A RIP experiments were performed using anti - myc antibodies extracts from Trt1 - myc expressing strains followed by qRT - PCR to detect G - rich TERRA , polyA + TERRA , ARRET , TER1 ACT1 . Values represent fractions of input detected in immunoprecipitated material expressed as fold increase over untagged ( unt ) wt . Bars error bars are averages SD from three independent experiments . * P < 0 . 05 ( relative to unt ; two - Student ' s t ) .

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 B-MEASURED_VAR O O 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 O O O O O O O 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 RIP experiments were performed using anti-myc antibodies and extracts from Trt1-myc expressing strains followed by qRT-PCR to detect G-rich TERRA, polyA+ TERRA, ARRET, TER1 and ACT1. Values represent fractions of input RNA detected in immunoprecipitated material expressed as fold increase over untagged (unt) wt strain. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to unt; two-tailed Student's t test).


B RIP experiments as in ( A ) where , prior to extraction , washed beads were resuspended in DNaseI buffer ( DNbuff ) incubated for 1 hour at 30 ° C in presence or absence of DNaseI . Values represent fractions of input polyA + TERRA detected in immunoprecipitated material expressed as fold increase over an untagged ( unt ) wt . Bars error bars are averages SD from three independent experiments . * P < 0 . 05 ( relative to unt ; two - Student ' s t ) .

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

B RIP experiments as in (A) where, prior to RNA extraction, washed beads were resuspended in DNaseI buffer (DNbuff) and incubated for 1 hour at 30° C in presence or absence of DNaseI. Values represent fractions of input polyA+ TERRA detected in immunoprecipitated material expressed as fold increase over an untagged (unt) wt strain. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to unt; two-tailed Student's t test).


C RIP experiments were performed using anti - myc antibodies extracts from Trt1 - myc expressing strains ( either ter1 + or ter1Δ ) cultured in YES medium for ~ 43 pds . Values represent polyA + TERRA detected in immunoprecipitated material expressed as a fraction of input ( graph on left ) or after normalization to ACT1 in corresponding input ( on right ) , expressed as fold increase over an untagged wt ( unt ter1 + ) . Bars error bars are averages SD from three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( relative to unt ter1 + ; two - Student ' s t ) .

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

C RIP experiments were performed using anti-myc antibodies and extracts from Trt1-myc expressing strains (either ter1+ or ter1Δ) cultured in YES medium for ~ 43 pds. Values represent polyA+ TERRA detected in immunoprecipitated material expressed as a fraction of the input (graph on the left) or after normalization to ACT1 RNA in the corresponding input (on the right), and expressed as fold increase over an untagged wt strain (unt ter1+). Bars and error bars are averages and SD from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (relative to unt ter1+; two-tailed Student's t test).


D Western blot of Trt1 - myc in used in ( C ) . Ponceau S serves as a loading control .

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

D Western blot analysis of Trt1-myc in cells used in (C). Ponceau S staining serves as a loading control.


B Genomic from wt ( CAF13 ) carrying two tiTELs ( CAF110 ) maintained in YES medium was digested with HindIII Southern blot - hybridized consecutively with indicated probes . RET refers to telomeric sequences ( tiTELs or natural telomeres , nTELs ) retained in upper part of gel ; REL refers to telomeric sequences released in lower part of gel . black arrow points to natural nmt1 + on chromosome III . Marker molecular weights are on left in kilobases .

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

B Genomic DNA from wt cells (CAF13) and cells carrying two tiTELs (CAF110) maintained in YES medium was digested with HindIII and Southern blot-hybridized consecutively with the indicated probes. RET refers to telomeric sequences (tiTELs or natural telomeres, nTELs) retained in the upper part of the gel; REL refers to telomeric sequences released in the lower part of the gel. The black arrow points to the natural nmt1+ sequence on chromosome III. Marker molecular weights are on the left in kilobases.


D qRT - PCR of tiTERRA in carrying no tiTEL ( CAF13 ) , one tiTEL ( CAF545 ) or two tiTELs ( CAF110 ) cultured in EMM medium in presence or absence of Thiamine ( THI ; repressed induced condition , respectively ) for 24 h . was reverse transcribed with oC cDNA was amplified with oF3 oR3 oligonucleotides to detect only tiTERRA ( graph on left ) or with oF1 oR1 oligonucleotides to detect simultaneously tiTERRA natural TERRA ( on right ) . Values are normalized to ACT1 mRNA expressed as fold increase over CAF545 in uninduced conditions ( THI + ) . Bars error bars are averages SD from 3 independent experiments . * P < 0 . 05 , * * P < 0 . 01 ( relative to CAF545 THI + ; two - Student ' s t ) .

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

D qRT-PCR analysis of tiTERRA in cells carrying no tiTEL (CAF13), one tiTEL (CAF545) or two tiTELs (CAF110) cultured in EMM medium in presence or absence of Thiamine (THI; repressed and induced condition, respectively) for 24 h. RNA was reverse transcribed with oC and cDNA was amplified with oF3 and oR3 oligonucleotides to detect only tiTERRA (graph on the left) or with oF1 and oR1 oligonucleotides to detect simultaneously tiTERRA and natural TERRA (on the right). Values are normalized to ACT1 mRNA and expressed as fold increase over CAF545 in uninduced conditions (THI+). Bars and error bars are averages and SD from 3 independent experiments. * P < 0.05, ** P < 0.01 (relative to CAF545 THI+; two-tailed Student's t test).


E Northern blot of total from wt carrying two tiTELs ( CAF110 ) treated with combinations of THI Trichostatin A ( TSA ) for 24 h . Two identical membranes were hybridized in parallel with oligonucleotides corresponding to C - rich ( oC ) or G - rich ( oG ) telomeric repeats . To confirm similar hybridization efficiencies of two oligonucleotides , northern blot membranes were simultaneously hybridized exposed along with blots of digested genomic ( upper right insets ) . After signal detection , RNAmembranes were stripped re - hybridized with U6 probes to assure equal loading . Marker molecular weights are on left in kilobases .

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E Northern blot analysis of total RNA from wt cells and cells carrying two tiTELs (CAF110) treated with combinations of THI and Trichostatin A (TSA) for 24 h. Two identical membranes were hybridized in parallel with oligonucleotides corresponding to C-rich (oC) or G-rich (oG) telomeric repeats. To confirm similar hybridization efficiencies of the two oligonucleotides, northern blot membranes were simultaneously hybridized and exposed along with blots of digested genomic DNA (upper right insets). After signal detection, RNAmembranes were stripped and re-hybridized with U6 probes to assure equal loading. Marker molecular weights are on the left in kilobases.


A Telomere restriction fragment of genomic from wt carrying two tiTELs ( CAF110 ) grown for 24 h in EMM medium containing THI TSA as indicated . was digested with HindIII hybridized first with a nmt1 probe successively with a telomeric probe . Marker molecular weights are on left in kilobases . Numbers at bottom indicate gel lanes .

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A Telomere restriction fragment analysis of genomic DNA from wt cells and cells carrying two tiTELs (CAF110) grown for 24 h in EMM medium containing THI and TSA as indicated. DNA was digested with HindIII and hybridized first with a nmt1 probe and successively with a telomeric probe. Marker molecular weights are on the left in kilobases. Numbers at the bottom indicate gel lanes.


B Genomic from carrying two tiTELs deleted for trt1 + ( CAF113 ) was analyzed as in ( A ) . Marker molecular weights are on left in kilobases . Numbers at bottom indicate gel lanes .

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

B Genomic DNA from cells carrying two tiTELs and deleted for trt1+ (CAF113) was analyzed as in (A). Marker molecular weights are on the left in kilobases. Numbers at the bottom indicate gel lanes.


C ter1 + carrying two tiTELs ( CAF110 , left panel ) or trt1Δ carrying one tiTEL ( MKSP2104 , central panel ) were cultured in EMM medium in presence of THI for 24 h ( THI + ) or in absence of THI for 24 , 48 or 72 h . In right panel , carrying two tiTELs ( CAF110 ) were treated with TSA for 24 h in presence or absence of THI . TiTEL telomeric sequences were amplified from genomic sequenced using a PacBio platform . statistical for each set of conditions is displayed above each panel . P values for each comparison were derived from Student ' s t between each condition THI + sample using Welch ' s correction when appropriate . new population of longer tiTELs appearing in trt1Δ upon prolonged transcription induction is indicated in red ( center panel ) .

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

C ter1+ cells carrying two tiTELs (CAF110, left panel) or trt1Δ cells carrying one tiTEL (MKSP2104, central panel) were cultured in EMM medium in presence of THI for 24 h (THI+) or in absence of THI for 24, 48 or 72 h. In the right panel, cells carrying two tiTELs (CAF110) were treated with TSA for 24 h in the presence or absence of THI. TiTEL telomeric sequences were amplified from genomic DNA and sequenced using a PacBio platform. The statistical analysis for each set of conditions is displayed above each panel. P values for each comparison were derived from the Student's t test between each condition and the THI+ sample using Welch's correction when appropriate. The new population of longer tiTELs appearing in trt1Δ cells upon prolonged transcription induction is indicated in red (center panel).


A qRT - PCR of G - rich polyA + tiTERRA in carrying one tiTEL expressing Trt1 - myc ( CAF610 ) cultured in EMM medium with indicated combinations of THI TSA for 24 h . Values are normalized to ACT1 mRNA expressed as fold increase over THI + TSA - samples . Bars error bars are averages SD from 4 independent experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ( relative to THI + TSA - ; two - Student ' s t ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 qRT-PCR analysis of G-rich and polyA+ tiTERRA in cells carrying one tiTEL and expressing Trt1-myc (CAF610) cultured in EMM medium with the indicated combinations of THI and TSA for 24 h. Values are normalized to ACT1 mRNA and expressed as fold increase over THI+ TSA- samples. Bars and error bars are averages and SD from 4 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, (relative to THI+ TSA-; two-tailed Student's t test).


B Telo - PCR of tiTELs using genomic extracted from as in ( A ) . Marker ( m ) molecular weights are on right in base pairs .

O O O O O 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 Telo-PCR analysis of tiTELs using genomic DNA extracted from cells as in (A). Marker (m) molecular weights are on the right in base pairs.


C RIP experiments performed using anti - myc antibodies extracts from as in ( A ) followed by qRT - PCR of G - rich polyA + tiTERRA . Values correspond to fraction of input expressed as fold increase over an untagged ( unt ) control tiTEL ( CAF545 ) . Bars error bars are averages SD from three independent experiments . * P < 0 . 05 , * * P < 0 . 01 ( relative to unt ; two - Student ' s t ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RIP experiments performed using anti-myc antibodies and extracts from cells as in (A) followed by qRT-PCR analysis of G-rich and polyA+ tiTERRA. Values correspond to fraction of input RNA expressed as fold increase over an untagged (unt) control tiTEL strain (CAF545). Bars and error bars are averages and SD from three independent experiments. * P < 0.05, ** P < 0.01 (relative to unt; two-tailed Student's t test).


D ChIP experiments performed using anti - myc antibodies extracts from as in ( A ) followed by qPCR using tiTEL specific oligonucleotides . Values correspond to fraction of input expressed as fold increase over THI + TSA - . Unt : untagged ( CAF545 ) . Bars error bars are averages SD from at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 ( relative to THI + TSA - ; two - Student ' s t ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ChIP experiments performed using anti-myc antibodies and extracts from cells as in (A) followed by qPCR using tiTEL specific oligonucleotides. Values correspond to fraction of input DNA expressed as fold increase over THI+ TSA-. Unt: untagged strain (CAF545). Bars and error bars are averages and SD from at least three independent experiments. * P < 0.05, ** P < 0.01 (relative to THI+ TSA-; two-tailed Student's t test).


A ) MALDI imaging was performed on - containing brain sections at a lateral resolution of 100µm ( M - H ) ions were analysed in negative mode with a FTICR Mass Spectrometer ( analytical range 100 - 1000Da ) . Hematoxylin - eosin ( HE ) of same sections was performed after MALDI . - derived glioma xenografts ( PDXs ) with IDH wildtype ( IDHwt ) , IDH mutant ( IDHm ) , control brain tissue ( CB ) were analyzed in a non - targeted targeted approach using regions of interest ( ROI ) of different sizes to compare ( T , yellow ) control brain ( CB , blue ) . non - targeted approach was used for statistical pair - wise comparisons between samples , while targeted approach on a selected set of metabolites allowed distribution analyses .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) MALDI imaging was performed on tumor-containing brain sections at a lateral resolution of 100µm and (M-H) ions were analysed in negative mode with a FTICR Mass Spectrometer (analytical range 100-1000Da). Hematoxylin-eosin (HE) staining of the same sections was performed after MALDI analysis. Patient-derived glioma xenografts (PDXs) with IDH wildtype (IDHwt), IDH mutant (IDHm), and control brain tissue (CB) were analyzed in a non-targeted and targeted approach using regions of interest (ROI) of different sizes to compare tumor (T, yellow line) and contralateral control brain (CB, blue line). The non-targeted approach was used for statistical pair-wise comparisons between samples, while the targeted approach on a selected set of metabolites allowed distribution and quantification analyses.


B ) Histological sections of 3 IDHwt PDXs ( P3 , T434 , P8 ) 3 IDHm PDXs ( ( T394 , E478 , T186 ) showing a large ROI ( boxed rectangle , > 500 pixels ) within applied for targeted of selected metabolites . Left rectangle in P3 PDX was used as control brain ( CB ) for quantifications . Middle panel shows tissue distribution of D - 2 - hydroxyglutarate ( D2HG ) by MSI , which is exclusively detected in IDHm PDXs ( D2HG in IDHwt tumours is below detection limit ) . Right panel shows tissue distribution of a key metabolite ( m / z 778 . 51 ) presenting strong accumulation in IDHm lower grade gliomas ( LGG ) versus glioblastomas ( GBM ) independent of IDH status . An - dependent color code indicates relative amount of a specific compound ( defined by m / z value ) throughout tissue section .

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

B) Histological sections of 3 IDHwt PDXs (P3, T434, P8) and 3 IDHm PDXs ((T394, E478, T186) showing a large ROI (boxed rectangle, >500 pixels) within the tumor area applied for targeted quantification of selected metabolites. Left rectangle in P3 PDX was used as control contralateral brain (CB) for quantifications. Middle panel shows tissue distribution of D-2-hydroxyglutarate (D2HG) by MSI, which is exclusively detected in IDHm PDXs (D2HG in IDHwt tumours is below detection limit). Right panel shows tissue distribution of a key metabolite (m/z 778.51) presenting strong accumulation in IDHm lower grade gliomas (LGG) versus glioblastomas (GBM) independent of IDH status. An intensity-dependent color code indicates the relative amount of a specific compound (defined by m/z value) throughout the tissue section.


C ) of several high mass metabolites differentially present in IDHm PDXs ( IDHwt glioblastomas ( GBM ) in blue , IDHm GBM in orange , IDHm lower grade gliomas ( LGG ) in red , control brain ( CB ) in grey ) . Box plots represent log values of metabolite intensities measured within a large ROI ( > 500 pixels ) . Many metabolites in this mass range ( m / z 700 - 900 ) have been putatively assigned to phospholipids ( Tables EV2 - 3 ) . m / z represents mass over charge of ionized metabolites , as measured by mass spectrometer . Except where otherwise indicated , statistical significance was calculated for IDHm ( n = 3 ) versus IDHwt ( n = 3 ) PDXs using T . pv : p - values .

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

C) Quantification of several high mass metabolites differentially present in IDHm PDXs (IDHwt glioblastomas (GBM) in blue, IDHm GBM in orange, IDHm lower grade gliomas (LGG) in red, contralateral control brain (CB) in grey). Box plots represent log values of metabolite intensities measured within a large ROI (>500 pixels). Many metabolites in this mass range (m/z 700-900) have been putatively assigned to phospholipids (Tables EV2-3). m/z represents the mass over charge ratio of ionized metabolites, as measured by the mass spectrometer. Except where otherwise indicated, statistical significance was calculated for IDHm (n=3) versus IDHwt (n=3) PDXs using T test. pv: p-values.


A ) MALDI imaging of indicated metabolites in IDHwt PDX ( P3 ) , IDHm PDX ( E478 ) control brain ( CB ) . Hex - P : hexose - phosphate ; AMP , ADP , ATP : adenosine mono - , di - triphosphate respectively .

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A) MALDI imaging of indicated metabolites in IDHwt PDX (P3), IDHm PDX (E478) and control brain (CB). Hex-P: hexose-phosphate; AMP, ADP, ATP: adenosine mono-, di- and triphosphate respectively.


B ) LC - MS of indicated metabolites in 6 PDXs . Relative quantities of metabolites are expressed in log scale normalized to tissue quantity used for extract preparation . IDHwt glioblastomas ( GBM ) in blue , IDHm GBM in orange , IDHm lower grade gliomas ( LGG ) in red , control brain ( CB ) in grey . Sdh7 - P : sedoheptulose - 7 - phosphate .

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B) LC-MS quantification of indicated metabolites in 6 PDXs. Relative quantities of metabolites are expressed in log scale and normalized to tissue quantity used for extract preparation. IDHwt glioblastomas (GBM) in blue, IDHm GBM in orange, IDHm lower grade gliomas (LGG) in red, contralateral control brain (CB) in grey. Sdh7-P: sedoheptulose-7-phosphate.


C ) Energetic charge defined as of ( ATP + 1 / 2ADP ) / ( ATP + ADP + AMP ) was calculated at each pixel to present its distribution throughout .

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C) Energetic charge defined as the ratio of (ATP+1/2ADP)/(ATP+ADP+AMP) was calculated at each pixel to present its distribution throughout the tumor.


( D ) energetic charge was confirmed by LC - MS data performed on separate extracts ( n = 3 / sample / group ) . Indicated statistical significant group differences are on T , pv : p - values .

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(D) The energetic charge ratio was confirmed by the LC-MS data performed on separate tumor extracts (n=3/ sample/ group). Indicated statistical significant group differences are based on T test, pv: p-values.


A ) LC - MS of steady - state level of glycolysis TCA cycle metabolites in three IDH1wt three IDH1m PDXs control brain ( CB ) ( n = 3 / sample / group ) . Metabolites showing statistically significant group differences between IDH1wt IDH1m are indicated ( pvalue : * < 0 . 05 ) . Color code as in Fig . 1 - 2 .

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A) LC-MS quantification of steady-state level of glycolysis and TCA cycle metabolites in three IDH1wt and three IDH1m PDXs and contralateral control brain (CB) (n=3/ sample/ group). Metabolites showing statistically significant group differences between IDH1wt and IDH1m are indicated (pvalue: * <0.05). Color code as in Fig. 1-2.


C ) In vivo flux of 13C - labelled glucose injected 20 minutes prior to sacrifice in one IDH1wt PDX ( P3 ) one IDH1m PDX ( E478 ) ( n = 3 / sample ) . of glucose - derived 13C label is shown in selected metabolites containing 0 to 6 13C atoms ( 13C0 - 13C6 as indicated by color code ) . Note absence of 13C2 label of αKG D2HG in IDHm tumours , while label was detectable in IDHwt PDX in brain of both tumors ( lower panel ) .

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C) In vivo flux analysis of 13C-labelled glucose injected 20 minutes prior to sacrifice in one IDH1wt PDX (P3) and one IDH1m PDX (E478) (n=3/ sample). The percentage of glucose-derived 13C label is shown in selected metabolites containing 0 to 6 13C atoms (13C0 - 13C6 as indicated by color code). Note the absence of 13C2 label of αKG and D2HG in IDHm tumours, while the label was detectable in IDHwt PDX and in contralateral brain of both tumors (lower panel).


D ) Quantities of 13C3 13C2 labeled metabolites from glucose tracer experiment in ( C ) . D2HG in IDH1m PDX shows a detectable 13C2 signature here , but represents a minor fraction of total D2HG pool in this . Hex - P : hexose phosphate , PEP : phosphoenolpyruvate , Pyr : pyruvate , Cit : citrate , Mal : malate , Asp : aspartate , Glu : glutamate , Gln : glutamine , α - KG : α - Ketoglutarate , D2HG : D2 - Hydroxyglutarate . Indicated statistical significant group differences are on T , pv : p - values .

D) Quantities of 13C3 and 13C2 labeled metabolites from glucose tracer experiment in (C). D2HG in IDH1m PDX shows a detectable 13C2 signature here, but represents a minor fraction of the total cellular D2HG pool in this tumor. Hex-P: hexose phosphate, PEP: phosphoenolpyruvate, Pyr: pyruvate, Cit: citrate, Mal: malate, Asp: aspartate, Glu: glutamate, Gln: glutamine, α-KG: α-Ketoglutarate, D2HG: D2-Hydroxyglutarate. Indicated statistical significant group differences are based on T test, pv: p-values.


A ) MALDI imaging of key metabolites in IDHwt ( P3 ) IDHm ( E478 ) glioma PDX , compared to control brain ( CB ) sections .

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A) MALDI imaging of key metabolites in IDHwt (P3) and IDHm (E478) glioma PDX, compared to control brain (CB) sections.


B ) of metabolite distribution by MSI in 6 PDX with IDH1wt IDH1m status ( n = 3 / group ) in control brain ( CB ) . No significant differences between IDH groups were detected , although significant differences are found between tumors control brain ( glutamate , apsartate ) . Color code as in Fig . 1 , NAA : N - acetylaspartic acid ; NAAG : N - acetylaspartylglutamic acid .

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B) Quantification of metabolite distribution by MSI in 6 PDX with IDH1wt and IDH1m status (n=3 / group) and in contralateral control brain (CB). No significant differences between IDH groups were detected, although significant differences are found between tumors and control brain (glutamate, apsartate). Color code as in Fig. 1, NAA: N-acetylaspartic acid; NAAG: N-acetylaspartylglutamic acid.


A ) NADPH are reduced in IDHm compared to wildtype tumors , resulting in a reduced NADPH / NAD + ( n = 3 / sample ) . p - value from T : * < 0 . 05 . NADPH , NADP : reduced , oxydized nicotinamide adenine dinucleotide phosphate , NADH , NAD : reduced , oxydized nicotinamide adenine dinucleotide .

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A) NADPH levels are reduced in IDHm compared to wildtype tumors, resulting in a reduced NADPH/NAD+ ratio (n=3/ sample). p-value from T test: *<0.05. NADPH, NADP: reduced, oxydized nicotinamide adenine dinucleotide phosphate, NADH, NAD: reduced, oxydized nicotinamide adenine dinucleotide.


B ) MALDI for glutathione ( GSH ) cystathionine on IDHwt ( P3 ) IDHm ( E478 ) PDXs .

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B) MALDI images for glutathione (GSH) and cystathionine on IDHwt (P3) and IDHm (E478) PDXs.


C ) of MSI signals for GSH cystathionine from 6 PDXs ( n = 3 / group ) .

C) Quantification of MSI signals for GSH and cystathionine from 6 PDXs (n=3 / group).


D ) LC - MS of independent samples for GSH , GSSG ( oxidized GSH ) , GSSG / GSH cystathionine from 6 PDXs ( n = 3 / sample / group ) . Statistical significance between IDH groups calculated by T , pv : p - value .

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D) LC-MS quantification of independent samples for GSH, GSSG (oxidized GSH), GSSG/GSH ratio and cystathionine from 6 PDXs (n=3/ sample / group). Statistical significance between IDH groups calculated by T test, pv: p-value.


A ) Comparative gene expression of key enzymes involved in GSH cysteine synthesis supply in IDHwt ( n = 220 ) , IDHm with 1p / 19q co - deletion ( n = 151 ) or IDHm without 1p / 19q co - deletion ( n = 226 ) on TCGA - seq data from a cohort of 597 . CBS : Cystathionine beta synthase , key enzyme of transsulfuration pathway . GCLC : catalytic subunit of glutamate - cysteine ligase . SLC7A11 : gene encoding xCT , cystine - glutamate antiporter .

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A) Comparative gene expression analysis of key enzymes involved in GSH and cysteine synthesis and supply in IDHwt (n=220), IDHm with 1p/19q co-deletion (n=151) or IDHm without 1p/19q co-deletion (n=226) patients based on TCGA RNA-seq data from a cohort of 597 patients. CBS: Cystathionine beta synthase, key enzyme of the transsulfuration pathway. GCLC: catalytic subunit of glutamate-cysteine ligase. SLC7A11: gene encoding xCT, the cystine-glutamate antiporter.


B ) Kaplan - Meier curves of CBS gene expression versus overall survival in with IDHwt , IDHm with or without 1p / 19q co - deletion . Statistical significance for survival is shown as : pv : p - value , ns = non significant .

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B) Kaplan-Meier curves of CBS gene expression versus overall survival in patients with IDHwt, IDHm with or without 1p/19q co-deletion. Statistical significance for the survival analysis is shown as: pv: p-value, ns= non significant.


B . structural predictions for S . pombe Rbd2 ( blue ) E . coli GlpG ( red ) . structures were predicted using Phyre2 server ( http : / / www . sbg . bio . ic . ac . uk / phyre2 ) . Rbd2 model structure was aligned to GlpG using PyMOL .

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B. Protein structural predictions for S. pombe Rbd2 (blue) and E. coli GlpG (red). Protein structures were predicted using the Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2). Rbd2 model structure was aligned to GlpG using PyMOL.


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E. Cells carrying a plasmid expressing rbd2-6xmCherry and either of two Golgi proteins dsc2-6xGFP(dsc2-6xGFPrbd2-6xmCherry) or Sec72-6xGFP (Sec72-6xGFPrbd2-6xmCherry) were imaged by confocal fluorescence microscopy.


A . Wild - type ( WT ) , rbd2Δ or sre1Δ yeast ( 200 ) containing either empty vector ( EV ) or a plasmid expressing sre1N ( Sre1amino 1 - 440 ) were grown on rich medium in presence or absence of cobalt chloride ( CoCl2 ) .

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A. Wild-type (WT), rbd2Δ or sre1Δ yeast (200 cells) containing either empty vector (EV) or a plasmid expressing sre1N (Sre1amino acids 1-440) were grown on rich medium in the presence or absence of cobalt chloride (CoCl2).


B . Western blot was probed with anti - Sre1 IgG of phosphatase - treated , whole lysates from WT indicated mutants grown for indicated time in absence of oxygen . Dsc5 serves as a loading control was detected by chemiluminescence . P N denote Sre1 precursor forms , respectively .

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B. Western blot was probed with anti-Sre1 IgG of phosphatase-treated, whole cell lysates from WT and the indicated mutants grown for the indicated time in the absence of oxygen. Dsc5 serves as a loading control and was detected by chemiluminescence. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.


C . Indicated yeast strains expressing rbd2 or chromosomal Flag - tagged rbd2 , rbd2 - S130A or rbd2 - H182A were analyzed for Sre1 cleavage . Western blot was performed using anti - Sre1 or anti - FlagIgG of phosphatase - treated , whole lysates from grown for 3 hr in presence or absence of oxygen .

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C. Indicated yeast strains expressing rbd2 or chromosomal Flag-tagged rbd2, rbd2-S130A or rbd2-H182A were analyzed for Sre1 cleavage. Western blot was performed using anti-Sre1 or anti-FlagIgG of phosphatase-treated, whole cell lysates from cells grown for 3 hr in the presence or absence of oxygen.


D . Indicated yeast strains expressing rbd2 or chromosomal Flag - tagged rbd2 , rbd2 - S130A or rbd2 - H182A were analyzed for Sre2 cleavage by western blot probed with anti - Sre2 serum or anti - FlagIgG of phosphatase - treated , whole lysates from grown in presence of oxygen .

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D. Indicated yeast strains expressing rbd2 or chromosomal Flag-tagged rbd2, rbd2-S130A or rbd2-H182A were analyzed for Sre2 cleavage by western blot probed with anti-Sre2 serum or anti-FlagIgG of phosphatase-treated, whole cell lysates from cells grown in the presence of oxygen.


E . Sre1 cleavage was performed as in ( C ) . G128V , A127D A186T mutant strains were obtained from our previous mutagenesis screening ( Stewart et al , 2012 ) . Dsc5 serves as a loading control .

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E. Sre1 cleavage assay was performed as in (C). G128V, A127D and A186T mutant strains were obtained from our previous mutagenesis screening (Stewart et al, 2012). Dsc5 serves as a loading control.


F . Sre2 cleavage for indicated yeast strains was performed as in ( D ) .

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F. Sre2 cleavage assay for indicated yeast strains was performed as in (D).


G . A model substrate of Sre2 containing 423 - 793 ( Sre2MS ) with an N - terminal 3xFlag epitope tag was expressed on a plasmid under control of a CaMV promoter in sre2Δ , dsc1Δsre2Δ or rbd2Δsre2Δ yeast strains . Whole lysates from indicated yeast strains were analyzed by western blot with anti - Flag antibody . Three independent isolates ( A - C ) are shown for each . Empty vector ( EV ) transformation serves as a negative control .

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G. A model substrate of Sre2 containing amino acids 423-793 (Sre2MS) with an N-terminal 3xFlag epitope tag was expressed on a plasmid under the control of a CaMV promoter in sre2Δ, dsc1Δsre2Δ or rbd2Δsre2Δ yeast strains. Whole cell lysates from indicated yeast strains were analyzed by western blot with anti-Flag antibody. Three independent isolates (A-C) are shown for each strain. Empty vector (EV) transformation serves as a negative control.


A . Wild - type ( WT ) indicated mutant strains were grown for 3 hr at non - permissive ( 36 ° C ) to inactivate Mts3 . Phosphatase - treated lysates were analyzed by western blot with anti - Sre2 serum imaged using chemiluminescence . P N denote Sre2 precursor forms , respectively .

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A. Wild-type (WT) and the indicated mutant strains were grown for 3 hr at the non-permissive temperature (36°C) to inactivate Mts3. Phosphatase-treated cell lysates were analyzed by western blot with anti-Sre2 serum and imaged using chemiluminescence. P and N denote Sre2 precursor and cleaved nuclear forms, respectively.


B . WT , rbd2Δ sre2Δ were treated with bortezomib ( Bz ) for 3 hr , phosphatase - treated , whole lysates were analyzed by western blot with anti - Sre2 serum .

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B. WT, rbd2Δ and sre2Δ cells were treated with bortezomib (Bz) for 3 hr, and phosphatase-treated, whole cell lysates were analyzed by western blot with anti-Sre2 serum.


C . WT , dsc1Δ rbd2Δ carrying either empty vector ( EV ) or a plasmid expressing 3xFlag - Sre2MS ( MS ) were treated with bortezomib ( Bz ) for 3 hr . Whole lysates were analyzed by western blot with anti - Flag antibody .

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C. WT, dsc1Δ and rbd2Δ cells carrying either empty vector (EV) or a plasmid expressing 3xFlag-Sre2MS (MS) were treated with bortezomib (Bz) for 3 hr. Whole cell lysates were analyzed by western blot with anti-Flag antibody.


D . Whole lysates from indicated strains were analyzed by western blot with anti - Sre2 serum .

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D. Whole cell lysates from the indicated strains were analyzed by western blot with anti-Sre2 serum.


E . Catalytically dead Dsc1 E3 ligase ( dsc1 - C634A ) mutant other indicated yeast strains were analyzed by western blot probed with anti - Sre2 serum imaged using chemiluminescence .

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E. Catalytically dead Dsc1 E3 ligase (dsc1-C634A) mutant and other indicated yeast strains were analyzed by western blot probed with anti-Sre2 serum and imaged using chemiluminescence.


A . HumanHEK293T were transfected with plasmids expressing different 3xHA - tagged rhomboid proteases : S . pomberbd2 , S . pomberbd2 recoded for human expression , humanRHBDL2 , D . melanogaster Rho1 , P . falciparumROM4 . Whole lysates were analyzed by western blot to detect HA - tagged rhomboid [ asterisks indicate loading control bands detected with antibody ab179726 ( abcam ) ] .

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A. HumanHEK293T cells were transfected with plasmids expressing different 3xHA-tagged rhomboid proteases: S. pomberbd2, S. pomberbd2 recoded for human expression, humanRHBDL2, D. melanogaster Rho1, and P. falciparumROM4. Whole cell lysates were analyzed by western blot to detect the HA-tagged rhomboid [asterisks indicate loading control bands detected with antibody ab179726 (abcam)].


B . HumanHEK293T were transfected with plasmids expressing indicated rhomboid substrates either recoded S . pombe rbd2 , D . melanogaster Rho1 , or P . falciparumROM4 . Conditioned media lysates were analyzed by western blot . P C denote precursor products , respectively .

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B. HumanHEK293T cells were transfected with plasmids expressing the indicated rhomboid substrates and either recoded S. pombe rbd2, D. melanogaster Rho1, or P. falciparumROM4. Conditioned media and cell lysates were analyzed by western blot. P and C denote precursor and cleaved products, respectively.


C . HumanHEK293T were transfected with plasmids expressing GFP - TatA - Flag substrate indicated rhomboid proteases . Whole lysates were analyzed by western blot . P , CN , CC denote precursor , N - terminal C - terminal products , respectively .

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C. HumanHEK293T cells were transfected with plasmids expressing the GFP-TatA-Flag substrate and the indicated rhomboid proteases. Whole cell lysates were analyzed by western blot. P, CN, CC denote precursor, N-terminal cleaved and C-terminal cleaved products, respectively.


D . HumanHEK293T were transfected with plasmids expressing indicated GFP - TatA - Flag substrates either wild - type or mutant recoded S . pomberbd2 . Whole lysates were analyzed by western blot .

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D. HumanHEK293T cells were transfected with plasmids expressing the indicated GFP-TatA-Flag substrates and either wild-type or mutant recoded S. pomberbd2. Whole cell lysates were analyzed by western blot.


E . HumanHEK293T were transfected with plasmids expressing indicated GFP - TatA - Flag substrates different rhomboid proteases . Whole lysates were analyzed by western blot .

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E. HumanHEK293T cells were transfected with plasmids expressing the indicated GFP-TatA-Flag substrates and different rhomboid proteases. Whole cell lysates were analyzed by western blot.


F . HumanHEK293T were transfected with plasmids expressing GFP - TatA ( A8L ) - Flag recoded S . pomberbd2 . C - terminal cleavage products were purified using anti - Flag antibodies purified was analyzed by mass spectrometry .

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F. HumanHEK293T cells were transfected with plasmids expressing the GFP-TatA (A8L)-Flag and recoded S. pomberbd2. C-terminal cleavage products were purified using anti-Flag antibodies and purified protein was analyzed by mass spectrometry.


B . Whole lysates from WT , rbd2Δ dsc1Δ carrying a plasmid expressing 3xFlag - Sre2MS WT or indicated mutants were analyzed by western blot with anti - Flag antibody . P N denote Sre2MS precursor forms , respectively .

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B. Whole cell lysates from WT, rbd2Δ and dsc1Δ cells carrying a plasmid expressing 3xFlag-Sre2MS WT or indicated mutants were analyzed by western blot with anti-Flag antibody. P and N denote Sre2MS precursor and cleaved forms, respectively.


D - E . Whole lysates from WT , rbd2Δ dsc1Δ carrying a plasmid expressing 3xFlag - Sre2MS WT or indicated mutants were analyzed by western blot with anti - Flag antibody . P N denote Sre2MS precursor forms , respectively . Asterisks indicate non - specific , loading control bands , which were used to normalize intensities of P N in Figure 5F . Two independent isolates , A B , are shown for each .

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D-E. Whole cell lysates from WT, rbd2Δ and dsc1Δ cells carrying a plasmid expressing 3xFlag-Sre2MS WT or indicated mutants were analyzed by western blot with anti-Flag antibody. P and N denote Sre2MS precursor and cleaved forms, respectively. Asterisks indicate non-specific, loading control bands, which were used to normalize the intensities of P and N in Figure 5F. Two independent isolates, A and B, are shown for each strain.


F . Intensities of P N bands from western blots were quantified using LI - COR software . Relative of P N to corresponding non - specific band ( asterisk ) in each lane was normalized to that of P level in isolate A of WT for each mutagenesis . of N to P or relative P level was presented in bar graphs .

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F. Intensities of individual P and N bands from western blots were quantified using LI-COR software. Relative intensity of P and N to corresponding non-specific band (asterisk) in each lane was normalized to that of P level in isolate A of WT strain for each mutagenesis. The ratio of N to P or relative P level was presented in bar graphs.


G . average of N to P level for different Sre2MS mutants in wild - type ( Appendix Fig S2 ) was plotted . Cleavage was scored as normal if was > 2 .

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G. The average ratio of N to P level for different Sre2MS mutants in wild-type cells (Appendix Fig S2) was plotted. Cleavage was scored as normal if the ratio was >2.


H . For Sre2MS mutants showing defects in cleavage , average fold - increase in P level in dsc1Δ compared to WT from two isolates ( Appendix Fig S2 ) was plotted . Mutants were grouped into three phenotypic classes ( intermediate , dsc1Δ , rbd2 ∆ ) according to observed precursor accumulation . Precursor accumulation was defined as dsc1Δ if < 1 . 5 ( orange ) , rbd2Δ if > 3 ( pink ) , intermediate if in between ( blue ) .

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H. For Sre2MS mutants showing defects in cleavage, the average fold-increase in P level in dsc1Δ cells compared to WT cells from two isolates (Appendix Fig S2) was plotted. Mutants were grouped into three phenotypic classes (intermediate, dsc1Δ, and rbd2∆) according to observed precursor accumulation. Precursor accumulation was defined as dsc1Δ if <1.5 (orange), rbd2Δ if >3 (pink), and intermediate if in between (blue).


B . Recombinant GST - fused Rbd2 C - terminus ( Rbd2200 - 251 ) , Dsc5 UBX ( Dsc5323 - 425 ) , Dsc2 UBA ( Dsc2298 - 372 ) , GST - HA - V5 control were bound to GST magnetic beads incubated with S . pombe cytosol fraction from wild - type . Equivalent amounts of unbound bound fractions were probed for anti - Cdc48 , anti - ubiquitin anti - GSTIgG .

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B. Recombinant proteins GST-fused Rbd2 C-terminus (Rbd2200-251), Dsc5 UBX (Dsc5323-425), Dsc2 UBA (Dsc2298-372), and GST-HA-V5 control were bound to GST magnetic beads and incubated with S. pombe cytosol fraction from wild-type cells. Equivalent amounts of unbound and bound fractions were probed for anti-Cdc48, anti-ubiquitin and anti-GSTIgG.


C . GSTpulldown was performed as in ( B ) using truncated forms of Rbd2 C - terminus ; Rbd2200 - 251 , Rbd2200 - 225 , Rbd2225 - 251 Rbd2200 - 240 . Unbound ( 1x ) bound ( 5x ) fractions were analyzed by western

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C. GSTpulldown assay was performed as in (B) using truncated forms of Rbd2 C-terminus; Rbd2200-251, Rbd2200-225, Rbd2225-251 and Rbd2200-240. Unbound (1x) and bound (5x) fractions were analyzed by western blotting


D . Single residue mutants of conserved glycine residues in Rbd2200 - 251 ( G244R G246R ) were tested for Cdc48 binding as in ( C ) . Western blots ( B - D ) were imaged using chemiluminescence .

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D. Single residue mutants of conserved glycine residues in Rbd2200-251 (G244R and G246R) were tested for Cdc48 binding as in (C). Western blots (B-D) were imaged using chemiluminescence.


A . Detergent - solubilized yeast extracts from containing chromosomal Flag - tagged rbd2 or rbd2 - G246R in rbd2Δ background were prepared , associated with Rbd2 - Flag were immunopurified using anti - Flag magnetic beads . Equal amount of total ( lanes 1 - 3 ) along with 25x bound fractions ( lanes 4 - 6 ) were analyzed by western blot with anti - Cdc48 serum anti - FlagIgG .

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A. Detergent-solubilized yeast extracts from cells containing chromosomal Flag-tagged rbd2 or rbd2-G246R in rbd2Δ background were prepared, and proteins associated with Rbd2-Flag were immunopurified using anti-Flag magnetic beads. Equal amount of total (lanes 1-3) along with 25x bound fractions (lanes 4-6) were analyzed by western blot with anti-Cdc48 serum and anti-FlagIgG.


C . Biotin - phenol labeling was performed for 1 min with H2O2 treatment as described in Materials Methods . WT or rbd2Δ carrying rbd2 - Flag - APEX2 ( rbd2 - F - AX ) or rbd2 - G246R - Flag - APEX2 ( G246R - F - AX ) plasmid were lysed after biotin - labeling reaction , were denatured by heating in lysis buffer containing 1 % SDS . Biotinylated were then enriched using streptavidin magnetic beads . Lysates 50x - enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin , anti - Cdc48 serum , or anti - FlagIgG .

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C. Biotin-phenol labeling was performed for 1 min with H2O2 treatment as described in Materials and Methods. WT cells or rbd2Δ cells carrying rbd2-Flag-APEX2 (rbd2-F-AX) or rbd2-G246R-Flag-APEX2 (G246R-F-AX) plasmid were lysed after biotin-labeling reaction, and proteins were denatured by heating the cells in lysis buffer containing 1%SDS. Biotinylated proteins were then enriched using streptavidin magnetic beads. Lysates and 50x-enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin, anti-Cdc48 serum, or anti-FlagIgG.


A . Yeast strains ( 200 ) containing chromosomal Flag - tagged rbd2 , rbd2 - G244R , rbd2 - G246R or rbd2 - S130A in rbd2Δ background sre1Δ yeast were grown on rich medium in absence or presence of cobalt chloride ( CoCl2 ) .

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A. Yeast strains (200 cells) containing chromosomal Flag-tagged rbd2, rbd2-G244R, rbd2-G246R or rbd2-S130A in rbd2Δ background and sre1Δ yeast were grown on rich medium in the absence or presence of cobalt chloride (CoCl2).


B . Western blot of phosphatase - treated , whole lysates from strains in ( A ) grown for 3 hr in presence or absence of oxygen was probed with anti - Sre1 or anti - Flag IgG . P N denote Sre1 precursor forms , respectively .

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B. Western blot of phosphatase-treated, whole cell lysates from strains in (A) grown for 3 hr in the presence or absence of oxygen was probed with anti-Sre1 or anti-Flag IgG. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.


C . Sre2 cleavage was assayed by western blot using anti - Sre2 serum or anti - Flag IgG of phosphatase - treated , whole lysates from indicated strains . N denotes Sre2 form .

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

C. Sre2 cleavage was assayed by western blot using anti-Sre2 serum or anti-Flag IgG of phosphatase-treated, whole cell lysates from the indicated strains. N denotes Sre2 cleaved nuclear form.


D . Whole lysates from indicated yeast strains carrying a plasmid expressing 3xFlag - Sre2MS were analyzed by western blot with anti - Flag antibody . Two independent isolates , A B , are shown for each . Asterisk denotes non - specific band .

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

D. Whole cell lysates from indicated yeast strains carrying a plasmid expressing 3xFlag-Sre2MS were analyzed by western blot with anti-Flag antibody. Two independent isolates, A and B, are shown for each strain. Asterisk denotes non-specific band.


F . Yeast strains containing Flag - tagged rbd2 - SHP , rbd2 - SHP * , rbd2 - G246R - SHP , rbd2 - G246R - SHP * were generated in rbd2Δ background by chromosomal integration of rbd2 fusion depicted in ( E ) . Indicated yeast strains were then assayed for Sre1 cleavage as in ( B ) .

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

F. Yeast strains containing Flag-tagged rbd2-SHP, rbd2-SHP*, rbd2-G246R-SHP, and rbd2-G246R-SHP* were generated in rbd2Δ background by chromosomal integration of the rbd2 fusion proteins depicted in (E). Indicated yeast strains were then assayed for Sre1 cleavage as in (B).


G . Indicated strains were assayed for Sre2 cleavage as in ( C ) .

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

G. Indicated strains were assayed for Sre2 cleavage as in (C).


H . Wild - type , sre2Δ or strains containing chromosomal Flag - tagged rbd2 , rbd2 - G246R or rbd2 - S130S in rbd2Δ background ( lane 7 - 12 ) were treated with bortezomib ( Bz ) for 2 hr , phosphatase - treated , whole lysates were analyzed by western blot with anti - Sre2 serum or anti - Flag IgG .

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

H. Wild-type, sre2Δ or strains containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130S in rbd2Δ background (lane 7-12) were treated with bortezomib (Bz) for 2 hr, and phosphatase-treated, whole cell lysates were analyzed by western blot with anti-Sre2 serum or anti-Flag IgG.


A . Western blot of phosphatase - treated whole lysates from yeast strains carrying rbd2 - Flag - APEX2 ( rbd2 - F ) , rbd2 - G246R - Flag - APEX2 ( G246R ) or rbd2 - S130A - Flag - APEX2 ( S130A ) plasmid in rbd2Δ background sre1Δ yeast was probed with anti - Sre1 or anti - Flag IgG . P N denote Sre1 precursor forms , respectively .

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

A. Western blot of phosphatase-treated whole cell lysates from yeast strains carrying rbd2-Flag-APEX2 (rbd2-F), rbd2-G246R-Flag-APEX2 (G246R) or rbd2-S130A-Flag-APEX2 (S130A) plasmid in rbd2Δ background and sre1Δ yeast was probed with anti-Sre1 or anti-Flag IgG. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.


B . Indicated yeast strains ( 200 ) containing chromosomal Flag - tagged rbd2 , rbd2 - G246R or rbd2 - S130A in rbd2Δ background ( lane 3 - 5 ) or yeast strains ( 200 ) carrying rbd2 - Flag - APEX2 , rbd2 - G246R - Flag - APEX2 or rbd2 - S130A - Flag - APEX2 plasmid in rbd2Δ background ( lane 6 - 8 ) were grown on rich medium in absence or presence of cobalt chloride ( CoCl2 ) .

O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 B-MEASURED_VAR 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 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. Indicated yeast strains (200 cells) containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130A in rbd2Δ background (lane 3-5) or yeast strains (200 cells) carrying rbd2-Flag-APEX2, rbd2-G246R-Flag-APEX2 or rbd2-S130A-Flag-APEX2 plasmid in rbd2Δ background (lane 6-8) were grown on rich medium in the absence or presence of cobalt chloride (CoCl2).


C . Comparison of Rbd2 from overexpression system to endogenous expression . Western blot of whole lysates from yeast strains containing chromosomal Flag - tagged rbd2 , rbd2 - G246R or rbd2 - S130A in rbd2Δ background yeast strains carrying rbd2 - Flag - APEX2 , rbd2 - G246R - Flag - APEX2 or rbd2 - S130A - Flag - APEX2 plasmid in rbd2Δ background was probed with anti - FlagIgG . For , of Flag band in each lane was normalized to non - specific band ( asterisk ) .

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

C. Comparison of Rbd2 protein levels from overexpression system to endogenous expression. Western blot of whole cell lysates from yeast strains containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130A in rbd2Δ background and yeast strains carrying rbd2-Flag-APEX2, rbd2-G246R-Flag-APEX2 or rbd2-S130A-Flag-APEX2 plasmid in rbd2Δ background was probed with anti-FlagIgG. For quantification, the intensity of Flag band in each lane was normalized to the non-specific band (asterisk).


D . rbd2Δ expressing indicated plasmids of GFP - Anp1 or GFP - Sre2MS together with rbd2 - Flag - APEX2 , rbd2 - S130A - Flag - APEX2 or rbd2 - S130A / G246R - Flag - APEX2 were treated with Bz at a of 1 mM for 2 hr prior to biotin - labeling reaction . After labeling termination , were lysed denatured as described in Materials Methods . Biotinylated were then enriched using streptavidin magnetic beads . Lysates ( 1x input ) 100x - enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin , anti - Flag IgG , anti - Cdc48 serum , anti - GFP IgG . of GFP band in enriched sample was normalized to that in corresponding input sample , then normalized to Flag in enriched sample . Relative intensities of biotinylated GFP level are shown in bar graph . Error bars denote standard deviation from three biological replicates .

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

D. rbd2Δ cells expressing indicated plasmids of GFP-Anp1 or GFP-Sre2MS together with rbd2-Flag-APEX2, rbd2-S130A-Flag-APEX2 or rbd2-S130A/G246R-Flag-APEX2 were treated with Bz at a concentration of 1 mM for 2 hr prior to biotin-labeling reaction. After labeling termination, cells were lysed and denatured as described in Materials and Methods. Biotinylated proteins were then enriched using streptavidin magnetic beads. Lysates (1x input) and 100x-enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin, anti-Flag IgG, anti-Cdc48 serum, and anti-GFP IgG. Intensity of GFP band in enriched sample was normalized to that in the corresponding input sample, then normalized to the Flag intensity in enriched sample. Relative intensities of biotinylated GFP level are shown in bar graph. Error bars denote standard deviation from three biological replicates.


A , B Representative time - lapse ( A ) kymograph ( B ) of mouse zygotes expressing H2B - RFP ( magenta ) labelled with SiR - Tubulin ( green ) . Shown is an example of marked chromosomal displacement to cortex . C Changes in chromosome - to - centre distances ( d ) over time relative to anaphase - onset ( n = 50 blastomeres ) . Data information : In ( A , time is shown as h post - metaphase scale bar = 20 µm . In ( C , data are shown as mean ± .

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

A, B Representative time-lapse images (A) and kymograph (B) of mouse zygotes expressing H2B-RFP (magenta) and labelled with SiR-Tubulin (green). Shown is an example of marked chromosomal displacement to the cortex. C Changes in chromosome-to-cell centre distances (d) over time relative to anaphase-onset (n = 50 blastomeres). Data information: In (A, time is shown as h post-metaphase and scale bar = 20 µm. In (C, data are shown as mean ± SEM.


B , C Representative time - lapse of in which DMSO ( B ) or nocodazole ( C ) was added after anaphase completion . D Changes in nucleus - to - centre distances ( d ) over time relative to time of drug addition following DMSO - ( n = 22 blastomeres ) or nocodazole - treatment ( n = 26 blastomeres ) at anaphase completion . Data information : In ( B , C , time is shown as h post - drug addition scale bar = 20 µm . In ( D , data are shown as mean ± . Statistical significance was assessed by an ordinary two - way ANOVA with Šidák ' s multiple comparisons ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , unremarked datapoints = not significant ) .

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

B, C Representative time-lapse images of embryos in which DMSO (B) or nocodazole (C) was added after anaphase completion. D Changes in nucleus-to-cell centre distances (d) over time relative to time of drug addition following DMSO- (n = 22 blastomeres) or nocodazole-treatment (n = 26 blastomeres) at anaphase completion. Data information: In (B,C, time is shown as h post-drug addition and scale bar = 20 µm. In (D, data are shown as mean ± SEM. Statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unremarked datapoints = not significant).


E - G Representative time - lapse of in which DMSO ( E ) , Nocodazole ( F ) or Cytochalasin D ( G ) was added when nuclei had travelled to cortex . H Changes in nucleus - to - centre distances ( d ) over time relative to time of drug addition DMSO - ( n = 20 blastomeres ) , nocodazole - ( n = 12 blastomeres ) or cytochalasin D - treatment ( n = 14 blastomeres ) after nuclei reached cortex . Data information : In E - G ) , time is shown as h post - drug addition scale bar = 20 µm . In H ) , data are shown as mean ± . Statistical significance was assessed by an ordinary two - way ANOVA with Šidák ' s multiple comparisons ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , unremarked datapoints = not significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G Representative time-lapse images of embryos in which DMSO (E), Nocodazole (F) or Cytochalasin D (G) was added when nuclei had travelled to the cortex. H Changes in nucleus-to-cell centre distances (d) over time relative to time of drug addition DMSO- (n = 20 blastomeres), nocodazole- (n = 12 blastomeres) or cytochalasin D-treatment (n = 14 blastomeres) after nuclei reached the cortex. Data information: In E - G), time is shown as h post-drug addition and scale bar = 20 µm. In H), data are shown as mean ± SEM. Statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unremarked datapoints = not significant).


A , B Representative time - lapse of that have been washed out of either nocodazole after nuclei were prevented from travelling to cortex after anaphase ( A , n = 15 ) or cytochalasin D after nuclei had been trapped at cortex by disrupting actin ( B , n = 22 ) . C Graph comparing distribution of maximal d values in that developed to blastocyst stage or not . Each data point represents mean maximal d value of two blastomeres from same ( n = 45 ) . D Graph of time taken to develop to blastocyst stage in relation to maximal d values ( n = 27 ) . E Graph comparing distribution of times taken for nuclei to re - centre in that developed to blastocyst stage or not ( n = 45 ) . F Graph of time taken to develop to blastocyst stage in relation to times taken for nuclei to re - centre ( n = 27 ) . Data information : In ( A , B ) , time is shown as h post - drug washout scale bar = 20 µm . In ( C - F ) , data are shown as mean ± . In ( C , E ) , statistical significance was assessed by Welch ' s t - , ns : not significant . In ( D , F ) , strength of correlation ( r ) statistical significance were assessed using Pearson correlation , ns : not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative time-lapse images of embryos that have been washed out of either nocodazole after nuclei were prevented from travelling to the cortex after anaphase (A, n = 15 embryos) or cytochalasin D after nuclei had been trapped at the cortex by disrupting actin (B, n = 22 embryos). C Graph comparing the distribution of maximal d values in embryos that developed to the blastocyst stage or not. Each data point represents the mean maximal d value of the two blastomeres from the same embryo (n = 45 embryos). D Graph of time taken to develop to the blastocyst stage in relation to maximal d values (n = 27 embryos). E Graph comparing the distribution of times taken for nuclei to re-centre in embryos that developed to the blastocyst stage or not (n = 45 embryos). F Graph of time taken to develop to the blastocyst stage in relation to times taken for nuclei to re-centre (n = 27 embryos). Data information: In (A, B), time is shown as h post-drug washout and scale bar = 20 µm. In (C - F), data are shown as mean ± SEM. In (C, E), statistical significance was assessed by Welch's t-test, ns: not significant. In (D, F), the strength of correlation (r) and statistical significance were assessed using the Pearson correlation test, ns: not significant.


A Representative time - lapse image of SiR - tubulin in DMSO - BFA - treated during first mitotic division subsequent interphase . Time is shown as h relative to first mitotic anaphase . B Changes in nucleus - to - centre distances ( d ) over time in BFA - treated , relative to maximal displacement to cortex ( n = 14 blastomeres ) . Data information : In ( A , scale bar = 20 µm .

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

A Representative time-lapse image of DNA and SiR-tubulin in DMSO- and BFA-treated embryos during the first mitotic division and subsequent interphase. Time is shown as h relative to the first mitotic anaphase. B Changes in nucleus-to-cell centre distances (d) over time in BFA-treated embryos, relative to maximal nuclear displacement to the cortex (n = 14 blastomeres). Data information: In (A, scale bar = 20 µm.


D Representative time - lapse of second mitotic division in DMSO - BFA - treated . Dashed lines highlight position of nuclei shortly before NEBD . Dashed yellow lines outline daughter - . Time is shown as h relative to second mitotic NEBD . E Dot plot of duration between first mitotic division anaphase - onset second mitotic division NEBD for DMSO - ( n = 47 blastomeres ) BFA - treated ( n = 47 blastomeres ) . F Plot of duration between NEBD anaphase of second mitotic division for DMSO - ( n = 44 blastomeres ) BFA - treated ( n = 43 blastomeres ) . G Plot of spindle - to - centre distances against pre - NEBD nucleus - to - centre ( n = 34 blastomeres ) . indicates mathematically regressed fit . Data information : In D ) , scale bar = 20 µm . In E - G , data are shown as mean ± . In ( E , F , statistical significance was assessed by Mann - Whitney , * * * * P < 0 . 0001 , ns : not significant . In ( G , strength of correlation ( r ) statistical significance were assessed using Pearson correlation . Two - p values are reported ( * * P < 0 . 01 ) .

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

D Representative time-lapse images of the second mitotic division in DMSO- and BFA-treated embryos. Dashed white lines highlight position of nuclei shortly before NEBD. Dashed yellow lines outline daughter-cells. Time is shown as h relative to the second mitotic NEBD. E Dot plot of the duration between first mitotic division anaphase-onset and second mitotic division NEBD for DMSO- (n = 47 blastomeres) and BFA-treated (n = 47 blastomeres) embryos. F Plot of the duration between NEBD and anaphase of the second mitotic division for DMSO- (n = 44 blastomeres) and BFA-treated (n = 43 blastomeres) embryos. G Plot of the spindle-to-cell centre distances against pre-NEBD nucleus-to-cell centre distance (n = 34 blastomeres). Line indicates mathematically regressed fit. Data information: In D), scale bar = 20 µm. In E - G, data are shown as mean ± SEM. In (E, F, statistical significance was assessed by Mann-Whitney test, ****P < 0.0001, ns: not significant. In (G, the strength of correlation (r) and statistical significance were assessed using the Pearson correlation test. Two-tailed p values are reported (**P < 0.01).


A Representative time - lapse of 2 - cultured with SiR - to label nuclei ( circles ) microinjected with oil droplets ( yellow circles ) . Time is shown as h : min relative to maximal displacement . B Plot of distances between oil droplets nuclei to - centre over time ( n = 14 oil - nucleus pairs ) . Data information : Scale bar = 20 µm in A , In ( B ) , data are shown as mean ± .

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

A Representative time-lapse images of 2-cell embryos cultured with SiR-DNA to label nuclei (white circles) and microinjected with oil droplets (yellow circles). Time is shown as h:min relative to maximal nuclear displacement. B Plot of the distances between oil droplets and nuclei to the cell-centre over time (n = 14 oil-nucleus pairs). Data information: Scale bar = 20 µm in A, In (B), data are shown as mean ± SEM.


E Top panel : an example of bead distribution in a 2 - . Lower panel : an example of region imaged for bead tracking at high temporal resolution . F , G Trajectories of bead movement over a 20 s - duration in oocytes ( F , n = 217 beads from 23 oocytes ) 2 - ( n = 277 beads from 26 ) . Each coloured represents a single bead track . Data information : Scale bar = 20 µm in E , except 2 µm in lower panel of 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

E Top panel: an example of fluorescent bead distribution in a 2-cell embryo. Lower panel: an example of region imaged for bead tracking at high temporal resolution. F, G Trajectories of bead movement over a 20 s-duration in oocytes (F, n = 217 beads from 23 oocytes) and 2-cell embryos (n = 277 beads from 26 embryos). Each coloured line represents a single bead track. Data information: Scale bar = 20 µm in E, except 2 µm in the lower panel of E.


B Shown is a representative 2 - immunostained for β - tubulin ( green ) ( magenta ) at anaphase completion ( n = 8 ) . C Dot plot of mean β - tubulin within regions that either lie ahead of nucleus ( cortex - facing aspect , dashed triangle in B ) or behind nucleus ( cleavage plane - facing aspect , dashed yellow triangle in B ) ( n = 14 ) . Data information : In ( B scale bar = 20 µm . In ( C , data are shown as mean ± . In ( C , statistical significance was assessed by Welch ' s t - , * * * * P < 0 . 0001 , ns = not significant .

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

B Shown is a representative 2-cell embryo immunostained for β-tubulin (green) and DNA (magenta) at anaphase completion (n = 8 embryos). C Dot plot of mean β-tubulin fluorescence intensity within cytoplasmic regions that either lie ahead of the nucleus (cortex-facing aspect, dashed white triangle in B) or behind the nucleus (cleavage plane-facing aspect, dashed yellow triangle in B) (n = 14 embryos). Data information: In (B scale bar = 20 µm. In (C, data are shown as mean ± SEM. In (C, statistical significance was assessed by Welch's t-test, ****P < 0.0001, ns = not significant.


D Representative time - lapse of expressing H2B - RFP labelled with SiR - tubulin following addition of dynarrestin at anaphase completion ( n = 14 ) . Data information : In ( D , time is shown as h post - drug addition . In D , scale bar = 20 µm .

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

D Representative time-lapse images of embryos expressing H2B-RFP and labelled with SiR-tubulin following the addition of dynarrestin at anaphase completion (n = 14 embryos). Data information: In (D, time is shown as h post-drug addition. In D, scale bar = 20 µm.


E Dot plot of maximal d values between DMSO - dynarrestin - treated ( n = 28 blastomeres per group ) . Data information : In E , data are shown as mean ± . In E ) , statistical significance was assessed by Welch ' s t - , * * * * P < 0 . 0001 , ns = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Dot plot of maximal d values between DMSO- and dynarrestin-treated embryos (n = 28 blastomeres per group). Data information: In E, data are shown as mean ± SEM. In E), statistical significance was assessed by Welch's t-test, ****P < 0.0001, ns = not significant.


G Representative time - lapse of expressing H2B - RFP labelled with SiR - tubulin following addition of taxol at maximal displacement . Data information : In G , scale bar = 20 µm .

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

G Representative time-lapse images of embryos expressing H2B-RFP and labelled with SiR-tubulin following the addition of taxol at maximal nuclear displacement. Data information: In G, scale bar = 20 µm.


H Changes in nucleus - to - centre distances ( d ) over time relative to time of drug addition in DMSO - ( n = 19 ) taxol - ( n = 19 ) treated 2 - blastomeres ( mean ± ) . Data information : In H ) , data are shown as mean ± . In ( H ) , statistical significance was assessed by an ordinary two - way ANOVA with Šidák ' s multiple comparisons ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , unremarked datapoints = not significant ) .

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

H Changes in nucleus-to-cell centre distances (d) over time relative to the time of drug addition in DMSO- (n = 19) and taxol- (n = 19) treated 2-cell blastomeres (mean ± SEM). Data information: In H), data are shown as mean ± SEM. In (H), statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, unremarked datapoints = not significant).


I Representative of immunostained for β - tubulin Hoechst following completion of live imaging ( shown in E ) in DMSO - taxol - treated ( n = 9 per group ) . Time is shown as h post - drug addition . Scale bar = 20 µm . Data information : In I ) , scale bar = 20 µm .

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

I Representative images of embryos immunostained for β-tubulin and Hoechst following the completion of live imaging (shown in E) in DMSO- and taxol-treated embryos (n = 9 embryos per group). Time is shown as h post-drug addition. Scale bar = 20 µm. Data information: In I), scale bar = 20 µm.


A . Hematoxylin eosin ( H & E ) - stained cross section of a human liver slice . Scale bar is representative of A - F . Image is also used in Fig . S3A . B . Spatial distribution relative abundance of glycogen ( represented by CL7 ) in an immediate adjacent tissue slice from A . image displays a gradient of from ( least abundant ) to red ( most abundant ) which is representative of B , D , F . C . H & E - stained cross section of a human lung slice . Image is also used in Fig . S3D . D . Spatial distribution relative abundance of glycogen ( represented by CL7 ) in an immediate adjacent tissue slice from C . E . H & E - stained cross section of frontal cortical region of a human brain tissue . Image is also used in Fig . S3G . F . Spatial distribution relative abundance of glycogen ( represented by CL7 ) in an immediate adjacent tissue slice from E . G . of relative abundance of total glycogen calculated from sum of CL3 - CL18 in human liver , lung , brain . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . * 0 . 01 < P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 , analyzed by one - way ANOVA with Tukey ' s multiple comparison for total glycogen . H . Glycogen structure represented by normalized chain distribution ( to CL3 ) of each human liver , lung brain from G . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Hematoxylin and eosin (H&E)-stained cross section of a human liver slice. Scale bar is representative of A-F. Image is also used in Fig. S3A. B. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from A. The image displays a heatmap gradient of intensity from white (least abundant) to red (most abundant) which is representative of B, D, and F. C. H&E-stained cross section of a human lung slice. Image is also used in Fig. S3D. D. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from C. E.H&E-stained cross section of frontal cortical region of a human brain tissue. Image is also used in Fig. S3G. F. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from E. G. Quantification of relative abundance of total glycogen calculated from the sum of CL3-CL18 in human liver, lung, and brain. Values are presented as mean +/- standard error (n=3 technical replicates per region). *0.01 < P < 0.05; ***P < 0.001; ****P<0.0001, analyzed by one-way ANOVA with Tukey's multiple comparison for total glycogen. H. Glycogen structure represented by normalized chain length distribution (ratio to CL3) of each human liver, lung and brain from G. Values are presented as mean +/- standard error (n=3 technical replicates per region).


A . Hematoxylin eosin ( H & E ) - stained cross section of adenocarcinoma of prostate , lung squamous carcinoma ( LUSC ) , lung adenocarcinoma ( LUAD ) , Ewing sarcoma ( ES ) of abdomen . Annotated regions are T - , S - stroma , D / G - / glandular , A - adipose tissue . Scale bar is representative of A B . are also used in Fig . S4A , 5D S6G . B . Spatial distribution relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section shown in A . image displays a gradient of with ( least abundant ) to red ( most abundant ) . are also used in Fig . S4E , 5D , S6G . C . Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain ( CL ) distribution from regions shown in A . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Hematoxylin and eosin (H&E)-stained cross section of adenocarcinoma of the prostate, lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), and Ewing sarcoma (ES) of the abdomen tumor. Annotated regions are T-tumor, S-stroma, D/G-duct/glandular, and A-adipose tissue. Scale bar is representative of A and B. Images are also used in Fig. S4A, 5D and S6G. B. Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section shown in A. The image displays a heatmap gradient of intensity with white (least abundant) to red (most abundant). Images are also used in Fig. S4E, 5D, and S6G. C. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from tumor regions shown in A. Values are presented as mean +/- standard error (n=3 technical replicates per region).


A . ( Left ) Hematoxylin eosin ( H & E ) - stained cross section of an Ewing sarcoma ( ES ) of shoulder . Annotated regions are annotated as T - M - muscle . ( Right ) Spatial distribution relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . image displays a gradient of from ( least abundant ) to red ( most abundant ) . Scale bar gradient below are representative of in A - E . B . ( Left ) H & E stained cross section of an ES of chest wall . Annotated regions are annotated as T - N - necrosis . ( Right ) Spatial distribution relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . C . ( Left ) H & E stained cross section of an ES of rib . Annotated regions are annotated as T - N - necrosis . ( Right ) Spatial distribution relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . D . ( Left ) H & E stained cross section of an ES of abdomen . Annotated regions are T - , S - stroma , A - adipose tissue . ( Right ) Spatial distribution relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . E . ( Left ) H & E stained cross section of an ES of bladder . Annotated regions are T - S - stroma . ( Right ) Spatial distribution relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . F . Magnified H & E cross section of ( top , left ) , ( top , right ) muscle tissue from ES rib with magnified image of CL7 ( m / z = 1175 ) of ( bottom , left ) , ( bottom , right ) muscle tissues . G . Magnified H & E cross section of ( top , left ) , ( top , middle ) stroma , ( top , right ) adipose tissues from ES abdomen with magnified image of CL7 ( m / z = 1175 ) of ( bottom , left ) , ( bottom , middle ) stroma ( bottom , right ) adipose tissues .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Hematoxylin and eosin (H&E)-stained cross section of an Ewing sarcoma (ES) of the shoulder tumor. Annotated regions are annotated as T-tumor and M-muscle. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. The image displays a heatmap gradient of intensity from white (least abundant) to red (most abundant). Scale bar and intensity gradient below are representative of all images in A-E. B. (Left) H&E stained cross section of an ES of the chest wall tumor. Annotated regions are annotated as T-tumor and N-necrosis. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. C. (Left) H&E stained cross section of an ES of the rib tumor. Annotated regions are annotated as T-tumor and N-necrosis. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. D. (Left) H&E stained cross section of an ES of the abdomen tumor. Annotated regions are T-tumor, S-stroma, and A-adipose tissue. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. E. (Left) H&E stained cross section of an ES of the bladder tumor. Annotated regions are T-tumor and S-stroma. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. F. Magnified H&E cross section of (top, left) tumor, and (top, right) muscle tissue from ES rib with magnified image of CL7 (m/z=1175) of (bottom, left) tumor, and (bottom, right) muscle tissues. G. Magnified H&E cross section of (top, left) tumor, (top, middle) stroma, and (top, right) adipose tissues from ES abdomen with magnified image of CL7 (m/z=1175) of (bottom, left) tumor, (bottom, middle) stroma and (bottom, right) adipose tissues.


A . ( Left ) Hematoxylin eosin ( H & E ) stained cross section of a normal human tibia ( Top ) resected at same time as Ewing sarcoma ( ES ) of tibia ( Bottom ) . Annotated regions are CT - connective tissue , B - decalcified bone , BM - bone marrow , T - , S - stroma . ( Right ) Spatial distribution relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section shown in A . image displays a gradient of with ( least abundant ) to red ( most abundant ) . Scale bar gradient below are representative of in A . B . Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain ( CL ) distribution from normal tibia in A . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . # 0 . 01 < P < 0 . 05 ; * * * * P < 0 . 0001 , analyzed by two - way ANOVA with Tukey ' s multiple comparison for each glycogen chain . C . Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain ( CL ) distribution from ES of tibia in A . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . * * 0 . 001 < P < 0 . 01 ; * * * * P < 0 . 0001 , analyzed by two - t - for each glycogen chain .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Hematoxylin and eosin (H&E) stained cross section of a normal human tibia (Top) resected at the same time as the Ewing sarcoma (ES) of the tibia tumor (Bottom). Annotated regions are CT-connective tissue, B-decalcified bone, BM-bone marrow, T-tumor, and S-stroma. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section shown in A. The image displays a heatmap gradient of intensity with white (least abundant) to red (most abundant). Scale bar and intensity gradient below are representative of all images in A. B. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from normal tibia in A. Values are presented as mean +/- standard error (n=3 technical replicates per region). #0.01 < P < 0.05; ****P<0.0001, analyzed by two-way ANOVA with Tukey's multiple comparison for each glycogen chain length.C. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from the ES of the tibia tumor in A. Values are presented as mean +/- standard error (n=3 technical replicates per region). **0.001 < P < 0.01; ****P<0.0001, analyzed by two-tailed t-test for each glycogen chain length.


F . In vivo growth of A673 EV or GYS1 - KO injected in flanks of athymic nude mice . Values are presented as mean + / - standard error ( n = 8 biological replicates per group ) . * * * * P < 0 . 0001 , analyzed by two - t - .

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

F. In vivo tumor growth of A673 EV or GYS1-KO injected in the flanks of athymic nude mice. Values are presented as mean +/- standard error (n=8 biological replicates per group). ****P<0.0001, analyzed by two-tailed t-test.


G . Half maximal effective ( EC50 ) of small molecular inhibitor guaiacol in A673 in vitro .

G. Half maximal effective concentration (EC50) of the small molecular inhibitor guaiacol in A673 cells in vitro.


J . Spatial distribution of phosphorylated CL5 of human tibia ( top ) tibial ES ( bottom ) . image displays a gradient of with black ( least abundant ) to yellow ( most abundant ) . K . Phosphorylated chain distribution in normal human tibia tibial ES in J . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . * * * * P < 0 . 0001 , analyzed by two - t - for each glycogen chain .

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

J. Spatial distribution of phosphorylated CL5 of human tibia (top) and tibial ES tumor (bottom). The image displays a heatmap gradient of intensity with black (least abundant) to yellow (most abundant). K. Phosphorylated chain length distribution in normal human tibia and tibial ES tumor in J. Values are presented as mean +/- standard error (n=3 technical replicates per region). ****P<0.0001, analyzed by two-tailed t-test for each glycogen chain length.


N . Representatives image of immunohistochemical of laforin in ES samples with annotation between ( T ) stroma ( S ) tissues . Tissues were scanned digitally using Axio Scan . Z1 side scanner . Scale bar is shown below image . O . of immunohistochemical laforin between normal tissue ( n = 3 technical replicates per region ) , tibial ES ( n = 7 technical replicates per region ) , stroma of five ES tissues ( n = 7 ) . Values are presented as mean + / - standard error . * * 0 . 001 < P < 0 . 0 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 , analyzed by one - way ANOVA with Tukey ' s multiple comparison .

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 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

N. Representatives image of immunohistochemical staining of laforin in ES patient samples with annotation between tumor (T) and stroma (S) tissues. Tissues were scanned digitally using the Axio Scan.Z1 side scanner. Scale bar is shown below the image. O. Quantification of immunohistochemical laforin staining between normal tissue (n=3 technical replicates per region), tibial ES tumor (n=7 technical replicates per region), and stroma of five ES tissues (n=7). Values are presented as mean +/- standard error. **0.001 < P < 0.0; ***P < 0.001; ****P<0.0001, analyzed by one-way ANOVA with Tukey's multiple comparison.


P . ( Top ) Western blot showing confirmation of laforin overexpression in A673 xenograft tumors . ( Bottom ) growth of xenografts of A673 empty vector ( EV ) laforin overexpression ( LAOE ) . Values are presented as mean + / - standard error ( n = 8 biological replicates per group ) . * * * * P < 0 . 0001 , analyzed by two - t - .

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-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

P. (Top) Western blot showing confirmation of laforin overexpression in A673 xenograft tumors. (Bottom) Tumor growth of xenografts of A673 empty vector (EV) and laforin overexpression (LAOE). Values are presented as mean +/- standard error (n=8 biological replicates per group). ****P<0.0001, analyzed by two-tailed t-test.


A . From left to right , Surface representation of three - dimensional structures of C3b - MCP ( CCP1 - 4 ) ( blue ) , C3b - SPICE ( CCP1 - 4 ) ( green ) , C3b - FH ( CCP1 - 4 ) ( orange ) , C3b - CR1 ( CCP15 - 17 ) ( red ) , C3b - DAF ( CCP2 - 4 ) ( purple ) .

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

A. From left to right, Surface representation of the three-dimensional structures of C3b-MCP (CCP1-4) (blue), C3b-SPICE (CCP1-4) (green), C3b-FH (CCP1-4) (orange), C3b-CR1 (CCP15-17) (dark red), C3b-DAF (CCP2-4) (purple).


B . Cartoon representation of five regulators . width of cartoon is proportional to B - factors observed in structural data . B - factor coloring ( from blue to red ) is in relative scale according to resolution of crystal structures .

O O O O O O O O O O O O O O O O O O O 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. Cartoon representation of the five regulators. The width of the cartoon is proportional to the B-factors observed in the structural data. The B-factor coloring (from blue to red) is in relative scale according to the resolution of the crystal structures.


C . Cartoon representation of five C3b molecules in C3b - regulator complexes . Coloring style as in B .

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

C. Cartoon representation of the five C3b molecules in the C3b-regulator complexes. Coloring and style as in B.


D . Highlight of electron density maps ( 2Fo - Fc , blue , contour level 1 . 2σ ; Fo - Fc , green red , contour level 3σ - 3σ , resp . ) for regions of C3b - regulator complexes showing flexibility . From left to right : putative site for MCP CCP2 , SPICE CCP4 , CR1 CCP15 , DAF CCP2 .

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

D. Highlight of electron density maps (2Fo-Fc, blue, contour level 1.2σ; Fo-Fc, green and red, contour level 3σ and -3σ, resp.) for the regions of the C3b-regulator complexes showing flexibility. From left to right: the putative site for MCP CCP2, SPICE CCP4, CR1 CCP15, and DAF CCP2.