VHL - 1 were performed in the presence or absence of other replisome factors as indicated . CMG was then isolated by immunoprecipitation of SLD - 5 and the indicated factors were monitored by immunoblotting .

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Reactions containing CMG and either CUL-2LRR-1 or CUL-2VHL-1 were performed in the presence or absence of other replisome factors as indicated. CMG was then isolated by immunoprecipitation of SLD-5 and the indicated factors were monitored by immunoblotting.


Reconstituted CMG - MCM - 7 ubiquitylation reactions were performed as above in the presence of the indicated factors . ' Neddylation ' indicates addition of the C . elegans ULA - 1 _ RFL - 1 E1 enzyme , the UBC - 12 E2 enzyme , the DCN - 1 E3 enzyme and NED - 8 .

Reconstituted CMG-MCM-7 ubiquitylation reactions were performed as above in the presence of the indicated factors. 'Neddylation' indicates addition of the C. elegans ULA-1_RFL-1 E1 enzyme, the UBC-12 E2 enzyme, the DCN-1 E3 enzyme and NED-8.


CMG was isolated by immunoprecipitation of SLD - 5 , after ubiquitylation in the presence of TIM - 1 _ TIPN - 1 , before incubation with the indicated factors . ' Neddylation ' indicates addition of the C . elegans ULA - 1 _ RFL - 1 E1 enzyme , the UBC - 12 E2 enzyme , the DCN - 1 E3 enzyme and NED - 8 . CMG disassembly was monitored by release of CDC - 45 or MCM - 2 - 7 proteins into the supernatant , whilst SLD - 5 and other GINS subunits remained on the beads . Asterisks indicate non - specific bands in the anti - MCM - 7 and CDC - 45 immunoblots .

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CMG was isolated by immunoprecipitation of SLD-5, after ubiquitylation in the presence of TIM-1_TIPN-1, before incubation with the indicated factors. 'Neddylation' indicates addition of the C. elegans ULA-1_RFL-1 E1 enzyme, the UBC-12 E2 enzyme, the DCN-1 E3 enzyme and NED-8. CMG disassembly was monitored by release of CDC-45 or MCM-2-7 proteins into the supernatant, whilst SLD-5 and other GINS subunits remained on the beads. Asterisks indicate non-specific bands in the anti-MCM-7 and CDC-45 immunoblots.


TAP - psf - 1 worms were fed on bacteria containing a single plasmid expressing the indicated RNAi treatments , before preparation of embryonic cell extracts and isolation of TAP - PSF - 1 by immunoprecipitation . The bound material was released from beads by cleavage of the TAP tag with TEV protease , before detection of the indicated factors by immunoblotting .

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TAP-psf-1 worms were fed on bacteria containing a single plasmid expressing the indicated RNAi treatments, before preparation of embryonic cell extracts and isolation of TAP-PSF-1 by immunoprecipitation. The bound material was released from beads by cleavage of the TAP tag with TEV protease, before detection of the indicated factors by immunoblotting.


Similar experiment comparing the impact of RNAi inactivation of lrr - 1 , tim - 1 and tipn - 1 upon CMG - MCM - 7 ubiquitylation and the association of CUL - 2LRR - 1 with the worm replisome . Asterisks indicate non - specific bands in the immunoblots .

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

Similar experiment comparing the impact of RNAi inactivation of lrr-1, tim-1 and tipn-1 upon CMG-MCM-7 ubiquitylation and the association of CUL-2LRR-1 with the worm replisome. Asterisks indicate non-specific bands in the immunoblots.


Embryonic viability was monitored as described in Materials and Methods , after feeding worms on the indicated proportions of bacteria expressing RNAi ( ' % RNAi ' ) to the denoted genes , mixed with bacteria containing empty vector ( to make a total of 100 % ) .

Embryonic viability was monitored as described in Materials and Methods, after feeding worms on the indicated proportions of bacteria expressing RNAi ('% RNAi') to the denoted genes, mixed with bacteria containing empty vector (to make a total of 100%).


The presence of GFP - PSF - 1 on mitotic chromatin ( indicated by white arrows ) was monitored by spinning disk confocal microscopy ( see Materials and Methods ) , in embryos derived from worms that were fed on bacteria containing a single plasmid expressing the indicated RNAi ( ' Control ' = empty vector ) . The scalebars correspond to 5 µm .

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

The presence of GFP-PSF-1 on mitotic chromatin (indicated by white arrows) was monitored by spinning disk confocal microscopy (see Materials and Methods), in embryos derived from worms that were fed on bacteria containing a single plasmid expressing the indicated RNAi ('Control' = empty vector). The scalebars correspond to 5 µm.