c : Detection of ADE - induced ZIKV infection in the presence of the ZIKV - specific neutralizing antibody 2B10 and IC - EVs derived from ZIKV - infected cells by ADE assay . K562 cells were incubated with a mixture of EV - free ZIKV , neutralizing antibody , and IC - EVs . Forty - eight hours post incubation ; cells were stained for ZIKV E and analyzed by FACS . Data represent the means from three biological replicates ; error bars represent standard deviations from the means . Differences between treatments were analyzed using the two - tailed , unpaired Student t test available in the GraphPad Prism 5 software package . P values of less than 0 . 05 ( indicated by asterisks ) were considered statistically significant . The following categories were used : * * * , P 0 . 001 ; * * , P 0 . 01 ; * , P 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Detection of ADE-induced ZIKV infection in the presence of the ZIKV-specific neutralizing antibody 2B10 and IC-EVs derived from ZIKV-infected cells by ADE assay. K562 cells were incubated with a mixture of EV-free ZIKV, neutralizing antibody, and IC-EVs. Forty-eight hours post incubation; cells were stained for ZIKV E and analyzed by FACS. Data represent the means from three biological replicates; error bars represent standard deviations from the means. Differences between treatments were analyzed using the two-tailed, unpaired Student t test available in the GraphPad Prism 5 software package. P values of less than 0.05 (indicated by asterisks) were considered statistically significant. The following categories were used: ***, P 0.001; **, P 0.01; *, P 0.05.


c / d : IC - EVs affected the ADE of DENV2 ( c ) or ZIKV ( d ) mediated by DENV3 patient serum . Diluted sera were mixed with EV - free viruses ( DENV2 or ZIKV ) and IC - EVs derived from DENV2 - or ZIKV - infected cells and then incubated with K562 cells as described in the methods . At 48 hours post incubation , cells were stained with an antibody against flavivirus E and analyzed by FACS . Data represent the means from three biological replicates ; error bars represent standard deviations from the means . Differences between treatments were analyzed using the two - tailed , unpaired Student t test available in the GraphPad Prism 5 software package . P values of less than 0 . 05 ( indicated by asterisks ) were considered statistically significant . The following categories were used : * * * , P 0 . 001 ; * * , P 0 . 01 ; * , P 0 . 05 .

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

c/d: IC-EVs affected the ADE of DENV2 (c) or ZIKV (d) mediated by DENV3 patient serum. Diluted sera were mixed with EV-free viruses (DENV2 or ZIKV) and IC-EVs derived from DENV2- or ZIKV-infected cells and then incubated with K562 cells as described in the methods. At 48 hours post incubation, cells were stained with an antibody against flavivirus E and analyzed by FACS. Data represent the means from three biological replicates; error bars represent standard deviations from the means. Differences between treatments were analyzed using the two-tailed, unpaired Student t test available in the GraphPad Prism 5 software package. P values of less than 0.05 (indicated by asterisks) were considered statistically significant. The following categories were used: ***, P 0.001; **, P 0.01; *, P 0.05.


The structure of SARS - CoV - 2 monomer .

The structure of SARS-CoV-2 monomer.


SDS - PAGE of SecYEG - ND sample after size - exclusion chromatography . Asterisks indicate translocon - enriched fractions used for forming the RNC FtsQ : SecYEG - ND complex . Lipid - loaded " empty " nanodiscs elute at larger volumes and so can be separated .

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

SDS-PAGE of SecYEG-ND sample after size-exclusion chromatography. Asterisks indicate translocon-enriched fractions used for forming the RNC FtsQ:SecYEG-ND complex. Lipid-loaded "empty" nanodiscs elute at larger volumes and so can be separated.


Representative cryo - EM micrograph of RNC FtsQ : SecYEG - ND . Exemplary individual ribosomes are encircled . Examples of two - dimensional classes of imaged particles . RNC : nanodisc assemblies can be seen at different view angles .

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

Representative cryo-EM micrograph of RNC FtsQ:SecYEG-ND. Exemplary individual ribosomes are encircled. Examples of two-dimensional classes of imaged particles. RNC:nanodisc assemblies can be seen at different view angles.


Western - blot against the hemagglutinin tag within the nascent chain FtsQC40 reveals a cross - linking product of ~ 80 kDa in presence of nanodisc - reconstituted SecYC282EG and SecYC283EG , presumably assigned to FtsQxSecY adduct . The double band for FtsQC40 likely indicates the incomplete cleavage of the hexa - histidine tag by 3C protease .

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

Western-blot against the hemagglutinin tag within the nascent chain FtsQC40 reveals a cross-linking product of ~80 kDa in presence of nanodisc-reconstituted SecYC282EG and SecYC283EG, presumably assigned to FtsQxSecY adduct. The double band for FtsQC40 likely indicates the incomplete cleavage of the hexa-histidine tag by 3C protease.


In - gel fluorescence imaging of SecYCF488A reveals a cross - linking product of ~ 80 kDa assigned to the FtsQC40xSecYC283 adduct ( " SecYxFtsQ " ) . Bands for covalently cross - linked SecY dimer ( " SecYxSecY " ) , and adducts of SecY and ribosomal proteins ( " SecYxRiboP " ) are indicated . The nascent chain - specific adduct " SecYxFtsQ " does not depend on the presence of PE lipids .

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

In-gel fluorescence imaging of SecYCF488A reveals a cross-linking product of ~80 kDa assigned to the FtsQC40xSecYC283 adduct ("SecYxFtsQ"). Bands for covalently cross-linked SecY dimer ("SecYxSecY"), and adducts of SecY and ribosomal proteins ("SecYxRiboP") are indicated. The nascent chain-specific adduct "SecYxFtsQ" does not depend on the presence of PE lipids.


FtsQ : SecY cross - linking is position specific . FtsQ TMH C - terminal residues 40 - 42 could be cross - linked to the lateral gate ( SecY residue 283 ) , while no adduct could be detected at the upstream position 35 .

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

FtsQ:SecY cross-linking is position specific. FtsQ TMH C-terminal residues 40-42 could be cross-linked to the lateral gate (SecY residue 283), while no adduct could be detected at the upstream position 35.


D . Relative FCR activities in the roots of wild - type plants and photoreceptor mutants grown under white light with either Fe - sufficient ( 20 µM , + Fe ) or Fe - deficient ( 2 µM , - Fe ) conditions for 7 d .

O O O O O O O O O O O O O O O O O O O O O O O O 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. Relative FCR activities in the roots of wild-type plants and photoreceptor mutants grown under white light with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.


D - F . Fe contents of whole plants , FER expression in roots and relative FCR activities in the roots of plants grown under Fe - sufficient ( 20 µM , + Fe ) or Fe - deficient ( 2 µM , - Fe ) conditions for 7 d . Plants were exposed to high ( H - , 2 . 0 ) or low ( L - , 0 . 5 ) red ( R ) to far red ( FR ) ratios .

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

D-F. Fe contents of whole plants, FER expression in roots and relative FCR activities in the roots of plants grown under Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d. Plants were exposed to high (H-, 2.0) or low (L-, 0.5) red (R) to far red (FR) ratios.


C . Accumulation of HY5 protein in leaves and roots of reciprocal grafts of phyB and WT plants grown under white light ( 200 µmol m - 2s - 1 ) for 7 d . Rubisco and β - Actin were used as loading controls for leaves and roots in the western blot analysis , respectively . The values shown above each lane indicate the relative abundance of the HY5 protein . D . Accumulation of HY5 protein in the roots of WT and phyB plants under high ( H , 2 . 0 ) or low ratio of ( L , 0 . 5 ) red ( R ) to far red ( FR ) light conditions . β - Actin was used as loading controls in the western blot analysis . The values shown above each lane indicate the relative abundance of the HY5 protein .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Accumulation of HY5 protein in leaves and roots of reciprocal grafts of phyB and WT plants grown under white light (200 µmol m-2s-1) for 7 d. Rubisco and β-Actin were used as loading controls for leaves and roots in the western blot analysis, respectively. The values shown above each lane indicate the relative abundance of the HY5 protein. D. Accumulation of HY5 protein in the roots of WT and phyB plants under high (H, 2.0) or low ratio of (L, 0.5) red (R) to far red (FR) light conditions. β-Actin was used as loading controls in the western blot analysis. The values shown above each lane indicate the relative abundance of the HY5 protein.


D . Fe content of whole plants . WT and hy5 plants were grown under high ( H ) or low ratios of ( L ) red ( R ) to far red ( FR ) light conditions with either Fe - sufficient ( 20 µM , + Fe ) or Fe - deficient ( 2 µM , - Fe ) conditions for 7 d .

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

D. Fe content of whole plants. WT and hy5 plants were grown under high (H) or low ratios of (L) red (R) to far red (FR) light conditions with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.


E . FER expression in roots . WT and hy5 plants were grown under high ( H ) or low ratios of ( L ) red ( R ) to far red ( FR ) light conditions with either Fe - sufficient ( 20 µM , + Fe ) or Fe - deficient ( 2 µM , - Fe ) conditions for 7 d .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. FER expression in roots. WT and hy5 plants were grown under high (H) or low ratios of (L) red (R) to far red (FR) light conditions with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.


E . Direct binding of HY5 to the FER promoter analyzed using ChIP - qPCR in 35S - HY5 - 3HA - overexpressing ( HY5 - OE ) tomato plants . WT and HY5 - OE plants were exposed to white light ( 200 µmol m - 2s - 1 ) grown under Fe - deficient ( 2 µM , - Fe ) conditions for 7 d . Input chromatin was isolated from root samples at 7 d . The epitope - tagged HY5 - chromatin complex was immunoprecipitated with an anti - HA antibody . A control reaction was processed side - by - side using mouse IgG . Input - and ChIP - DNA samples were quantified by RT - qPCR using primers specific for the promoter and exon fragment of the FER gene as indicated in ( D ) . The ChIP results are presented as percentage of the input DNA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Direct binding of HY5 to the FER promoter analyzed using ChIP-qPCR in 35S-HY5-3HA-overexpressing (HY5-OE) tomato plants. WT and HY5-OE plants were exposed to white light (200 µmol m-2s-1) grown under Fe-deficient (2 µM, -Fe) conditions for 7 d. Input chromatin was isolated from root samples at 7 d. The epitope-tagged HY5-chromatin complex was immunoprecipitated with an anti-HA antibody. A control reaction was processed side-by-side using mouse IgG. Input- and ChIP-DNA samples were quantified by RT-qPCR using primers specific for the promoter and exon fragment of the FER gene as indicated in (D). The ChIP results are presented as percentage of the input DNA.


A - C . Fe content of whole plants ( A ) and the photochemical efficiencies of PSI and PSII [ Y ( I ) and Y ( II ) ] ( B and C ) in young leaves of grafted tomato plants with grafts between WT / WT and WT / fer grown under H - R / FR ( 2 . 0 ) or L - R / FR ( 0 . 5 ) conditions for 7 d .

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

A-C. Fe content of whole plants (A) and the photochemical efficiencies of PSI and PSII [Y(I) and Y(II)] (B and C) in young leaves of grafted tomato plants with grafts between WT/WT and WT/fer grown under H-R/FR (2.0) or L-R/FR (0.5) conditions for 7 d.


D - F . Transcript levels of FER , FRO1 and IRT1 in the roots of grafted tomato plants with grafts between WT / WT and WT / fer grown under H - R / FR ( 2 . 0 ) or L - R / FR ( 0 . 5 ) conditions for 7 d .

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

D-F. Transcript levels of FER, FRO1 and IRT1 in the roots of grafted tomato plants with grafts between WT/WT and WT/fer grown under H-R/FR (2.0) or L-R/FR (0.5) conditions for 7 d.


B . The abundance of FER transcripts in the roots of WT and phyB plants grown under 20 μM Fe - EDTA in nutrient solution in a growth room .

O O O O O O 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. The abundance of FER transcripts in the roots of WT and phyB plants grown under 20 μM Fe-EDTA in nutrient solution in a growth room.


D . Fe content in the fruits of WT and phyB plants . Plants were grown in soil in a greenhouse .

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

D. Fe content in the fruits of WT and phyB plants. Plants were grown in soil in a greenhouse.


( A ) Coomassie ‐ stained gels showing the results of liposome co ‐ sedimentation assays using Folch liposomes and the indicated proteins . Liposome binding allows the protein to be pelleted . The star indicates a degradation product of the Atg5 - Atg12 conjugate .

(A) Coomassie‐stained gels showing the results of liposome co‐sedimentation assays using Folch liposomes and the indicated proteins. Liposome binding allows the protein to be pelleted. The star indicates a degradation product of the Atg5-Atg12 conjugate.


( B , D ) Yeast cells of the indicated genotype were transformed with plasmids coding for Atg5 and Atg16 , respectively , and subjected to fractionation experiments . The proteins were detected by anti ‐ Myc western blotting . The presence of the protein in the pellet fraction indicated membrane binding .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D) Yeast cells of the indicated genotype were transformed with plasmids coding for Atg5 and Atg16, respectively, and subjected to fractionation experiments. The proteins were detected by anti‐Myc western blotting. The presence of the protein in the pellet fraction indicated membrane binding.


( C ) Quantification showing the relative amounts of Atg5 - 9 × Myc and Atg5 - 9 × Myc - Atg12 in the pellet fractions relative to Pex30 . The amount of Atg5 - 9 × Myc and Atg5 - 9 × Myc - Atg12 in atg5Δ cells was set to 1 . See Supplementary Figure 1 for graph based on non ‐ normed data . The quantification is based on three independent experiments and the averages and the standard deviations are shown . The numbers next to the blots indicate the molecular weight in kDa . P , pellet ; S , supernatant . Figure source data can be found with the Supplementary data .

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

(C) Quantification showing the relative amounts of Atg5-9 × Myc and Atg5-9 × Myc-Atg12 in the pellet fractions relative to Pex30. The amount of Atg5-9 × Myc and Atg5-9 × Myc-Atg12 in atg5Δ cells was set to 1. See Supplementary Figure 1 for graph based on non‐normed data. The quantification is based on three independent experiments and the averages and the standard deviations are shown. The numbers next to the blots indicate the molecular weight in kDa. P, pellet; S, supernatant. Figure source data can be found with the Supplementary data.


( A ) Coomassie ‐ stained gels of liposome co ‐ sedimentation assays using either Atg5 - Atg12 / Atg16 ( upper panel ) or Atg5 / Atg16 ( lower panel ) and liposomes with the indicated lipid composition ( Folch : Folch lipids , PE / PI3P : 40 % POPC , 35 % POPS , 20 % POPE , 5 % PI3P , PE / PI : 40 % POPC , 35 % POPS , 20 % POPE , 5 % PI , DAG / PI3P : 40 % POPC , 35 % POPS , 20 % DAG , 5 % PI3P , DAG / PI : 40 % POPC , 35 % POPS , 20 % DAG , 5 % PI ) . Note that the Atg5 - Atg12 / Atg16 and the Atg5 / Atg16 complexes show almost identical binding behaviours .

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

(A) Coomassie‐stained gels of liposome co‐sedimentation assays using either Atg5-Atg12/Atg16 (upper panel) or Atg5/Atg16 (lower panel) and liposomes with the indicated lipid composition (Folch: Folch lipids, PE/PI3P: 40% POPC, 35% POPS, 20% POPE, 5% PI3P, PE/PI: 40% POPC, 35% POPS, 20% POPE, 5% PI, DAG/PI3P: 40% POPC, 35% POPS, 20% DAG, 5% PI3P, DAG/PI: 40% POPC, 35% POPS, 20% DAG, 5% PI). Note that the Atg5-Atg12/Atg16 and the Atg5/Atg16 complexes show almost identical binding behaviours.


( B ) Quantification showing the amounts of Atg5 - Atg12 / Atg16 and Atg5 / Atg16 in the pellet fractions . The amount of protein in the pellet fraction in the absence of liposomes ( background ) was subtracted from the amount of protein in the pellet fractions in the presence of liposomes . Data were obtained from three independent experiments , one of which is shown in ( A ) . The Atg5 - Atg12 and Atg5 bands were used for quantification .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification showing the amounts of Atg5-Atg12/Atg16 and Atg5/Atg16 in the pellet fractions. The amount of protein in the pellet fraction in the absence of liposomes (background) was subtracted from the amount of protein in the pellet fractions in the presence of liposomes. Data were obtained from three independent experiments, one of which is shown in (A). The Atg5-Atg12 and Atg5 bands were used for quantification.


( C ) Coomassie ‐ stained gel of a liposome co ‐ sedimentation assay showing the lipid requirements of the Atg5 - Atg12 / Atg16 complex . Liposomes were composed of 40 % POPC , 35 % POPS , 20 % POPE and 5 % PI3P . In the liposomes containing no POPE or POPS , these lipids were replaced with POPC .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Coomassie‐stained gel of a liposome co‐sedimentation assay showing the lipid requirements of the Atg5-Atg12/Atg16 complex. Liposomes were composed of 40% POPC, 35% POPS, 20% POPE and 5% PI3P. In the liposomes containing no POPE or POPS, these lipids were replaced with POPC.


( D ) Quantification of Atg5 - Atg12 / Atg16 in the pellet fraction in liposome co ‐ sedimentation , one of which is shown in ( C ) . Data were obtained from six independent experiments . The Atg5 - Atg12 was used for quantification . The average value is shown and the error bars represent the standard deviation . P ‐ values were calculated using Student ' s t ‐ test . The numbers next to the gels indicate the molecular weight in kDa . DAG , diacylglycerol ; P , pellet ; PI , phosphatidylinositol ; PI3P , phosphatidylinositol ‐ 3 ‐ phosphate ; ( PO ) PC , ( 1 ‐ palmitoyl ‐ 2 ‐ oleoyl ‐ ) phosphatidylcholine ; ( PO ) PE , ( 1 ‐ palmitoyl ‐ 2 ‐ oleoyl ‐ ) phosphatidylethanolamine ; ( PO ) PS , ( 1 ‐ palmitoyl ‐ 2 ‐ oleoyl ‐ ) phosphatidylserine ; S , supernatant . Figure source data can be found with the Supplementary data .

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

(D) Quantification of Atg5-Atg12/Atg16 in the pellet fraction in liposome co‐sedimentation, one of which is shown in (C). Data were obtained from six independent experiments. The Atg5-Atg12 was used for quantification. The average value is shown and the error bars represent the standard deviation. P‐values were calculated using Student's t‐test. The numbers next to the gels indicate the molecular weight in kDa. DAG, diacylglycerol; P, pellet; PI, phosphatidylinositol; PI3P, phosphatidylinositol‐3‐phosphate; (PO)PC, (1‐palmitoyl‐2‐oleoyl‐) phosphatidylcholine; (PO)PE, (1‐palmitoyl‐2‐oleoyl‐) phosphatidylethanolamine; (PO)PS, (1‐palmitoyl‐2‐oleoyl‐) phosphatidylserine; S, supernatant. Figure source data can be found with the Supplementary data.


( A ) Coomassie ‐ stained gels showing co ‐ sedimentation assays using Folch liposomes and the indicated proteins . Atg5 - Atg12 / Atg16 was most efficient in recruiting Atg3 to the pellet fraction .

O O O O O O O O O O O O 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) Coomassie‐stained gels showing co‐sedimentation assays using Folch liposomes and the indicated proteins. Atg5-Atg12/Atg16 was most efficient in recruiting Atg3 to the pellet fraction.


( B ) Quantification of Atg3 in the pellet fraction in the presence of the indicated proteins . The quantification is based on three independent experiments .

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

(B) Quantification of Atg3 in the pellet fraction in the presence of the indicated proteins. The quantification is based on three independent experiments.


( C ) Coomassie ‐ stained gels showing the results of Atg8 - PE conjugation assays using the indicated proteins and E . coli lipids derived liposomes . Atg8 - PE conjugation was detected as characteristic band shift as the conjugated form runs faster on urea ‐ SDS gels . The numbers above the gels indicate the time in minutes . Note that at least under these conditions almost no Atg8 - PE conjugation was detected in the absence of the Atg5 - Atg12 conjugate or the Atg5 - Atg12 / Atg16 complex . The numbers next to the gels indicate the molecular weight in kDa . The averages and the error bars representing the standard deviations are shown . P ‐ values were calculated using Student ' s t ‐ test . NS , non ‐ significant ; P , pellet ; S , supernatant . Figure source data can be found with the Supplementary data .

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 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 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

(C) Coomassie‐stained gels showing the results of Atg8-PE conjugation assays using the indicated proteins and E. coli lipids derived liposomes. Atg8-PE conjugation was detected as characteristic band shift as the conjugated form runs faster on urea‐SDS gels. The numbers above the gels indicate the time in minutes. Note that at least under these conditions almost no Atg8-PE conjugation was detected in the absence of the Atg5-Atg12 conjugate or the Atg5-Atg12/Atg16 complex. The numbers next to the gels indicate the molecular weight in kDa. The averages and the error bars representing the standard deviations are shown. P‐values were calculated using Student's t‐test. NS, non‐significant; P, pellet; S, supernatant. Figure source data can be found with the Supplementary data.


( A ) Confocal microscopy images showing eGFP - Atg8 recruitment to GUVs is dependent on the Atg5 - Atg12 / Atg16 complex and PE . The membrane was labelled by incorporation of rhodamine ‐ PE .

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

(A) Confocal microscopy images showing eGFP-Atg8 recruitment to GUVs is dependent on the Atg5-Atg12/Atg16 complex and PE. The membrane was labelled by incorporation of rhodamine‐PE.


( B ) Single confocal section showing colocalization of Atg5 - Atg12 / Atg16 - eGFP and mCherry - Atg8 on GUVs .

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

(B) Single confocal section showing colocalization of Atg5-Atg12/Atg16-eGFP and mCherry-Atg8 on GUVs.


( C ) Quantification of GUV ‐ associated eGFP - Atg8 in the presence of the Atg5 - Atg12 / Atg16 complex and the Atg5 - Atg12 conjugate . The first graph shows the percentage of GUVs associated with eGFP - Atg8 fluorescence . The second graph shows the intensity of eGFP - Atg8 on the GUVs . The quantification is based on four independent experiments . Inset : anti ‐ Atg12 western blot using the rabbit anti ‐ Atg12 antiserum showing the amounts of Atg5 - Atg12 / Atg16 complex and Atg5 - Atg12 conjugate in one of the assays used for the quantification shown in ( C ) .

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(C) Quantification of GUV‐associated eGFP-Atg8 in the presence of the Atg5-Atg12/Atg16 complex and the Atg5-Atg12 conjugate. The first graph shows the percentage of GUVs associated with eGFP-Atg8 fluorescence. The second graph shows the intensity of eGFP-Atg8 on the GUVs. The quantification is based on four independent experiments. Inset: anti‐Atg12 western blot using the rabbit anti‐Atg12 antiserum showing the amounts of Atg5-Atg12/Atg16 complex and Atg5-Atg12 conjugate in one of the assays used for the quantification shown in (C).


( D ) Quantification of membrane recruitment of Atg3 - mCherry by the Atg5 - Atg12 / Atg16 complex , the Atg5 - Atg12 conjugate and a buffer control . The graph is based on two independent experiments .

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(D) Quantification of membrane recruitment of Atg3-mCherry by the Atg5-Atg12/Atg16 complex, the Atg5-Atg12 conjugate and a buffer control. The graph is based on two independent experiments.


( E ) Widefield images showing individual frames of a time series of GUVs incubated with Atg3 , Atg7 , Atg8 and the Atg5 - Atg12 / Atg16 complex ( see Materials and methods for details ) . The membrane was labelled by incorporation of rhodamine ‐ PE . The time is indicated in minutes .

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(E) Widefield images showing individual frames of a time series of GUVs incubated with Atg3, Atg7, Atg8 and the Atg5-Atg12/Atg16 complex (see Materials and methods for details). The membrane was labelled by incorporation of rhodamine‐PE. The time is indicated in minutes.


( F ) Widefield images of GUVs incubated with Atg3 , Atg7 , eGFP - Atg8 and the Atg5 - Atg12 / Atg16 complex for 35 min ( left ) or 70 min ( right ) . The membrane was labelled by incorporation of rhodamine ‐ PE . Note the massive accumulation of eGFP - Atg8 at the interface between individual GUVs . The arrows point to some of these interfaces . For the quantifications , the averages and standard deviations are shown . P ‐ values were calculated using Student ' s t ‐ test . Scale bars : 10 μm . Figure source data can be found with the Supplementary data .

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(F) Widefield images of GUVs incubated with Atg3, Atg7, eGFP-Atg8 and the Atg5-Atg12/Atg16 complex for 35 min (left) or 70 min (right). The membrane was labelled by incorporation of rhodamine‐PE. Note the massive accumulation of eGFP-Atg8 at the interface between individual GUVs. The arrows point to some of these interfaces. For the quantifications, the averages and standard deviations are shown. P‐values were calculated using Student's t‐test. Scale bars: 10 μm. Figure source data can be found with the Supplementary data.


( A ) Frames of widefield images taken from a time series of rhodamine ‐ PE labelled GUVs in the presence of the Atg5 - Atg12 / Atg16 complex . For the control condition buffer but no protein was added . Note the increase in clustered GUVs over time . The numbers indicate the time in minutes .

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(A) Frames of widefield images taken from a time series of rhodamine‐PE labelled GUVs in the presence of the Atg5-Atg12/Atg16 complex. For the control condition buffer but no protein was added. Note the increase in clustered GUVs over time. The numbers indicate the time in minutes.


( B ) Confocal images of rhodamine ‐ PE labelled GUVs incubated with Atg5 - Atg12 / Atg16 - eGFP complex . Note the accumulation of the complex at the interface between GUVs ( arrows ) .

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(B) Confocal images of rhodamine‐PE labelled GUVs incubated with Atg5-Atg12/Atg16-eGFP complex. Note the accumulation of the complex at the interface between GUVs (arrows).


( C ) Individual confocal microscopy frames of a time series of rhodamine ‐ PE labelled GUVs incubated with Atg5 - Atg12 / Atg16 - eGFP . The arrows point to the GUV - GUV interface where the protein initially accumulated .

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(C) Individual confocal microscopy frames of a time series of rhodamine‐PE labelled GUVs incubated with Atg5-Atg12/Atg16-eGFP. The arrows point to the GUV-GUV interface where the protein initially accumulated.


( D ) Confocal microscopy images of rhodamine ‐ PE labelled GUVs incubated with Atg5 - Atg12 / Atg16 - eGFP . The arrows point to the concentrations of Atg5 - Atg12 / Atg16 on the GUV membrane . Scale bars : 10 μm ( A - C ) and 3 μm ( D ) .

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(D) Confocal microscopy images of rhodamine‐PE labelled GUVs incubated with Atg5-Atg12/Atg16-eGFP. The arrows point to the concentrations of Atg5-Atg12/Atg16 on the GUV membrane. Scale bars: 10 μm (A-C) and 3 μm (D).


( A ) Widefield images of rhodamine ‐ PE labelled GUVs 30 min GUVs cluster only in the presence of the Atg5 - Atg12 / Atg16 and Atg5 / Atg16 complexes .

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(A) Widefield images of rhodamine‐PE labelled GUVs 30 min GUVs cluster only in the presence of the Atg5-Atg12/Atg16 and Atg5/Atg16 complexes.


( B ) SUV aggregation assay using Folch liposomes and the indicated proteins . The proteins were added after a short delay . The symbols above , below the images or next to the curves represent Atg5 ( blue oval ) , Atg12 ( red circle ) and Atg16 ( green Y ) . The overall shape of the Atg5 - Atg12 / Atg16 complex is according to the structural model of the complex as proposed in Fujioka et al ( 2010 ) . Scale bars : 10 μm .

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(B) SUV aggregation assay using Folch liposomes and the indicated proteins. The proteins were added after a short delay. The symbols above, below the images or next to the curves represent Atg5 (blue oval), Atg12 (red circle) and Atg16 (green Y). The overall shape of the Atg5-Atg12/Atg16 complex is according to the structural model of the complex as proposed in Fujioka et al (2010). Scale bars: 10 μm.


( A ) Liposome co ‐ sedimentation assay using Folch liposomes and the indicated Atg5 proteins stabilized with an N ‐ terminal fragment of Atg16 ( amino acids 1 - 46 ) .

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(A) Liposome co‐sedimentation assay using Folch liposomes and the indicated Atg5 proteins stabilized with an N‐terminal fragment of Atg16 (amino acids 1-46).


( B ) Quantification based on three co ‐ sedimentation assays as shown in ( A ) . The amount of protein pelleted in the absence of liposomes was subtracted . The averages and the standard deviations are shown .

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(B) Quantification based on three co‐sedimentation assays as shown in (A). The amount of protein pelleted in the absence of liposomes was subtracted. The averages and the standard deviations are shown.


( C ) Yeast cells were transformed with the indicated expression constructs and Atg5 - 2 × Myc was immunoprecipitated with an anti ‐ Myc antibody . The precipitated protein was subjected to anti ‐ Atg5 and anti ‐ Atg12western blotting . The substitution of R171 and K160 to glutamate caused the mutant protein to run higher . The same phenomenon was observed for the recombinant protein ( Supplementary Figure 3 ) .

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(C) Yeast cells were transformed with the indicated expression constructs and Atg5-2 × Myc was immunoprecipitated with an anti‐Myc antibody. The precipitated protein was subjected to anti‐Atg5 and anti‐Atg12western blotting. The substitution of R171 and K160 to glutamate caused the mutant protein to run higher. The same phenomenon was observed for the recombinant protein ( Supplementary Figure 3).


( D ) Anti ‐ Atg12 western blot of cell fractions prepared from rapamycin ‐ treated yeast cells . The anti ‐ Pgk1 and anti ‐ Pex30 served as control for the cytosolic and membrane fractions , respectively .

(D) Anti‐Atg12 western blot of cell fractions prepared from rapamycin‐treated yeast cells. The anti‐Pgk1 and anti‐Pex30 served as control for the cytosolic and membrane fractions, respectively.


( E ) Anti ‐ Myc and anti ‐ Pex30western blots of yeast cell fractions from cells of the indicated genotype and expressing Atg16 and Atg5 or Atg5 K160E , R171E .

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(E) Anti‐Myc and anti‐Pex30western blots of yeast cell fractions from cells of the indicated genotype and expressing Atg16 and Atg5 or Atg5 K160E, R171E.


( F ) Starvation assay with the indicated yeast strains and the indicated rescue constructs . GFP was detected by western blotting . Pgk1 served as loading control .

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(F) Starvation assay with the indicated yeast strains and the indicated rescue constructs. GFP was detected by western blotting. Pgk1 served as loading control.


( G ) Same assay as shown in ( F ) but using an anti ‐ Ape1 antiserum for western blotting .

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(G) Same assay as shown in (F) but using an anti‐Ape1 antiserum for western blotting.


( H ) Western blots of yeast cell lysates using an anti ‐ Atg8 antiserum .

(H) Western blots of yeast cell lysates using an anti‐Atg8 antiserum.


( I ) Ape1 protease protection assay with the indicated yeast strains . The percentages below the blots indicate the percent of protected ApeI in the + protease K , − Triton X ‐ 100 lanes and are the average of three independent experiments ± the standard deviation . The numbers next to the blots indicate the molecular weight in kDa . Figure source data can be found with the Supplementary data .

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(I) Ape1 protease protection assay with the indicated yeast strains. The percentages below the blots indicate the percent of protected ApeI in the +protease K, −Triton X‐100 lanes and are the average of three independent experiments±the standard deviation. The numbers next to the blots indicate the molecular weight in kDa. Figure source data can be found with the Supplementary data.


( A ) Yeast cells of the indicated genotype expressing Atg5 - 3 × mCherry or Atg5 K160E , R171E - 3 × mCherry were treated with rapamycin and imaged using a Deltavision microscope . Maximum projections of z ‐ stacks are shown .

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(A) Yeast cells of the indicated genotype expressing Atg5-3 × mCherry or Atg5 K160E, R171E-3 × mCherry were treated with rapamycin and imaged using a Deltavision microscope. Maximum projections of z‐stacks are shown.


( B ) Graph showing the percentage of mCherry ‐ positive cells that display Atg5 - 3 × mCherry dots . In total , 3 independent experiments were conducted and > 400 cells were counted .

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(B) Graph showing the percentage of mCherry‐positive cells that display Atg5-3 × mCherry dots. In total, 3 independent experiments were conducted and >400 cells were counted.


( C ) Graph showing the percentage of cells that display Atg16 - 3 × mCherry dots . In total , 3 independent experiments were conducted and > 750 cells were counted . The graphs show the averages and the error bars representing the standard deviations . P ‐ values were calculated using Student ' s t ‐ test . Scale bars : 5 μm .

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(C) Graph showing the percentage of cells that display Atg16-3 × mCherry dots. In total, 3 independent experiments were conducted and >750 cells were counted. The graphs show the averages and the error bars representing the standard deviations. P‐values were calculated using Student's t‐test. Scale bars: 5 μm.


A . Frequencies and total numbers ( mean + SEM ) of CD11b + myeloid cells isolated from the spleen and peripheral LN of naïve non - immunized mice .

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A. Frequencies and total numbers (mean +SEM) of CD11b+myeloid cells isolated from the spleen and peripheral LN of naïve non-immunized mice.


B . Total numbers ( mean + SEM ) of CD11b + cells expressing IL - 1β shown in ( A ) and restimulated with indicated stimuli for 4 h .

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B. Total numbers (mean +SEM) of CD11b+ cells expressing IL-1β shown in (A) and restimulated with indicated stimuli for 4 h.


C . Quantification ( mean + SEM ) of myeloid cell populations expressing IL - 1β , isolated from LN and restimulated with GM - CSF shown in ( B ) .

C. Quantification (mean +SEM) of myeloid cell populations expressing IL-1β, isolated from LN and restimulated with GM-CSF shown in (B).


D . Frequencies and total numbers ( mean + SEM ) of CD11b + myeloid cells isolated from the spleen of immunized mice .

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D. Frequencies and total numbers (mean +SEM) of CD11b+myeloid cells isolated from the spleen of immunized mice.


E . Analysis of IL - 1R1 expression by CD11b + cells isolated from dLN of mice treated as in ( D ) . Data are representative FACS histogram overlays , gated on VD - CD11b + cells with mean frequencies per group .

E. Analysis of IL-1R1 expression by CD11b+cells isolated from dLN of mice treated as in (D). Data are representative FACS histogram overlays, gated on VD-CD11b+cells with mean frequencies per group.


F . Quantification ( mean + SEM ) of IL - 1β expression by CD11b + cells shown in ( E ) .

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F. Quantification (mean +SEM) of IL-1β expression by CD11b+ cells shown in (E).


G . Analysis of IL - 1β expressing CD11b + cells shown in ( E ) . Data are representative FACS plots , gated on VD - CD11b + IL - 1β + cells with mean frequencies per group . H . Quantification ( mean + SEM ) of myeloid cell populations expressing IL - 1β shown in ( G ) .

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G. Analysis of IL-1β expressing CD11b+cells shown in (E). Data are representative FACS plots, gated on VD- CD11b+IL-1β+cells with mean frequencies per group.H. Quantification (mean +SEM) of myeloid cell populations expressing IL-1β shown in (G).


A - D . Analysis of IL - 1R1 expression by different populations of murine ( A ) CD4 T cells and ( B ) Th17 cells isolated from the dLN 9 days after immunization ; and ( C ) CD4T cells and ( D ) Th17 cells isolated from the CNS at the peak of EAE . Cells ( A - D ) were restimulated in the presence of MOG for 6 h . Data consist of n = 4 wild type mice immunized with MOG / CFA / PTx . Data ( A , C ) are representative FACS histogram overlay of VD - TCRβ + CD4 + cells gated on IL - 17A + , IL - 17A - IFNγ + ( IFNγ + ) , IL - 17A - GM - CSF + ( GM - CSF + ) or IL - 17A - FoxP3 + ( FoxP3 + ) cells ( also see Appendix Fig S1 ) and quantifications shown as bar diagram ( mean + SEM ) . Data ( B , D , F ) are FACS plots of Th17 cells and histograms of IL - 1R1 staining of indicated cell populations with mean fluorescent intensity ( MFI ) ± SEM or mean frequencies per group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; two - tailed unpaired t - test . Experiments were performed twice with similar results .

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A-D. Analysis of IL-1R1 expression by different populations of murine (A) CD4 T cells and (B) Th17 cells isolated from the dLN 9 days after immunization; and (C) CD4T cells and (D) Th17 cells isolated from the CNS at the peak of EAE.Cells (A-D) were restimulated in the presence of MOG for 6 h. Data consist of n=4 wild type mice immunized with MOG/CFA/PTx.Data (A, C) are representative FACS histogram overlay of VD-TCRβ+CD4+ cells gated on IL-17A+, IL-17A-IFNγ+ (IFNγ+), IL-17A-GM-CSF+ (GM-CSF+) or IL-17A-FoxP3+ (FoxP3+) cells (also see Appendix Fig S1) and quantifications shown as bar diagram (mean +SEM). Data (B, D, F) are FACS plots of Th17 cells and histograms of IL-1R1 staining of indicated cell populations with mean fluorescent intensity (MFI) ±SEM or mean frequencies per group. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed unpaired t-test. Experiments were performed twice with similar results.


E - F . Analysis of IL - 1R1 expression by different populations of human ( E ) CD4 T cells and ( F ) Th17 cells isolated from the peripheral blood . Cells ( E - F ) were restimulated with PMA / Ionomycin for 4 h . Data consist of PBMC isolated from n = 4 healthy individuals . Data ( E ) are FACS plots of cells gated on CD14 - CD3 + CD4 + cells with mean frequencies per group and quantifications shown as bar diagram ( mean + SEM ) . Data ( B , D , F ) are FACS plots of Th17 cells and histograms of IL - 1R1 staining of indicated cell populations with mean fluorescent intensity ( MFI ) ± SEM or mean frequencies per group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; two - tailed unpaired t - test . Experiments were performed twice with similar results .

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E-F. Analysis of IL-1R1 expression by different populations of human (E) CD4 T cells and (F) Th17 cells isolated from the peripheral blood.Cells (E-F) were restimulated with PMA/Ionomycin for 4 h. Data consist of PBMC isolated from n=4 healthy individuals.Data (E) are FACS plots of cells gated on CD14-CD3+CD4+cells with mean frequencies per group and quantifications shown as bar diagram (mean +SEM). Data (B, D, F) are FACS plots of Th17 cells and histograms of IL-1R1 staining of indicated cell populations with mean fluorescent intensity (MFI) ±SEM or mean frequencies per group. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed unpaired t-test. Experiments were performed twice with similar results.


A . Analysis of cytokine expression by CD4T cells isolated from the spleen . Data are representative FACS plots , gated on VD - TCRβ + CD4 + CD44 + cells ( upper row ) with mean frequencies among CD4T cells per group ± SEM , and ( lower row ) with mean frequencies per group . Cells ( A - D ) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h . Data consist of n = 5 WT PBS , n = 4 WT PTx , n = 4 IL - 1R1 - / - PBS , n = 4 IL - 1R1 - / - PTx , MOG / CFA immunized mice . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed at least twice with similar results .

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A. Analysis of cytokine expression by CD4T cells isolated from the spleen. Data are representative FACS plots, gated on VD-TCRβ+CD4+CD44+cells (upper row) with mean frequencies among CD4T cells per group ± SEM, and (lower row) with mean frequencies per group.Cells (A-D) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h. Data consist of n=5 WT PBS, n=4 WT PTx, n=4 IL-1R1-/- PBS, n=4 IL-1R1-/- PTx, MOG/CFA immunized mice. *p < 0.05, **p < 0.01, ***p < 0.001, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed at least twice with similar results.


B . Frequencies and total numbers ( mean + SEM ) of MOG - specific Th17 cells isolated from the dLN . Cells ( A - D ) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h . Data consist of n = 5 WT PBS , n = 4 WT PTx , n = 4 IL - 1R1 - / - PBS , n = 4 IL - 1R1 - / - PTx , MOG / CFA immunized mice . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed at least twice with similar results .

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B. Frequencies and total numbers (mean +SEM) of MOG-specific Th17 cells isolated from the dLN.Cells (A-D) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h. Data consist of n=5 WT PBS, n=4 WT PTx, n=4 IL-1R1-/- PBS, n=4 IL-1R1-/- PTx, MOG/CFA immunized mice. *p < 0.05, **p < 0.01, ***p < 0.001, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed at least twice with similar results.


C - D . Frequencies and total numbers ( mean + SEM ) of MOG - specific GM - CSF + Th17 cells isolated from the spleen ( C ) and dLN ( D ) . Cells ( A - D ) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h . Data consist of n = 5 WT PBS , n = 4 WT PTx , n = 4 IL - 1R1 - / - PBS , n = 4 IL - 1R1 - / - PTx , MOG / CFA immunized mice . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed at least twice with similar results .

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C-D. Frequencies and total numbers (mean +SEM) of MOG-specific GM-CSF+ Th17 cells isolated from the spleen (C) and dLN (D).Cells (A-D) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h. Data consist of n=5 WT PBS, n=4 WT PTx, n=4 IL-1R1-/- PBS, n=4 IL-1R1-/- PTx, MOG/CFA immunized mice. *p < 0.05, **p < 0.01, ***p < 0.001, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed at least twice with similar results.


A . Analysis of CD4T cells isolated from the dLN and restimulated with PMA / Ionomycin for 4 h . Data are representative FACS plots , gated on VD - CD4 + cells with mean frequencies per group . Cells ( A - D ) were isolated at day 9 after MOG / CFA / PTx immunization . Data consist of n = 5 mice of each genotype . * p < 0 . 05 , * * p < 0 . 01 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed twice with similar results .

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A. Analysis of CD4T cells isolated from the dLN and restimulated with PMA/Ionomycin for 4 h. Data are representative FACS plots, gated on VD-CD4+cells with mean frequencies per group.Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.


B . Analysis of cytokine expression by CD4T cells isolated from the spleen and restimulated with MOG for 6 h . Data are representative FACS plots , gated on VD - TCRβ + CD4 + CD44 + cells ( upper row ) with mean frequencies among CD4T cells per group ± SEM , and ( lower row ) with mean frequencies per group . Cells ( A - D ) were isolated at day 9 after MOG / CFA / PTx immunization . Data consist of n = 5 mice of each genotype . * p < 0 . 05 , * * p < 0 . 01 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed twice with similar results .

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B. Analysis of cytokine expression by CD4T cells isolated from the spleen and restimulated with MOG for 6 h. Data are representative FACS plots, gated on VD-TCRβ+CD4+CD44+cells (upper row) with mean frequencies among CD4T cells per group ± SEM, and (lower row) with mean frequencies per group.Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.


C . Total cell numbers ( mean + SEM ) of MOG - specific Th17 cells isolated from the spleen , shown in ( B , upper row ) . Cells ( A - D ) were isolated at day 9 after MOG / CFA / PTx immunization . Data consist of n = 5 mice of each genotype . * p < 0 . 05 , * * p < 0 . 01 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed twice with similar results .

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C. Total cell numbers (mean +SEM) of MOG-specific Th17 cells isolated from the spleen, shown in (B, upper row).Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.


D . Frequencies and total numbers ( mean + SEM ) of MOG - specific GM - CSF + IFNγ + / - Th17 cells isolated from the spleen , shown in ( B , lower row ) . Cells ( A - D ) were isolated at day 9 after MOG / CFA / PTx immunization . Data consist of n = 5 mice of each genotype . * p < 0 . 05 , * * p < 0 . 01 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed twice with similar results .

D. Frequencies and total numbers (mean +SEM) of MOG-specific GM-CSF+IFNγ+/- Th17 cells isolated from the spleen, shown in (B, lower row).Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.


A - C . ( A ) Mean , ( B ) maximal EAE clinical scores and ( C ) area under the curve ( AUC ) of mice immunized with MOG / CFA / PTx . Data shown as ( A ) mean ( + SEM ) , ( B , C ) individual plots with mean and are combined from at least four independent experiments and consist of n = 41 WT , n = 24 IL - 1R1ΔT , n = 18 IL - 1R1 - / - mice . * p < 0 . 05 , * * * p < 0 . 001 ; two - tailed unpaired t - test . Black ( * ) signs in ( A ) represent statistical analysis of IL - 1R1ΔT vs WT and blue ( * ) of IL - 1R1ΔT vs IL - 1R1 - / - groups .

A-C. (A) Mean, (B) maximal EAE clinical scores and (C) area under the curve (AUC) of mice immunized with MOG/CFA/PTx. Data shown as (A) mean (+SEM), (B, C) individual plots with mean and are combined from at least four independent experiments and consist of n=41 WT, n=24 IL-1R1ΔT, n=18 IL-1R1-/-mice. *p < 0.05, ***p < 0.001; two-tailed unpaired t-test. Black (*) signs in (A) represent statistical analysis of IL-1R1ΔT vs WT and blue (*) of IL-1R1ΔT vs IL-1R1-/- groups.


D - E . ( D ) Maximal EAE clinical scores ( individual plots with mean ) of selected

D-E. (D) Maximal EAE clinical scores (individual plots with mean) of selected mice


D - E . ( E ) total cell numbers ( mean + SEM ) of infiltrates isolated from CNS and used for further analysis .

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D-E. (E) total cell numbers (mean +SEM) of infiltrates isolated from CNS and used for further analysis.


F - H . Analysis of cytokine expression by CD4 T cells isolated from the CNS of mice shown in ( D ) . Data ( F ) are representative FACS plots , gated on VD - CD90 + CD4 + CD44 + cells ( upper row ) with mean frequencies among CD4 T cells per group ± SEM , and ( lower row ) with mean frequencies per group . ( G ) Mean fluorescent intensity ( mean + SEM ) of IL - 17A staining by MOG - specific Th17 cells , shown in ( F , upper row ) . ( H ) Quantification of MOG - specific GM - CSF + Th17 cells ( mean + SEM ) shown in ( F , lower row ) .

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F-H. Analysis of cytokine expression by CD4 T cells isolated from the CNS of mice shown in (D). Data (F) are representative FACS plots, gated on VD-CD90+CD4+CD44+cells (upper row) with mean frequencies among CD4 T cells per group ± SEM, and (lower row) with mean frequencies per group. (G) Mean fluorescent intensity (mean +SEM) of IL-17A staining by MOG-specific Th17 cells, shown in (F, upper row). (H) Quantification of MOG-specific GM-CSF+ Th17 cells (mean +SEM) shown in (F, lower row).


I - K . Analysis of Treg cells isolated from mice shown in ( D ) . ( I ) Total Treg cell numbers ( mean + SEM ) isolated from CNS and dLN . ( J ) Ratio of Treg and Th17cells isolated from the CNS . Data are representative FACS plots , gated on VD - CD90 + CD4 + cells with mean frequencies per group . ( K ) Quantification of Treg / Th17 cell ratio ( mean + SEM ) within CNS and dLN . Cells were restimulated with MOG for 6 h and ( I - N ) in the presence of PMA / Ionomycin for 4 h .

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I-K. Analysis of Treg cells isolated from mice shown in (D). (I) Total Treg cell numbers (mean +SEM) isolated from CNS and dLN. (J) Ratio of Treg and Th17cells isolated from the CNS. Data are representative FACS plots, gated on VD-CD90+CD4+cells with mean frequencies per group. (K) Quantification of Treg/Th17 cell ratio (mean +SEM) within CNS and dLN.Cells were restimulated with MOG for 6 h and (I-N) in the presence of PMA/Ionomycin for 4 h.


L - N . Analysis of CCR6 expression by CD4 T cells isolated from the dLN and CNS of EAE diseased mice . Data ( L ) are representative FACS histogram overlays of ( black ) control , ( blue ) IL - 1R1ΔTcells , gated on CD3 + CD4 + IL - 17A + cells ; and of ( black filled ) control , ( blue filled ) IL - 1R1ΔTcells , gated on CD3 + CD4 + IL - 17A - cells . ( M ) Mean fluorescent intensity ( mean + SEM ) of CCR6 staining by CD4 T cells , shown in ( L ) . ( N ) Mean fluorescent intensity ( mean + SEM ) of CCR6 staining by GM - CSF + and GM - CSF - Th17 cells isolated from CNS , shown in ( L ) .

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L-N. Analysis of CCR6 expression by CD4 T cells isolated from the dLN and CNS of EAE diseased mice. Data (L) are representative FACS histogram overlays of (black) control, (blue) IL-1R1ΔTcells, gated on CD3+CD4+IL-17A+cells; and of (black filled) control, (blue filled) IL-1R1ΔTcells, gated on CD3+CD4+IL-17A-cells. (M) Mean fluorescent intensity (mean +SEM) of CCR6 staining by CD4 T cells, shown in (L). (N) Mean fluorescent intensity (mean +SEM) of CCR6 staining by GM-CSF+ and GM-CSF-Th17 cells isolated from CNS, shown in (L).


A - C . Analysis of ( A ) IL - 17A expression by wild type CD4T cells , followed with the analysis of ( B ) GM - CSF and ( C ) GM - CSF and IFNγ co - expression by Th17 cells .

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A-C. Analysis of (A) IL-17A expression by wild type CD4T cells, followed with the analysis of (B) GM-CSF and (C) GM-CSF and IFNγ co-expression by Th17 cells.


D - G . Analysis of cytokine expression by cells isolated from WT ( black ) , IL - 1R1ΔT ( blue ) and IL - 1R1 - / - ( red ) mice and cultured with or without IL - 23 . Data ( D ) are representative FACS plots , gated on VD - TCRβ + cells with mean frequencies per group . Cells ( A - G ) were isolated from dLN and the spleen of MOG / CFA immunized mice , cultured under indicated conditions for four days and restimulated with MOG for 6 h prior to analysis . Data consist of n = 4 of each genotype .

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D-G. Analysis of cytokine expression by cells isolated from WT (black), IL-1R1ΔT (blue) and IL-1R1-/- (red) mice and cultured with or without IL-23. Data (D) are representative FACS plots, gated on VD-TCRβ+cells with mean frequencies per group.Cells (A-G) were isolated from dLN and the spleen of MOG/CFA immunized mice, cultured under indicated conditions for four days and restimulated with MOG for 6 h prior to analysis. Data consist of n=4 of each genotype.


D - G . Analysis of cytokine expression by cells isolated from WT ( black ) , IL - 1R1ΔT ( blue ) and IL - 1R1 - / - ( red ) mice and cultured with or without IL - 23 . Data ( D ) are representative FACS plots , gated on VD - TCRβ + cells with mean frequencies per group . Quantification ( mean + SEM ) of ( E ) MOG - specific Th17 cells , ( F ) GM - CSF + Th17 cells and ( G ) GM - CSF + IFNγ + Th17 cells . Cells ( A - G ) were isolated from dLN and the spleen of MOG / CFA immunized mice , cultured under indicated conditions for four days and restimulated with MOG for 6 h prior to analysis . Data consist of n = 4 of each genotype .

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D-G. Analysis of cytokine expression by cells isolated from WT (black), IL-1R1ΔT (blue) and IL-1R1-/- (red) mice and cultured with or without IL-23. Data (D) are representative FACS plots, gated on VD-TCRβ+cells with mean frequencies per group. Quantification (mean +SEM) of (E) MOG-specific Th17 cells, (F) GM-CSF+Th17 cells and (G) GM-CSF+IFNγ+Th17 cells.Cells (A-G) were isolated from dLN and the spleen of MOG/CFA immunized mice, cultured under indicated conditions for four days and restimulated with MOG for 6 h prior to analysis. Data consist of n=4 of each genotype.


H - I . ( H ) Mean EAE clinical scores ( + SEM ) of Rag1 - / - mice which received 1 x 105 IL - 17A + cells of indicated genotypes and ( I ) total cell numbers ( mean + SEM ) of infiltrates isolated from CNS and used for further analysis . Data ( H - L ) consist of Rag1 - / - mice which received WT ( n = 6 ) , IL - 1R1ΔT ( n = 3 ) and IL - 1R1 - / - ( n = 4 ) cells polarized in the presence of IL - 23 prior to transfer . All experiments were performed at least twice with similar results .

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H-I. (H) Mean EAE clinical scores (+SEM) of Rag1-/- mice which received 1 x 105 IL-17A+cells of indicated genotypes and (I) total cell numbers (mean +SEM) of infiltrates isolated from CNS and used for further analysis.Data (H-L) consist of Rag1-/-mice which received WT (n=6), IL-1R1ΔT (n=3) and IL-1R1-/- (n=4) cells polarized in the presence of IL-23 prior to transfer. All experiments were performed at least twice with similar results.


H - I . Rag1 - / - mice which received 1 x 105 IL - 17A + cells of indicated genotypes and ( I ) total cell numbers ( mean + SEM ) of infiltrates isolated from CNS and used for further analysis . Data ( H - L ) consist of Rag1 - / - mice which received WT ( n = 6 ) , IL - 1R1ΔT ( n = 3 ) and IL - 1R1 - / - ( n = 4 ) cells polarized in the presence of IL - 23 prior to transfer . All experiments were performed at least twice with similar results .

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H-I. Rag1-/- mice which received 1 x 105 IL-17A+cells of indicated genotypes and (I) total cell numbers (mean +SEM) of infiltrates isolated from CNS and used for further analysis.Data (H-L) consist of Rag1-/-mice which received WT (n=6), IL-1R1ΔT (n=3) and IL-1R1-/- (n=4) cells polarized in the presence of IL-23 prior to transfer. All experiments were performed at least twice with similar results.


J - L . Analysis of cytokine expression by CD4 T cells isolated from CNS of mice shown in ( H ) and restimulated with MOG for 6 h . ( C ) Data are representative FACS plots , gated on VD - TCRβ + cells ( upper row ) with mean frequencies ± SEM . Data ( H - L ) consist of Rag1 - / - mice which received WT ( n = 6 ) , IL - 1R1ΔT ( n = 3 ) and IL - 1R1 - / - ( n = 4 ) cells polarized in the presence of IL - 23 prior to transfer . All experiments were performed at least twice with similar results .

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J-L. Analysis of cytokine expression by CD4 T cells isolated from CNS of mice shown in (H) and restimulated with MOG for 6 h. (C) Data are representative FACS plots, gated on VD-TCRβ+cells (upper row) with mean frequencies ± SEM.Data (H-L) consist of Rag1-/-mice which received WT (n=6), IL-1R1ΔT (n=3) and IL-1R1-/- (n=4) cells polarized in the presence of IL-23 prior to transfer. All experiments were performed at least twice with similar results.


J - L . Analysis of cytokine expression by CD4T cells isolated from CNS of mice shown in ( H ) and restimulated with MOG for 6 h . ( C ) Data are representative FACS plots , gated on VD - TCRβ + cells ( upper row ) with mean frequencies ± SEM . Data ( H - L ) consist of Rag1 - / - mice which received WT ( n = 6 ) , IL - 1R1ΔT ( n = 3 ) and IL - 1R1 - / - ( n = 4 ) cells polarized in the presence of IL - 23 prior to transfer . All experiments were performed at least twice with similar results .

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J-L. Analysis of cytokine expression by CD4T cells isolated from CNS of mice shown in (H) and restimulated with MOG for 6 h. (C) Data are representative FACS plots, gated on VD-TCRβ+cells (upper row) with mean frequencies ± SEM.Data (H-L) consist of Rag1-/-mice which received WT (n=6), IL-1R1ΔT (n=3) and IL-1R1-/- (n=4) cells polarized in the presence of IL-23 prior to transfer. All experiments were performed at least twice with similar results.


A . Mean EAE clinical scores ( + SEM ) . Data consist of ( A ) n = 10 WT , n = 4 IL - 1R1 - / - PBS , n = 4 WT , n = 4 IL - 1R1ΔT and n = 5 IL - 1R1 - / - PC61 treated mice .

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A. Mean EAE clinical scores (+SEM).Data consist of (A) n=10 WT, n=4 IL-1R1-/- PBS, n = 4 WT, n=4 IL-1R1ΔT and n=5 IL-1R1-/- PC61 treated mice.


B . Maximal EAE clinical scores ( individual plots with mean ) of selected mice .

B. Maximal EAE clinical scores (individual plots with mean) of selected mice.


C . Total cell numbers ( mean + SEM ) of infiltrates isolated from the CNS of mice , shown in ( B ) .

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C. Total cell numbers (mean +SEM) of infiltrates isolated from the CNS of mice, shown in (B).


D . Analysis of IL - 1R1 expression by Th17 cells isolated from CNS of EAE diseased mice , shown in ( B ) . Data are representative FACS histogram overlays of cells isolated from ( grey ) wild type mice treated with PBS , ( black ) wild type , ( blue ) IL - 1R1ΔT and ( red ) IL - 1R1 - / - mice treated with PC61 , gated on VD - TCRβ + CD4 + IL - 17A + cells .

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D. Analysis of IL-1R1 expression by Th17 cells isolated from CNS of EAE diseased mice, shown in (B). Data are representative FACS histogram overlays of cells isolated from (grey) wild type mice treated with PBS, (black) wild type, (blue) IL-1R1ΔT and (red) IL-1R1-/-mice treated with PC61, gated on VD-TCRβ+CD4+IL-17A+cells.


( E ) Frequencies of Th17 cells positive for IL - 1R1 , shown in ( D ) .

(E) Frequencies of Th17 cells positive for IL-1R1, shown in (D).


( a ) Synchronization dynamics of a population of HeLa cells within almost 3 complete cell cycles measured via PI staining followed by FACS analysis . The increase in cell number following each mitosis is shown by an overlaid curve ( in orange ; mean and s . d . of n = 3 ) .

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(a) Synchronization dynamics of a population of HeLa cells within almost 3 complete cell cycles measured via PI staining followed by FACS analysis. The increase in cell number following each mitosis is shown by an overlaid curve (in orange; mean and s.d. of n=3).


( b ) Computational modelling of the synchronization loss , considering 11 % cell - cell variation in doubling time , shows that the simulated fraction of the cells in G1 , S , and G2 / M in the synchronized cells throughout the cell cycle ( straight lines ) match experimental measurements ( marked with asterisk ) .

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(b) Computational modelling of the synchronization loss, considering 11% cell-cell variation in doubling time, shows that the simulated fraction of the cells in G1, S, and G2/M in the synchronized cells throughout the cell cycle (straight lines) match experimental measurements (marked with asterisk).


( c ) The measured average cell volume in the synchronized cell population ( red ; mean and s . d . of n = 3 ; in Equation 6 ) , the deconvoluted signal ( in case of no synchronization loss ; green ; in Equation 6 ) , and the simulated average cell volume considering the loss in synchronization ( black ; matching the measured concentration data ; Equation 6 ) .

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(c) The measured average cell volume in the synchronized cell population (red; mean and s.d. of n=3; in Equation 6), the deconvoluted signal (in case of no synchronization loss; green; in Equation 6), and the simulated average cell volume considering the loss in synchronization (black; matching the measured concentration data; Equation 6).


( d ) The measured concentration of CTP in synchronized cells shown in red ( mean and s . d . of n = 5 ; ) in Equation 7 ; the deconvoluted concentration dynamics , in case of no synchronization loss ( green ; in Equation 7 ) ; and the expected concentration dynamics based on the deconvoluted concentrations and considering the loss in synchronization , matching the measured concentrations ( black ; Equation 7 ) . The measured concentrations converge towards the steady - state concentrations measured in non - synchronized cells ( horizontal blue line ) .

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(d) The measured concentration of CTP in synchronized cells shown in red (mean and s.d. of n=5; ) in Equation 7; the deconvoluted concentration dynamics, in case of no synchronization loss (green; in Equation 7); and the expected concentration dynamics based on the deconvoluted concentrations and considering the loss in synchronization, matching the measured concentrations (black; Equation 7). The measured concentrations converge towards the steady-state concentrations measured in non-synchronized cells (horizontal blue line).


Figure 2 : Oscillation in metabolite concentrations throughout the cell cycle in HeLa cells . The figure shows metabolites found to significantly oscillate throughout the cell cycle ( concentrations normalized per metabolite ; maximal concentration in red ; minimal concentration in blue ) . The amplitude of the oscillations is shown on the right . Metabolites are color - coded according to metabolic pathways : Energy / redox cofactors ( purple ) ; amino acids ( blue ) ; glycolytic metabolites ( red ) ; TCA cycle metabolites ( green ) .

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Figure 2: Oscillation in metabolite concentrations throughout the cell cycle in HeLa cells. The figure shows metabolites found to significantly oscillate throughout the cell cycle (concentrations normalized per metabolite; maximal concentration in red; minimal concentration in blue). The amplitude of the oscillations is shown on the right. Metabolites are color-coded according to metabolic pathways: Energy/redox cofactors (purple); amino acids (blue); glycolytic metabolites (red); TCA cycle metabolites (green).


( c - e ) Measured relative fraction of the m + 2 labeling of TCA cycle intermediates after feeding [ U - 13C ] - glucose ( red ; mean and s . d . of n = 3 ) , the deconvoluted signal ( green ) , and the expected labeling dynamics considering the loss in synchronization ( black ; representing TCA cycle oxidation of glucose - derived acetyl - CoA ) .

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(c-e) Measured relative fraction of the m+2 labeling of TCA cycle intermediates after feeding [U-13C]-glucose (red; mean and s.d. of n=3), the deconvoluted signal (green), and the expected labeling dynamics considering the loss in synchronization (black; representing TCA cycle oxidation of glucose-derived acetyl-CoA).


( f ) Oscillations in citrate m + 4 labeling after feeding [ U - 13C ] - glutamine ( mean and s . d . of n = 3 ) .

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(f) Oscillations in citrate m+4 labeling after feeding [U-13C]-glutamine (mean and s.d. of n=3).


( g ) Oscillations in the total citrate concentration throughout the cell cycle ( mean and s . d . of n = 5 ) .

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(g) Oscillations in the total citrate concentration throughout the cell cycle (mean and s.d. of n=5).


( h ) The measured lactate secretion flux in synchronized cells shown in red ( mean and s . d . of n = 5 ; in Equation 9 ; ) ; the deconvoluted secretion flux dynamics , in case of no synchronization loss ( green ; in Equation 10 ) ; and the expected secretion flux based on the deconvoluted fluxes and considering the loss in synchronization , matching the measured fluxes ( black ; Equation 10 ) .

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

(h) The measured lactate secretion flux in synchronized cells shown in red (mean and s.d. of n=5; in Equation 9;); the deconvoluted secretion flux dynamics, in case of no synchronization loss (green; in Equation 10); and the expected secretion flux based on the deconvoluted fluxes and considering the loss in synchronization, matching the measured fluxes (black; Equation 10).


Oscillations in aspartate ( a ) concentrations throughout the cell cycle when feeding [ U - 13C ] - glutamine ( representing oxidative TCA cycle activity ) .

O O B-MEASURED_VAR 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

Oscillations in aspartate (a)concentrations throughout the cell cycle when feeding [U-13C]-glutamine (representing oxidative TCA cycle activity).


Oscillations in malate ( b ) concentrations throughout the cell cycle when feeding [ U - 13C ] - glutamine ( representing oxidative TCA cycle activity ) .

Oscillations in malate (b) concentrations throughout the cell cycle when feeding [U-13C]-glutamine (representing oxidative TCA cycle activity).


( c ) Uniform malate m + 4 labeling throughout the cell cycle ( combined with the increase in malate concentration in S phase representing increased oxidative TCA cycle flux in S phase ) .

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

(c) Uniform malate m+4 labeling throughout the cell cycle (combined with the increase in malate concentration in S phase representing increased oxidative TCA cycle flux in S phase).


( d ) Oscillations in pyrimidines m + 3 labeling throughout the cell cycle when feeding [ U - 13C ] - glutamine ( representing de novo pyrimidine biosynthesis ) .

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

(d) Oscillations in pyrimidines m+3 labeling throughout the cell cycle when feeding [U-13C]-glutamine (representing de novo pyrimidine biosynthesis).


( e ) Oscillations in lactate m + 3 labeling throughout the cell cycle when feeding [ U - 13C ] - glutamine ( representing malic enzyme activity ) .

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

(e) Oscillations in lactate m+3 labeling throughout the cell cycle when feeding [U-13C]-glutamine (representing malic enzyme activity).


( f ) Oscillations in malate m + 3 throughout the cell cycle when feeding [ U - 13C ] - glucose ( representing pyruvate carboxylase activity ) .

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

(f) Oscillations in malate m+3 throughout the cell cycle when feeding [U-13C]-glucose (representing pyruvate carboxylase activity).


( g ) Oscillations in citrate m + 5 when feeding [ U - 13C ] - glutamine throughout the cell cycle ( representing reductive IDH flux ) .

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

(g) Oscillations in citrate m+5 when feeding [U-13C]-glutamine throughout the cell cycle (representing reductive IDH flux).


( h ) Oscillations in acetyl - CoA m + 2 when feeding [ U - 13C ] - glucose , representing oxidative glucose metabolism .

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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O

(h) Oscillations in acetyl-CoA m+2 when feeding [U-13C]-glucose, representing oxidative glucose metabolism.


Oscillations in acetyl - CoA m + 2 when feeding [ U - 13C ] - glutamine , representing reductive glutamine metabolism ( i ) . For measurements of metabolite concentrations ( a - b ) , red marks represent mean and s . d . of n = 5 . For measurements of fractional isotopic labeling ( c - i ) , red marks represent mean and s . d . of n = 3 .

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

Oscillations in acetyl-CoA m+2 when feeding [U-13C]-glutamine, representing reductive glutamine metabolism (i). For measurements of metabolite concentrations (a-b), red marks represent mean and s.d. of n=5. For measurements of fractional isotopic labeling (c-i), red marks represent mean and s.d. of n=3.


Figure 5 : Complementary oscillations of glucose versus glutamine - derived fluxes in TCA cycle . ( a - i ) Oscillations in metabolic flux throughout the cell cycle ( in mM / h ) , computed based on metabolic modelling of measured oscillations in metabolite concentrations and isotopic labeling ( red and green marks represent optimal estimates of transient flux with 95 % c . i . ) . Blue lines represent average fluxes inferred in a non - synchronized cell population . As shown , glucose - derived flux into TCA cycle peak in late G1 phase , while oxidative and reductive glutamine metabolism dominates S phase .

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

Figure 5: Complementary oscillations of glucose versus glutamine-derived fluxes in TCA cycle. (a-i) Oscillations in metabolic flux throughout the cell cycle (in mM/h), computed based on metabolic modelling of measured oscillations in metabolite concentrations and isotopic labeling (red and green marks represent optimal estimates of transient flux with 95% c.i.). Blue lines represent average fluxes inferred in a non-synchronized cell population. As shown, glucose-derived flux into TCA cycle peak in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase.


( a - b ) One hour treatment of synchronized cells with DCA inhibits the oscillations in citrate m + 2 ( a ) from isotopic glucose ( representing glycolytic flux into TCA cycle ) .

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

(a-b) One hour treatment of synchronized cells with DCA inhibits the oscillations in citrate m+2 (a) from isotopic glucose (representing glycolytic flux into TCA cycle).


( a - b ) One hour treatment of synchronized cells with DCA inhibits the oscillations in acetyl - CoA m + 2 ( b ) from isotopic glucose ( representing glycolytic flux into TCA cycle ) .

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

(a-b) One hour treatment of synchronized cells with DCA inhibits the oscillations in acetyl-CoA m+2 (b) from isotopic glucose (representing glycolytic flux into TCA cycle).


It further inhibits oscillations in citrate m + 5 / m + 4 ratio ( c ) from isotopic glutamine ( representing oxidative versus reductive TCA cycle flux ) .

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

It further inhibits oscillations in citrate m+5/m+4 ratio (c) from isotopic glutamine (representing oxidative versus reductive TCA cycle flux).


It further inhibits oscillations in acetyl - CoA m + 2 ( d ) from isotopic glutamine ( representing oxidative versus reductive TCA cycle flux ) .

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

It further inhibits oscillations in acetyl-CoA m+2 (d) from isotopic glutamine (representing oxidative versus reductive TCA cycle flux).


( e ) The fraction of cells in G1 , S , and G2 / M phases in synchronized HeLa cells after 3 hour treatment with DCA ( normalized by measurements in untreated control cells ) .

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(e) The fraction of cells in G1, S, and G2/M phases in synchronized HeLa cells after 3 hour treatment with DCA (normalized by measurements in untreated control cells).


( f ) The fraction of cells in G1 , S , and G2 / M phases in non - synchronized HeLa and HCT - 116 cells after 24 hour treatment with DCA ( normalized by measurements in untreated control cells ) . As shown , DCA significantly increases the fraction of cells in S phase , inhibiting cellular progression into G2 phase ; showing mean and s . d . of n = 3 for all isotopic labeling forms and FACS measurements ( a - f ) . Statistical significance of changes in the fraction of cells in each cell cycle phase following DCA treatment is calculated based on two - tailed , unequal variance t - test ( e - f ) .

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

(f) The fraction of cells in G1, S, and G2/M phases in non-synchronized HeLa and HCT-116 cells after 24 hour treatment with DCA (normalized by measurements in untreated control cells). As shown, DCA significantly increases the fraction of cells in S phase, inhibiting cellular progression into G2 phase; showing mean and s.d. of n=3 for all isotopic labeling forms and FACS measurements (a-f). Statistical significance of changes in the fraction of cells in each cell cycle phase following DCA treatment is calculated based on two-tailed, unequal variance t-test (e-f).


( A ) Representative images of H & E staining of colon from 3m C57Bl ( n = 4 ) and mdx ( n = 4 ) mice . High magnification ( scale bar : 20 μm ) and low magnification ( scale bar : 200 μm ) .

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(A) Representative images of H&E staining of colon from 3m C57Bl (n=4) and mdx (n=4) mice. High magnification (scale bar: 20 μm) and low magnification (scale bar: 200 μm).


( B ) Mucus layer , area between yellow dash lines ; crypt length , yellow headed arrow . Scale bar : 100 μm . ( C ) Mucus layer thickness and crypt length were quantified for n = 4 mice per group ( with pooled samples of n = 60 for mucus layer thickness and n = 80 for crypt length ) .

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(B) Mucus layer, area between yellow dash lines; crypt length, yellow headed arrow. Scale bar: 100 μm. (C) Mucus layer thickness and crypt length were quantified for n=4 mice per group (with pooled samples of n=60 for mucus layer thickness and n=80 for crypt length).


( D ) SCFA faecal quantification of 3m C57Bl ( n = 3 ) and mdx ( n = 4 - 5 ) mice . Data information : Data are presented as mean ± SD ( * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; Student t - test ) .

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(D) SCFA faecal quantification of 3m C57Bl (n=3) and mdx (n=4-5) mice. Data information: Data are presented as mean ± SD (*p<0.05; **p<0.01, ***p<0.001, ****p<0.0001; Student t-test).


( E ) Colon images captured with the iMScope TRIO described altered pattern of expression of different Phosphatidylcholines ( PC ) and Lysophosphatidylcholines ( LysoPC ) ( as indicated by m / z values ) in 3m mdx mice ( n = 3 ) . Scale bar : 50 μm . For each lipid , the mean intensities measured at 12 positions throughout colon images are shown on the right side where bars are mean ± SEM ( n = 3 ) . Data information : Data are presented as mean ± SD ( * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; Student t - test ) .

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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Colon images captured with the iMScope TRIO described altered pattern of expression of different Phosphatidylcholines (PC) and Lysophosphatidylcholines (LysoPC) (as indicated by m/z values) in 3m mdx mice (n=3). Scale bar: 50 μm. For each lipid, the mean intensities measured at 12 positions throughout colon images are shown on the right side where bars are mean ± SEM (n = 3). Data information: Data are presented as mean ± SD (*p<0.05; **p<0.01, ***p<0.001, ****p<0.0001; Student t-test).


( F ) Cropped images of representative WB showing the expression of proteins involved in inflammation and fibrosis in colon tissues of 3m C57Bl ( n = 3 ) and 3m mdx ( n = 5 ) . Densitometric analyses of protein expression was shown as ratio to actin . Data information : Data are presented as mean ± SD ( * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; Student t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cropped images of representative WB showing the expression of proteins involved in inflammation and fibrosis in colon tissues of 3m C57Bl (n=3) and 3m mdx (n=5). Densitometric analyses of protein expression was shown as ratio to actin. Data information: Data are presented as mean ± SD (*p<0.05; **p<0.01, ***p<0.001, ****p<0.0001; Student t-test).


( E ) Faecal content quantification of SCFAs in 3m mdx and 3m mdx + ABX mice ( n = 5 per group ) . Data equal to 0 . Data are presented as mean ± SD ( * * p < 0 . 01 , * * * p < 0 . 001 ; Student t - test ) .

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(E) Faecal content quantification of SCFAs in 3m mdx and 3m mdx+ABX mice (n=5 per group). Data equal to 0. Data are presented as mean ± SD (**p< 0.01, ***p< 0.001; Student t-test).


( A - D ) FACS analysis of spleen and muscle homogenates from 3m C57Bl ( n = 4 ) , mdx ( n = 5 ) and mdx + ABX ( n = 7 ) mice demonstrates no significant alteration of CD45 + CD11b + CD4 - CD8 - myeloid cells ( A and C ) and few differences in CD4 + or CD8 + naïve ( CD62L + CD44 - ) , central memory ( CD62L + CD44 + ) and effector ( CD62L - CD44 + ) T cells ( B and D ) . Data information : Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ordinary one - way ANOVA , Tukey ' s multiple comparison test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) FACS analysis of spleen and muscle homogenates from 3m C57Bl (n=4), mdx (n=5) and mdx+ABX (n=7) mice demonstrates no significant alteration of CD45+CD11b+CD4-CD8- myeloid cells (A and C) and few differences in CD4+ or CD8+ naïve (CD62L+ CD44-), central memory (CD62L+ CD44+) and effector (CD62L- CD44+) T cells (B and D). Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 ordinary one-way ANOVA, Tukey's multiple comparison test.


( E ) FACS analysis of spleen of 3m C57Bl ( n = 7 ) , mdx ( n = 9 ) and GFmdx ( n = 5 ) mice revealed similar proportions of CD4 + and CD8 + T cells but reduced activated CD44 + T cells in GFmdx mice . Representative plots are depicted . Data information : Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ordinary one - way ANOVA , Tukey ' s multiple comparison test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FACS analysis of spleen of 3m C57Bl (n=7), mdx (n=9) and GFmdx (n=5) mice revealed similar proportions of CD4+ and CD8+ T cells but reduced activated CD44+ T cells in GFmdx mice. Representative plots are depicted. Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 ordinary one-way ANOVA, Tukey's multiple comparison test.


( F ) Graphs show cumulative frequencies of CD4 + and CD8 + T cells on live cells of 3m C57Bl ( n = 7 ) , mdx ( n = 4 ) and GFmdx ( n = 5 ) mice . Representative dot plots and cumulative frequencies of splenic CD4 + GITR + CD25 + Treg . Frequencies of effector CD44 + T cells were significantly decreased in spleen of GFmdx mice . Data information : Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ordinary one - way ANOVA , Tukey ' s multiple comparison test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Graphs show cumulative frequencies of CD4+ and CD8+ T cells on live cells of 3m C57Bl (n=7), mdx (n=4) and GFmdx (n=5) mice. Representative dot plots and cumulative frequencies of splenic CD4+GITR+CD25+ Treg. Frequencies of effector CD44+ T cells were significantly decreased in spleen of GFmdx mice. Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 ordinary one-way ANOVA, Tukey's multiple comparison test.


( G ) Representative dot - plots showing the proportion of muscle - infiltrating CD45 + cells of 3m C57Bl ( n = 6 ) , mdx ( n = 6 ) and GFmdx ( n = 5 ) mice . Cumulative frequencies of muscle - infiltrating CD45 + cells are shown . Data information : Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ordinary one - way ANOVA , Tukey ' s multiple comparison test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 dot-plots showing the proportion of muscle-infiltrating CD45+ cells of 3m C57Bl (n=6), mdx (n=6) and GFmdx (n=5) mice. Cumulative frequencies of muscle-infiltrating CD45+ cells are shown. Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 ordinary one-way ANOVA, Tukey's multiple comparison test.


( H ) Representative images of TA muscles from 3m mdx and GFmdx mice stained for CD45 ( in green ) , isolectin ( in red ) and phalloidin ( in purple ) . Nuclei were counterstained with DAPI ( in blue ) . Scale bar : 10 μm .

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

(H) Representative images of TA muscles from 3m mdx and GFmdx mice stained for CD45 (in green), isolectin (in red) and phalloidin (in purple). Nuclei were counterstained with DAPI (in blue). Scale bar: 10 μm.


( I ) Absolute number of CD3 + inflammatory cells ( white arrows ) were quantified in n = 12 images of TA of 3m C57Bl , 3m mdx and 3m GFmdx mice ( n = 6 each ) . CD3 staining is shown in green and DAPI in blue . Scale bars : 50 μm . Data information : Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 The comparisons among the averages of CD3 + cells were evaluated using unpaired t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Absolute number of CD3+ inflammatory cells (white arrows) were quantified in n=12 images of TA of 3m C57Bl, 3m mdx and 3m GFmdx mice (n=6 each). CD3 staining is shown in green and DAPI in blue. Scale bars: 50 μm. Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 The comparisons among the averages of CD3+ cells were evaluated using unpaired t-test.


( D ) RT - qPCR analysis of TA muscles of two independent experiments with 3m mdx ( n = 4 ) , mdx + ABX ( n = 4 ) and GFmdx ( n = 5 ) mice determined the expression of myogenic markers . Data information : Data are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , ordinary one - way ANOVA and non - parametric test followed by Kruskal - Wallis test for RT - qPCR ) .

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(D) RT-qPCR analysis of TA muscles of two independent experiments with 3m mdx (n=4), mdx+ABX (n=4) and GFmdx (n=5) mice determined the expression of myogenic markers. Data information: Data are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, ordinary one-way ANOVA and non-parametric test followed by Kruskal-Wallis test for RT-qPCR).