( H ) Left : Representative immunohistochemistry pictures of phosphorylated FAK on tyrosine ( Y ) 397 and of CD206 in serial sections of human PDAC tissues . Scale bar : 200µm Middle : Correlation plot between fibroblastic pY397 FAK score and CD45 or CD206 + cell number within human PDAC samples . Fibroblastic pY397 FAK score and percentage of CD206 positive cell quantifications were performed on a 47 patient cohort based on IHC staining . Spearman r = 0 . 4145 , p = 0 . 0019 . Right : Percentage of CD206 positive cells in PDAC patient expressing either low or high fibroblastic FAK activity score .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left: Representative immunohistochemistry pictures of phosphorylated FAK on tyrosine (Y) 397 and of CD206 in serial sections of human PDAC tissues. Scale bar: 200µm Middle: Correlation plot between fibroblastic pY397 FAK score and CD45 or CD206+ cell number within human PDAC samples. Fibroblastic pY397 FAK score and percentage of CD206 positive cell quantifications were performed on a 47 patient cohort based on IHC staining. Spearman r = 0.4145, p=0.0019. Right: Percentage of CD206 positive cells in PDAC patient expressing either low or high fibroblastic FAK activity score.


( A ) Representative images of three independent scratch wound assays of co - cultivated red - labelled pancreatic tumour cells with either FAK - WT ( wild - type ) or FAK - KD ( Kinase - dead ) green - labelled fibroblasts at time zero and 46 hours ( corresponding to the wound closure of co - cultured FAK - WT fibroblasts with tumour cells ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of three independent scratch wound assays of co-cultivated red-labelled pancreatic tumour cells with either FAK-WT (wild-type) or FAK-KD (Kinase-dead) green-labelled fibroblasts at time zero and 46 hours (corresponding to the wound closure of co-cultured FAK-WT fibroblasts with tumour cells).


Cell migration ( has been analyzed in detail by tracking the location of cells ( n > 16 cells per condition ) over time for fibroblasts or tumour cells ( B ) ,

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

Cell migration (has been analyzed in detail by tracking the location of cells (n >16 cells per condition) over time for fibroblasts or tumour cells (B),


measuring fibroblast velocity ( C ) , directionality ( a cell with a directionality equal to 1 is moving straightforward ) ( D ) and migration distance ( E ) or tumour cell velocity ( F ) , directionality ( G ) and distance ( H ) . Values are means ± SEM of 16 tracked cells from two independent experiments .

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

measuring fibroblast velocity (C), directionality (a cell with a directionality equal to 1 is moving straightforward) (D) and migration distance (E) or tumour cell velocity (F), directionality (G) and distance (H). Values are means ± SEM of 16 tracked cells from two independent experiments.


( I ) 3D reconstitution of cell invasion into collagen matrix from spheroid composed of green - labelled FAK - WT or FAK - KD fibroblasts plus red - labelled pancreatic tumour cells ( ratio 1 / 1 ) . Shown are representative 3D reconstitution of spheroid invasion into 2mg / ml collagen I matrix obtained using Zen software . ( J - K ) Quantification of fibroblast ( J ) or tumour cell ( K ) invasion within collagen matrix . Values are means ± SEM of 60 tracked cells from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 3D reconstitution of cell invasion into collagen matrix from spheroid composed of green-labelled FAK-WT or FAK-KD fibroblasts plus red-labelled pancreatic tumour cells (ratio 1/1). Shown are representative 3D reconstitution of spheroid invasion into 2mg/ml collagen I matrix obtained using Zen software. (J-K) Quantification of fibroblast (J) or tumour cell (K) invasion within collagen matrix. Values are means ± SEM of 60 tracked cells from three independent experiments.


( A ) Representative images of CK19 and α - SMA immunohistochemistry and Sirius red staining in serial sections of primary mouse tumour ( graft of FAK - WT or FAK - KD fibroblasts + tumours cells at 38 days after injection ) at the invasive front . Scale bar , 100 µm . Bottom : schematic organization of the cells ( tumour cells in brown , CAFs in yellow and non - transformed epithelial cells in beige ) .

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(A) Representative images of CK19 and α-SMA immunohistochemistry and Sirius red staining in serial sections of primary mouse tumour (graft of FAK-WT or FAK-KD fibroblasts + tumours cells at 38 days after injection) at the invasive front. Scale bar, 100 µm. Bottom: schematic organization of the cells (tumour cells in brown, CAFs in yellow and non-transformed epithelial cells in beige).


( B ) Representative images of ECM deposition ( stained using Alexa Fluor 488 succinimidyl ester ( NHS ) ) generated by FAK - WT or - KD activated fibroblasts during migration .

(B) Representative images of ECM deposition (stained using Alexa Fluor 488 succinimidyl ester (NHS)) generated by FAK-WT or -KD activated fibroblasts during migration.


( C ) Quantification of collagen I gel contraction ( calculated based on photographed gels using the formula 100 * ( well diameter - gel diameter ) / well diameter ) induced by fibroblasts in presence or not of tumour cells , or by hCAF in presence or not of FAK inhibitor ( FAK - I , 1µM )

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(C) Quantification of collagen I gel contraction (calculated based on photographed gels using the formula 100*(well diameter - gel diameter)/well diameter) induced by fibroblasts in presence or not of tumour cells , or by hCAF in presence or not of FAK inhibitor (FAK-I, 1µM)


( D ) Quantification of the percentage change of Y397 FAK , total FAK , collagen I ( coll I ) , collagen III ( coll III ) , collagen IV ( coll IV ) , LOXL2 , periostin ( POSTN ) , osteopontin ( OPN ) expression by human CAFs treated or not ( NT ) with FAK inhibitor ( 1µM ) , based on immunofluorescence analyses . Values are means ± SEM obtained from at least 5 primary hCAFs , isolated from 5 to 12 different fresh human PDAC samples .

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(D) Quantification of the percentage change of Y397 FAK, total FAK, collagen I (coll I), collagen III (coll III), collagen IV (coll IV), LOXL2, periostin (POSTN), osteopontin (OPN) expression by human CAFs treated or not (NT) with FAK inhibitor (1µM), based on immunofluorescence analyses. Values are means ± SEM obtained from at least 5 primary hCAFs, isolated from 5 to 12 different fresh human PDAC samples.


( E ) Collagen I , III and IV and periostin deposition are shown using heat map of the IF staining , and cell shapes are identified by white line .

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(E) Collagen I, III and IV and periostin deposition are shown using heat map of the IF staining, and cell shapes are identified by white line.


Left : Normalized peptide abundance for collagens from hCAFs ( each CAF duplicate is represented , CAF1 in blue , 2 in red and 3 in black ) treated or not with FAK inhibitor . Right : Heatmap of the collagen modifications in the 3 hCAFs upon FAK inhibition in hCAFs . The value of log2 fold - change between FAK inhibitor treated CAFs and non - treated CAFs for each protein was indicated by the colored scale , with red indicating increased expression while blue implies decreased expression . For each CAFs and each protein , log2 fold - change was calculated based on 2 technical replicates per

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Left: Normalized peptide abundance for collagens from hCAFs (each CAF duplicate is represented, CAF1 in blue, 2 in red and 3 in black) treated or not with FAK inhibitor. Right: Heatmap of the collagen modifications in the 3 hCAFs upon FAK inhibition in hCAFs. The value of log2 fold-change between FAK inhibitor treated CAFs and non-treated CAFs for each protein was indicated by the colored scale, with red indicating increased expression while blue implies decreased expression. For each CAFs and each protein, log2 fold-change was calculated based on 2 technical replicates per condition


Left : Normalized peptide abundance for proteoglycans ( H ) from hCAFs ( each CAF duplicate is represented , CAF1 in blue , 2 in red and 3 in black ) treated or not with FAK inhibitor . Right : Heatmap of the proteoglycan ( H ) modifications in the 3 hCAFs upon FAK inhibition in hCAFs . The value of log2 fold - change between FAK inhibitor treated CAFs and non - treated CAFs for each protein was indicated by the colored scale , with red indicating increased expression while blue implies decreased expression . For each CAFs and each protein , log2 fold - change was calculated based on 2 technical replicates per

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Left: Normalized peptide abundance for proteoglycans (H) from hCAFs (each CAF duplicate is represented, CAF1 in blue, 2 in red and 3 in black) treated or not with FAK inhibitor. Right: Heatmap of the proteoglycan (H) modifications in the 3 hCAFs upon FAK inhibition in hCAFs. The value of log2 fold-change between FAK inhibitor treated CAFs and non-treated CAFs for each protein was indicated by the colored scale, with red indicating increased expression while blue implies decreased expression. For each CAFs and each protein, log2 fold-change was calculated based on 2 technical replicates per condition


( I ) Normalized peptide abundance for glycoproteins from hCAFs treated or not with FAK - I .

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(I) Normalized peptide abundance for glycoproteins from hCAFs treated or not with FAK-I.


( J ) Heatmap of the glycoprotein modifications upon FAK - I in hCAFs .

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(J) Heatmap of the glycoprotein modifications upon FAK-I in hCAFs.


( K ) Normalized peptide abundance for " matrisome - associated " protein division : ECM - affiliated proteins , Secreted factors , and ECM regulators from hCAFs treated or not with FAK inhibitor .

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(K) Normalized peptide abundance for "matrisome-associated" protein division: ECM-affiliated proteins, Secreted factors, and ECM regulators from hCAFs treated or not with FAK inhibitor.


( L ) Left : Representative pictures of β1 integrin activation ( red ) of tumour cells , upon adhesion to either ECM deposited from non - treated CAFs or treated with FAK - I for 7 days , analyzed by IF . Merge : activated β1 integrin ( red ) , phalloidin ( green ) , dapi ( blue ) . Right : quantification of the number of integrin β1 activated cluster per cells and size of those clusters .

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(L) Left: Representative pictures of β1 integrin activation (red) of tumour cells, upon adhesion to either ECM deposited from non-treated CAFs or treated with FAK-I for 7 days, analyzed by IF. Merge: activated β1 integrin (red), phalloidin (green), dapi (blue). Right: quantification of the number of integrin β1 activated cluster per cells and size of those clusters.


( A ) Schematic overview of FMR1 , FXR1 , FXR2 , FMR2 and position of the 8 selected single nucleotide polymorphisms ( SNPs ) . Line represents introns , grey box at the beginning and end of each gene stands for UTR region and red boxes represent exons . Gene structure plots generated using FancyGene ( Rambaldi Ciccarelli , 2009 )

(A) Schematic overview of FMR1, FXR1, FXR2, FMR2 and position of the 8 selected single nucleotide polymorphisms (SNPs). Line represents introns, grey box at the beginning and end of each gene stands for UTR region and red boxes represent exons. Gene structure plots generated using FancyGene (Rambaldi Ciccarelli, 2009)


( B ) Distribution of repeat polymorphism lengths in FMR1 of male GRAS schizophrenia patients and healthy controls . ( C ) Distribution of repeat polymorphism lengths in FMR2 of male GRAS schizophrenia patients and healthy controls .

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(B) Distribution of repeat polymorphism lengths in FMR1 of male GRAS schizophrenia patients and healthy controls. (C) Distribution of repeat polymorphism lengths in FMR2 of male GRAS schizophrenia patients and healthy controls.


Figure 2 : SNP overview and unbiased selection according to the standard operating procedure ( SOP ) developed for phenotype - based genetic association study ( PGAS ) approaches . ( A ) Genes from the ' broader fragile X family ' and their chromosomal position . ( B ) SNP numbers available through direct genotyping in the here used semi - custom genotyping array ( Affymetrix , Santa Clara , CA , USA ) . ( C ) SNPs fulfilling some of the first round of selection criteria ( ' functional ' = SNPs , i . e . located in promoter region , 3 ' UTR or coding sequence ; MAF = MAF ≥ 0 . 2 ; LD = SNPs that ' survive ' after linkage disequilibrium pruning : r² < 0 . 8 ) . Underlined are the 13 SNPs selected for the PGAS approach using the PAUSS ( selection requirements : fulfilled 2 of the above criteria or were functional ) . Not more than 3 SNPs per gene are selected to avoid overrepresentation of one gene . ( D ) SNPs with a tendency in PGAS ( see Fig 3A ) at single SNP basis went into the final accumulation model .

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

Figure 2: SNP overview and unbiased selection according to the standard operating procedure (SOP) developed for phenotype-based genetic association study (PGAS) approaches. (A) Genes from the 'broader fragile X family' and their chromosomal position. (B) SNP numbers available through direct genotyping in the here used semi-custom genotyping array (Affymetrix, Santa Clara, CA, USA). (C) SNPs fulfilling some of the first round of selection criteria ('functional'=SNPs, i.e. located in promoter region, 3'UTR or coding sequence; MAF=MAF≥0.2; LD=SNPs that 'survive' after linkage disequilibrium pruning: r²<0.8). Underlined are the 13 SNPs selected for the PGAS approach using the PAUSS (selection requirements: fulfilled 2 of the above criteria or were functional). Not more than 3 SNPs per gene are selected to avoid overrepresentation of one gene. (D) SNPs with a tendency in PGAS (see Fig 3A) at single SNP basis went into the final accumulation model.


( A - B ) A total of 13 SNPs pre - selected according to the SOP presented in Fig 2 underwent PGAS screening as exemplified here : ( A ) PAUSS association pattern of an exemplary fictitious autosomal ( upper panel ) and a sex - chromosomal ( lower panel ) SNP which are eligible for the accumulation model . The genotype associated with the highest average PAUSS ( in this example CC ) is the ' proautistic genotype ' ( indicated by the black arrow ) and is assigned a score of 1 . The heterozygous genotype is assigned a score of 0 . 5 and the homozygous genotype associated with the lowest PAUSS receives a score of 0 . Please note that the difference between genotypes does not have to be statistically significant . ( B ) PAUSS association pattern of an exemplary fictitious autosomal ( upper panel ) and a sex - chromosomal ( lower panel ) SNP which would not be selected for the accumulation model because of unclear phenotypical / biological relevance .

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(A-B) A total of 13 SNPs pre-selected according to the SOP presented in Fig 2 underwent PGAS screening as exemplified here: (A) PAUSS association pattern of an exemplary fictitious autosomal (upper panel) and a sex-chromosomal (lower panel) SNP which are eligible for the accumulation model. The genotype associated with the highest average PAUSS (in this example CC) is the 'proautistic genotype' (indicated by the black arrow) and is assigned a score of 1. The heterozygous genotype is assigned a score of 0.5 and the homozygous genotype associated with the lowest PAUSS receives a score of 0. Please note that the difference between genotypes does not have to be statistically significant. (B) PAUSS association pattern of an exemplary fictitious autosomal (upper panel) and a sex-chromosomal (lower panel) SNP which would not be selected for the accumulation model because of unclear phenotypical/biological relevance.


( C - D ) The specificity of the association of the 8SNP accumulation model with an autistic phenotype ( as determined using PAUSS ; compare Fig 4B ) is controlled by applying an unrelated ( or ' non - sense ' ) phenotype , e . g . delusional - depression : ( C ) Intercorrelation pattern of single items included in the delusional - depression composite score , used here as example control phenotype . Cronbach ' s alpha is presented as measure of internal consistency . ( D ) The delusional - depression composite score is not associated with the number of proautistic genotypes of the 8SNP risk model in the discovery sample .

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(C-D) The specificity of the association of the 8SNP accumulation model with an autistic phenotype (as determined using PAUSS; compare Fig 4B) is controlled by applying an unrelated (or 'non-sense') phenotype, e.g. delusional-depression: (C) Intercorrelation pattern of single items included in the delusional-depression composite score, used here as example control phenotype. Cronbach's alpha is presented as measure of internal consistency. (D) The delusional-depression composite score is not associated with the number of proautistic genotypes of the 8SNP risk model in the discovery sample.


( A ) PAUSS ( PANSS autism severity score ) composition and item intercorrelation pattern in the GRAS sample of male schizophrenic individuals ( discovery sample ) . Cronbach ' s alpha is presented as measure of internal consistency and also provided for the male replication samples I and II .

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(A) PAUSS (PANSS autism severity score) composition and item intercorrelation pattern in the GRAS sample of male schizophrenic individuals (discovery sample). Cronbach's alpha is presented as measure of internal consistency and also provided for the male replication samples I and II.


( B ) Association of PAUSS with the number of proautistic genotypes of the 8SNP risk model in the discovery sample .

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(B) Association of PAUSS with the number of proautistic genotypes of the 8SNP risk model in the discovery sample.


( C ) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in the discovery sample ; binary logistic regression analysis with non - z - standardized PAUSS as dependent variable ; mean ± SEM .

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(C) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in the discovery sample; binary logistic regression analysis with non-z-standardized PAUSS as dependent variable; mean±SEM.


( D ) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in replication sample I of male schizophrenia patients ; binary logistic regression analysis ; mean ± SEM .

(D) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in replication sample I of male schizophrenia patients; binary logistic regression analysis; mean±SEM.


( E ) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in replication sample II of male disease control patients ; Mann - Whitney U test ; mean ± SEM .

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(E) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in replication sample II of male disease control patients; Mann-Whitney U test; mean±SEM.


F ) The highly significant correlation of PAUSS and social support underlines the validity of social support as an autism proxy phenotype ; mean ± SEM .

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F) The highly significant correlation of PAUSS and social support underlines the validity of social support as an autism proxy phenotype; mean±SEM.


( G ) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the discovery sample ; binary logistic regression analysis ; mean ± SEM .

(G) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the discovery sample; binary logistic regression analysis; mean±SEM.


( H ) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the male replication sample III from general population ; binary logistic regression analysis ; mean ± SEM . For all replications p - values for one - sided tests are shown .

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

(H) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the male replication sample III from general population; binary logistic regression analysis; mean±SEM. For all replications p-values for one-sided tests are shown.


( A ) FXR1 expression in PBMC of individuals carrying the rs2601 GG ( low risk ; N = 16 ) versus AA genotype ( high risk ; N = 27 ) .

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(A) FXR1 expression in PBMC of individuals carrying the rs2601 GG (low risk; N=16) versus AA genotype (high risk; N=27).


( B ) PBMC microRNA expression was normalized and data is plotted from all 4 miR - 181 - 5p members ( miR - 181a , b , c , d - 5p ) .

(B) PBMC microRNA expression was normalized and data is plotted from all 4 miR-181-5p members (miR-181a, b, c, d-5p).


( C ) Sequence homology of all 4 human mature miR - 181 species shown together with sequences in the broader fragile X gene family containing binding sites for the miR - 181 family ( seed matches identified using Target Scan Human and SFOLD ) . The red letters specify seed sequences and seed matches , respectively . Chromosome positions for each seed sequence and seed match are denoted ( human genome assembly GRCh38 / hg38 ) .

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(C) Sequence homology of all 4 human mature miR-181 species shown together with sequences in the broader fragile X gene family containing binding sites for the miR-181 family (seed matches identified using Target Scan Human and SFOLD). The red letters specify seed sequences and seed matches, respectively. Chromosome positions for each seed sequence and seed match are denoted (human genome assembly GRCh38/hg38).


( D ) Denoted are ΔΔG values for binding of each of the miR - 181 family members to different 3 ' UTR positions . Positions were identified using Target Scan Human and SFOLD and then processed using PITA algorithm to yield the denoted ΔΔG values . ΔΔG is an energetic score , and the more negative its value , the stronger is the expected binding of the microRNA to the given

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(D) Denoted are ΔΔG values for binding of each of the miR-181 family members to different 3'UTR positions. Positions were identified using Target Scan Human and SFOLD and then processed using PITA algorithm to yield the denoted ΔΔG values. ΔΔG is an energetic score, and the more negative its value, the stronger is the expected binding of the microRNA to the given site


Representative images of the size of Mex3a mutant mice and control littermates at postnatal day ( P ) 15 . Scale bar , 1 cm . Genotypes were confirmed by Mex3a mRNA ISH in intestinal tissue ( right panels ) . Scale bars , 50 μm . The offspring number ( n ) and observed genotype frequencies ( % ) resulting from heterozygous crosses are indicated below .

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Representative images of the size of Mex3a mutant mice and control littermates at postnatal day (P)15. Scale bar, 1 cm. Genotypes were confirmed by Mex3a mRNA ISH in intestinal tissue (right panels). Scale bars, 50 μm. The offspring number (n) and observed genotype frequencies (%) resulting from heterozygous crosses are indicated below.


Absolute weight of Mex3a KO mice and control littermates at different ages . Data is represented in a box and whiskers plot as mean ( middle line ) with the minimum and maximum distribution values . Each point depicts one animal ( WT : P1 , n = 15 ; P8 , n = 36 ; P15 , n = 43 ; P22 , n = 14 ; Adult , n = 14 ; Mex3a + / - : P1 , n = 24 ; P8 , n = 90 ; P15 , n = 90 ; P22 , n = 37 ; Adult , n = 21 ; Mex3a - / - : P1 , n = 9 ; P8 , n = 24 ; P15 , n = 26 ; P22 , n = 14 ; Adult , n = 8 ) * * * P = 0 . 0003 , * * * * P < 0 . 0001 , two - way ANOVA test .

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Absolute weight of Mex3a KO mice and control littermates at different ages. Data is represented in a box and whiskers plot as mean (middle line) with the minimum and maximum distribution values. Each point depicts one animal (WT: P1, n = 15; P8, n = 36; P15, n = 43; P22, n = 14; Adult, n = 14; Mex3a+/-: P1, n = 24; P8, n = 90; P15, n = 90; P22, n = 37; Adult, n = 21; Mex3a-/-: P1, n = 9; P8, n = 24; P15, n = 26; P22, n = 14; Adult, n = 8) ***P = 0.0003, ****P < 0.0001, two-way ANOVA test.


Kaplan - Meier survival curves for the different Mex3a genotypes ( n = 80 for each genotype ) * * * * P < 0 . 0001 , Log - rank ( Mantel - Cox ) test .

Kaplan-Meier survival curves for the different Mex3a genotypes (n = 80 for each genotype) ****P < 0.0001, Log-rank (Mantel-Cox) test.


Macroscopic assessment of the gastrointestinal tract of Mex3a KO and WT mice . These images are representative of the phenotype observed in mutant animals euthanized at different postnatal days . Boxed areas depict the distal small intestinal section ( ileum ) used for subsequent immunohistochemical analyses .

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Macroscopic assessment of the gastrointestinal tract of Mex3a KO and WT mice. These images are representative of the phenotype observed in mutant animals euthanized at different postnatal days. Boxed areas depict the distal small intestinal section (ileum) used for subsequent immunohistochemical analyses.


H & E staining of a Mex3a KO and WT littermate at P19 . Inserts depict high magnification of the boxed areas .

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H&E staining of a Mex3a KO and WT littermate at P19. Inserts depict high magnification of the boxed areas.


Average crypt depth ( C ) of WT animals ( n = 3 , > 40 crypts counted per animal ) and Mex3a mutants ( n = 6 , > 20 crypts counted per animal ) . Also shown is the average villi height ( D ) of WT animals ( n = 3 , > 25 villi counted per animal ) and Mex3a mutants ( n = 6 , > 15 villi counted per animal ) . Data is represented as mean ± standard error between P16 and P18 . * P = 0 . 03 , * * * P = 0 . 0006 , Student ` s t test .

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Average crypt depth (C) of WT animals (n = 3, > 40 crypts counted per animal) and Mex3a mutants (n = 6, > 20 crypts counted per animal). Also shown is the average villi height (D) of WT animals (n = 3, > 25 villi counted per animal) and Mex3a mutants (n = 6, > 15 villi counted per animal). Data is represented as mean ± standard error between P16 and P18. *P = 0.03, ***P = 0.0006, Student`s t test.


Immunohistochemistry staining for ( E ) the tuft cell marker DCLK1 ( black arrowheads indicate positive cells ) , ( F ) the Paneth cell marker

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Immunohistochemistry staining for (E) the tuft cell marker DCLK1 (black arrowheads indicate positive cells), (F) the Paneth cell marker LYZ1


Immunohistochemistry staining for the proliferation marker KI67 .

Immunohistochemistry staining for the proliferation marker KI67.


mRNA ISH staining for the stem cell markers ( H ) Lgr5 , ( I ) Olfm4 , ( J ) Lrig1 , and ( K ) Hopx in ileal sections of Mex3a mutants and littermate controls at P19 . Inserts depict high magnification of the boxed areas .

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mRNA ISH staining for the stem cell markers (H) Lgr5, (I) Olfm4, (J) Lrig1, and (K) Hopx in ileal sections of Mex3a mutants and littermate controls at P19. Inserts depict high magnification of the boxed areas.


qPCR analysis of the expression level of the indicated stem cell markers in freshly isolated crypt fractions from Mex3a KO and WT animals ( n = 3 of each genotype ) . Data is represented as mean fold - change plus standard error in Mex3a KO mice relatively to WT animals ( dashed line ) . * P = 0 . 0208 , * * P < 0 . 01 , * * * P = 0 . 0007 , Student ` s t test .

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qPCR analysis of the expression level of the indicated stem cell markers in freshly isolated crypt fractions from Mex3a KO and WT animals (n = 3 of each genotype). Data is represented as mean fold-change plus standard error in Mex3a KO mice relatively to WT animals (dashed line). *P = 0.0208, **P < 0.01, ***P = 0.0007, Student`s t test.


Quantification of the size ( diameter length ) of Mex3a KO and WT organoid - initiating structures 2 days after crypt isolation ( n = 3 for each genotype , > 150 structures counted in total ) . Data is represented in a box and whiskers plot as mean ( middle line ) with the minimum and maximum distribution values . Each point depicts one organoid - initiating structure . * * * * P < 0 . 0001 , Student ` s t test . Phase contrast microscopy images of intestinal organoids generated by Mex3a - / - and WT crypts 6 days after crypt isolation . Scale bars , 100 μm .

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

Quantification of the size (diameter length) of Mex3a KO and WT organoid-initiating structures 2 days after crypt isolation (n = 3 for each genotype, > 150 structures counted in total). Data is represented in a box and whiskers plot as mean (middle line) with the minimum and maximum distribution values. Each point depicts one organoid-initiating structure. ****P < 0.0001, Student`s t test. Phase contrast microscopy images of intestinal organoids generated by Mex3a-/- and WT crypts 6 days after crypt isolation. Scale bars, 100 μm.


Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids at days 2 and 4 of culture after 10 weekly passages . Scale bars , 100 μm .

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Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids at days 2 and 4 of culture after 10 weekly passages. Scale bars, 100 μm.


qPCR analysis of crypt lineage marker genes in organoid cultures at day 2 . Data is presented as the mean fold - change plus standard error ( n = 4 for each genotype ) for target gene expression in Mex3a KO intestinal organoids relative to WT ( dashed line ) . * P < 0 . 05 , Student ` s 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

qPCR analysis of crypt lineage marker genes in organoid cultures at day 2. Data is presented as the mean fold-change plus standard error (n = 4 for each genotype) for target gene expression in Mex3a KO intestinal organoids relative to WT (dashed line). *P < 0.05, Student`s t test.


Immunohistochemistry staining for the Paneth cell marker LYZ1 in Mex3a KO and WT intestinal organoid sections at day 2 of culture . Scale bars , 50 μm .

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Immunohistochemistry staining for the Paneth cell marker LYZ1 in Mex3a KO and WT intestinal organoid sections at day 2 of culture. Scale bars, 50 μm.


qPCR analysis of crypt lineage marker genes in organoid cultures at day 4 . Data is presented as the mean fold - change plus standard error ( n = 4 for each genotype ) for target gene expression in Mex3a KO intestinal organoids relative to WT ( dashed line ) .

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qPCR analysis of crypt lineage marker genes in organoid cultures at day 4. Data is presented as the mean fold-change plus standard error (n = 4 for each genotype) for target gene expression in Mex3a KO intestinal organoids relative to WT (dashed line).


mRNA ISH staining for the stem cell markers Mex3a , Lgr5 , Hopx and Lrig1 in Mex3a KO and WT intestinal organoid sections at day 2 of culture . Scale bars , 50 μm .

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mRNA ISH staining for the stem cell markers Mex3a, Lgr5, Hopx and Lrig1 in Mex3a KO and WT intestinal organoid sections at day 2 of culture. Scale bars, 50 μm.


Representative images of immunofluorescence staining for the proliferation marker KI67 and BrdU incorporation in Mex3a KO and WT animals . Mice were injected at P17 and sacrificed at P20 ( n = 3 for each genotype ) . Scale bars , 50 μm .

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Representative images of immunofluorescence staining for the proliferation marker KI67 and BrdU incorporation in Mex3a KO and WT animals. Mice were injected at P17 and sacrificed at P20 (n = 3 for each genotype). Scale bars, 50 μm.


Hierarchical clustering of samples ( n = 3 for each genotype ) and heatmap of differentially expressed genes between the Mex3a - / - and WT mice . The red colour represents over - expression , while the blue colour under - expression .

Hierarchical clustering of samples (n = 3 for each genotype) and heatmap of differentially expressed genes between the Mex3a-/- and WT mice. The red colour represents over-expression, while the blue colour under-expression.


GSEA pre - ranked analysis comparing the Mex3a - / - transcriptional profile against ( E ) the

GSEA pre-ranked analysis comparing the Mex3a-/- transcriptional profile against (E) the Mex3ahigh


GSEA pre - ranked analysis comparing the Mex3a - / - transcriptional profile against the Lgr5high and ( G ) the Wnt signalling pathway gene signatures .

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GSEA pre-ranked analysis comparing the Mex3a-/- transcriptional profile against the Lgr5high and (G) the Wnt signalling pathway gene signatures.


qPCR analysis of selected genes in a cohort of independent mice . Data is presented as the mean fold - change of target gene expression in 2 biological replicates ( Mex3a - / - Vs WT # 1 and Mex3a - / - Vs WT # 2 ) of Mex3a KO intestinal crypts relative to the respective WT controls ( dashed line ) .

qPCR analysis of selected genes in a cohort of independent mice. Data is presented as the mean fold-change of target gene expression in 2 biological replicates (Mex3a-/- Vs WT #1 and Mex3a-/- Vs WT #2) of Mex3a KO intestinal crypts relative to the respective WT controls (dashed line).


Immunohistochemistry staining for PPARγ in Mex3a KO and WT mice ileum . Inserts depict high magnification of the boxed areas . Scale bars , 50 μm .

Immunohistochemistry staining for PPARγ in Mex3a KO and WT mice ileum. Inserts depict high magnification of the boxed areas. Scale bars, 50 μm.


Pparg mRNA ISH in ileal sections of Mex3a mutants and littermate controls at P18 . Inserts depict high magnification of the boxed areas . Scale bars , 50 μm .

Pparg mRNA ISH in ileal sections of Mex3a mutants and littermate controls at P18. Inserts depict high magnification of the boxed areas. Scale bars, 50 μm.


qPCR analysis of PPARγ transcriptional targets in organoid cultures at day 4 . Data is presented as the mean fold - change plus standard error ( n = 3 for each genotype ) for target gene expression in Mex3a KO intestinal organoids relative to WT ( dashed line ) .

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qPCR analysis of PPARγ transcriptional targets in organoid cultures at day 4. Data is presented as the mean fold-change plus standard error (n = 3 for each genotype) for target gene expression in Mex3a KO intestinal organoids relative to WT (dashed line).


Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids after 5 days of treatment with 50 μM Pioglitazone or vehicle ( DMSO ) . Scale bars , 100 μm .

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Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids after 5 days of treatment with 50 μM Pioglitazone or vehicle (DMSO). Scale bars, 100 μm.


qPCR analysis of Lgr5 , Pcna and Angptl4 expression in organoid cultures after 5 days of treatment with 50 μM Pioglitazone . Data is presented as the mean fold - change plus standard error ( n = 4 for each genotype ) for target gene expression in Pioglitazone - treated organoids relative to the DMSO vehicle ( dashed line ) . * P = 0 . 02 , Student ` s t test .

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qPCR analysis of Lgr5, Pcna and Angptl4 expression in organoid cultures after 5 days of treatment with 50 μM Pioglitazone. Data is presented as the mean fold-change plus standard error (n = 4 for each genotype) for target gene expression in Pioglitazone-treated organoids relative to the DMSO vehicle (dashed line). * P = 0.02, Student`s t test.


Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids at day 1 after passaging Pioglitazone - or vehicle ( DMSO ) - treated organoids . Scale bars , 100 μm .

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Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids at day 1 after passaging Pioglitazone- or vehicle (DMSO)-treated organoids. Scale bars, 100 μm.


B ) Localization of the GFP - tagged PE synthesizing enzyme ( Psd ) targeted to the indicated organelles in choppΔ cells , grown in SD medium supplemented with 10 mM choline . Images shown are either a single Z - slice ( ER construct ) or a maximum intensity projection of several Z - sections ( other constructs ) ; LD , lipid droplets ; MM , mitochondrial matrix . A scheme depicting each chimeric Psd construct is shown under the corresponding microscopy image . C ) Localization of the GFP - tagged PC synthesizing enzyme ( Pmt ) targeted to the indicated organelles in choppΔ cells , grown in SD medium supplemented with 10 mM ethanolamine . Images shown are either a single Z - slice ( ER construct ) or a maximum intensity projection of several Z - sections ( other constructs ) . A schematic depicting each chimeric Pmt construct is shown under the corresponding microscopy image .

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B) Localization of the GFP-tagged PE synthesizing enzyme (Psd) targeted to the indicated organelles in choppΔ cells, grown in SD medium supplemented with 10 mM choline. Images shown are either a single Z-slice (ER construct) or a maximum intensity projection of several Z-sections (other constructs); LD, lipid droplets; MM, mitochondrial matrix. A scheme depicting each chimeric Psd construct is shown under the corresponding microscopy image. C) Localization of the GFP-tagged PC synthesizing enzyme (Pmt) targeted to the indicated organelles in choppΔ cells, grown in SD medium supplemented with 10 mM ethanolamine. Images shown are either a single Z-slice (ER construct) or a maximum intensity projection of several Z-sections (other constructs). A schematic depicting each chimeric Pmt construct is shown under the corresponding microscopy image.


B ) Five - fold serial dilutions of strains of the indicated genotypes ( wt cells and choppΔ cells harboring empty vectors or choppΔ cells expressing chimeric Psd and Pmt enzymes ) . Cells were grown on SD - HIS - LEU ( - HL ) medium ( KennedyOFF ) or SD - HL containing ethanolamine and choline ( KennedyON ) .

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B) Five-fold serial dilutions of strains of the indicated genotypes (wt cells and choppΔ cells harboring empty vectors or choppΔ cells expressing chimeric Psd and Pmt enzymes). Cells were grown on SD-HIS-LEU (-HL) medium (KennedyOFF) or SD-HL containing ethanolamine and choline (KennedyON).


C ) Thin layer chromatography ( TLC ) analysis of steady - state phospholipid profiles of cells of the indicated genotypes . Where Psd and / or Pmt are missing ( - ) , choppΔ cells were grown in the presence of ethanolamine and / or choline . Bands corresponding to the phospholipids PA , PS , PE , PI and PC are indicated .

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C) Thin layer chromatography (TLC) analysis of steady-state phospholipid profiles of cells of the indicated genotypes. Where Psd and/or Pmt are missing (-), choppΔ cells were grown in the presence of ethanolamine and/or choline. Bands corresponding to the phospholipids PA, PS, PE, PI and PC are indicated.


C ) VPS13 is required in KennedyOFF conditions when chimeric enzymes are targeted to mitochondria or endosomes . Volcano plots show the fold change of number of transposon insertions per gene of libraries grown in KennedyOFF versus ON conditions . This comparison included all six libraries where the enzymes are targeted to endosomes and / or mitochondria ( top left panel ) or six libraries with enzymes targeted to other organelles ( bottom left panel ) . Data - points for VPS13 are highlighted in red . Schematic illustrating Vps13 - mediated lipid transport at endosomes and mitochondria contact sites ( right panel ) .

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C) VPS13 is required in KennedyOFF conditions when chimeric enzymes are targeted to mitochondria or endosomes. Volcano plots show the fold change of number of transposon insertions per gene of libraries grown in KennedyOFF versus ON conditions. This comparison included all six libraries where the enzymes are targeted to endosomes and/or mitochondria (top left panel) or six libraries with enzymes targeted to other organelles (bottom left panel). Data-points for VPS13 are highlighted in red. Schematic illustrating Vps13-mediated lipid transport at endosomes and mitochondria contact sites (right panel).


B ) Volcano plot comparing the number of transposon insertions per gene of all 12 libraries in KennedyON and OFF conditions . Genes significantly required in KennedyOFF conditions are highlighted in red ( p - value < 0 . 05 , log2 ( fold change ) < - 0 . 5 ) . Genes highlighted in the clusterogram in A ( top inset ) are indicated with blue arrows .

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B) Volcano plot comparing the number of transposon insertions per gene of all 12 libraries in KennedyON and OFF conditions. Genes significantly required in KennedyOFF conditions are highlighted in red (p-value < 0.05, log2(fold change) < -0.5). Genes highlighted in the clusterogram in A (top inset) are indicated with blue arrows.


B ) Requirement for mitochondrial lipid transport and biosynthesis genes in rewired libraries . Left panel , schematic depicting PE and cardiolipin ( CL ) synthesis in mitochondria . The inner mitochondrial putative lipid transporter Mdm31 is depicted in yellow . Grey arrows indicate the need for lipid transport from the ER or other organelles to mitochondria and transport between the MOM and MIM . Right panel , transposon numbers ( normalized to wt libraries ) for the indicated ( set of ) libraries for MDM31 , PGS1 , and CRD1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Requirement for mitochondrial lipid transport and biosynthesis genes in rewired libraries. Left panel, schematic depicting PE and cardiolipin (CL) synthesis in mitochondria. The inner mitochondrial putative lipid transporter Mdm31 is depicted in yellow. Grey arrows indicate the need for lipid transport from the ER or other organelles to mitochondria and transport between the MOM and MIM. Right panel, transposon numbers (normalized to wt libraries) for the indicated (set of) libraries for MDM31, PGS1, and CRD1.


B ) Five - fold serial dilutions of strains of the indicated genotypes grown at 30 ° C on SD medium supplemented with 10 mM ethanolamine and / or 10 mM choline . Growth assays are representative images of biological replicates .

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B) Five-fold serial dilutions of strains of the indicated genotypes grown at 30°C on SD medium supplemented with 10 mM ethanolamine and/or 10 mM choline. Growth assays are representative images of biological replicates.


A ) Top : Co - localization of Csf1 - GFP cells in log phase with genomically integrated Pex10 - mCherry ( peroxisome marker ) and mtBFP plasmid ( mito marker ) . Bottom : Co - localization of log growing Csf1 - GFP cells with mCherry - Ubc6 ( tail - anchor ) ( ER marker ) and mtBFP plasmid ( mito marker ) . Scale bar , 2 µm .

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A) Top: Co-localization of Csf1-GFP cells in log phase with genomically integrated Pex10-mCherry (peroxisome marker) and mtBFP plasmid (mito marker). Bottom: Co-localization of log growing Csf1-GFP cells with mCherry-Ubc6 (tail-anchor) (ER marker) and mtBFP plasmid (mito marker). Scale bar, 2 µm.


C ) Five - fold serial dilutions of strains of the indicated genotypes grown at indicated temperatures , in the presence or absence of 10 mM ethanolamine ( etn ) .

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C) Five-fold serial dilutions of strains of the indicated genotypes grown at indicated temperatures, in the presence or absence of 10 mM ethanolamine (etn).


E ) Bar graphs depicting the fraction of more saturated species ( defined as fatty acid chains with one double bond for ≤ 30 carbon chains or with two double bonds for ≥ 32 ) , and less unsaturated species ( defined as fatty acid chains with zero double bond for ≤ 30 carbon chains or with one double bond for ≥ 32 ) for each phospholipid class with different head groups . Bars represent the means of two or three biological replicates ( individual measurements are shown as points and triangles ) . p - values are calculated with a two - sample t - test .

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E) Bar graphs depicting the fraction of more saturated species (defined as fatty acid chains with one double bond for ≤30 carbon chains or with two double bonds for ≥32), and less unsaturated species (defined as fatty acid chains with zero double bond for ≤30 carbon chains or with one double bond for ≥32) for each phospholipid class with different head groups. Bars represent the means of two or three biological replicates (individual measurements are shown as points and triangles). p-values are calculated with a two-sample t-test.


( a ) Intracellular levels of spermidine ( Spd ) in chronologically ageing wild - type yeast . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 05 and * P 0 . 001 ) .

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(a) Intracellular levels of spermidine (Spd) in chronologically ageing wild-type yeast. Data represent means ± s.e.m. (n = 3; *P 0.05 and *P 0.001).


( b ) Survival determined by clonogenicity during chronological ageing of wild - type yeast ( BY4741 ) cells with ( o ) and without ( ▪ ) addition of spermidine ( 4 mM ) at day 1 . Data represent means ± s . e . m . ( n = 5 ) .

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(b) Survival determined by clonogenicity during chronological ageing of wild-type yeast (BY4741) cells with (o) and without (▪) addition of spermidine (4 mM) at day 1. Data represent means ± s.e.m. (n = 5).


( c ) Intracellular levels of spermidine in chronologically ageing wild - type yeast cells cultured with ( open bar ) or without ( closed bar ) spermidine ( 4 mM ) for 5 days . Data represent means ± s . e . m . ( n = 3 ; * * P 0 . 001 ) .

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(c) Intracellular levels of spermidine in chronologically ageing wild-type yeast cells cultured with (open bar) or without (closed bar) spermidine (4 mM) for 5 days. Data represent means ± s.e.m. (n = 3; **P 0.001).


( d ) Replicative lifespan analysis of BY4741 wild - type yeast cells after separation into old ( fraction V ) and young ( fraction II ) cells by elutriation centrifugation . The remaining lifespan with or without ( control ) spermidine ( 1 mM , applied after elutriation ) on 2 % glucose synthetic complete medium is shown .

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(d) Replicative lifespan analysis of BY4741 wild-type yeast cells after separation into old (fraction V) and young (fraction II) cells by elutriation centrifugation. The remaining lifespan with or without (control) spermidine (1 mM, applied after elutriation) on 2% glucose synthetic complete medium is shown.


( e ) Survival determined by the age - specific number of dead individuals of female Drosophila with and without ( control ) supplementation of food with various concentrations of spermidine ( as indicated ) . A representative ageing experiment of at least 50 flies per sample is shown .

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(e) Survival determined by the age-specific number of dead individuals of female Drosophila with and without (control) supplementation of food with various concentrations of spermidine (as indicated). A representative ageing experiment of at least 50 flies per sample is shown.


( f ) Endogenous spermidine of female Drosophila fed for 48 h with food supplemented by 1 mM spermidine , compared with normal food ( control ) . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 01 ) .

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(f) Endogenous spermidine of female Drosophila fed for 48 h with food supplemented by 1 mM spermidine, compared with normal food (control). Data represent means ± s.e.m. (n = 3; *P 0.01).


( g ) Survival determined by annexin V / 7 - AAD co - staining ( unstained cells were considered as viable ) of human immune cells ( PBMC ) cultured for 6 and 12 days in the absence ( black bar ) or presence ( white bars ) of various spermidine concentrations ( as indicated ) . Data represent means ± s . e . m . of 3 independent experiments ( each performed on PBMC from different donors ) . * P 0 . 05 and * * P 0 . 01 .

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(g) Survival determined by annexin V/7-AAD co-staining (unstained cells were considered as viable) of human immune cells (PBMC) cultured for 6 and 12 days in the absence (black bar) or presence (white bars) of various spermidine concentrations (as indicated). Data represent means ± s.e.m. of 3 independent experiments (each performed on PBMC from different donors). *P 0.05 and **P 0.01.


( h , i ) Free thiol group ( RSH ) concentration in serum ( h ) and intracellular spermidine concentration in hepatocytes ( i ) of ageing mice with ( open bars ) or without ( closed bar ) supplementation of drinking water with spermidine ( 0 . 3 and 3 mM ) for 200 days . Data represent mean ± s . e . m . ( n = 3 ; * P 0 . 05 and * * P 0 . 01 ) .

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(h, i) Free thiol group (RSH) concentration in serum (h) and intracellular spermidine concentration in hepatocytes (i) of ageing mice with (open bars) or without (closed bar) supplementation of drinking water with spermidine (0.3 and 3 mM) for 200 days. Data represent mean ± s.e.m. (n = 3; *P 0.05 and **P 0.01).


( a ) Intracellular spermidine of five - day - oldΔspe1 cells , compared with wild - type cells . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 001 ) .

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(a) Intracellular spermidine of five-day-oldΔspe1 cells, compared with wild-type cells. Data represent means ± s.e.m. (n = 3; *P 0.001).


( b ) Chronological ageing of wild - type ( ▪ , o ) and polyamine - depleted Δspe1 ( ▴ , ▿ ) yeast cells with ( open symbols ) and without ( closed symbols ) supplementation of low doses of spermidine . Data represent means ± s . e . m . ( n = 4 ) . Cells were tested for cell death markers at day 3 .

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(b) Chronological ageing of wild-type (▪, o) and polyamine-depleted Δspe1 (▴, ▿) yeast cells with (open symbols) and without (closed symbols) supplementation of low doses of spermidine. Data represent means ± s.e.m. (n = 4). Cells were tested for cell death markers at day 3.


( c ) Fluorescence microscopy of DHE → Eth conversion in wild - type and Δspe1 cells indicating ROS production . Scale bars , 10 μm .

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(c) Fluorescence microscopy of DHE→Eth conversion in wild-type and Δspe1 cells indicating ROS production. Scale bars, 10 μm.


( d ) Quantification ( FACS analysis ) of ROS accumulation using DHE → Eth conversion of wild - type ( WT ) and Δspe1 cells with and without supplementation of low doses of spermidine . Data represent means ± s . e . m . ( n = 4 ; * P 0 . 001 ) .

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(d) Quantification (FACS analysis) of ROS accumulation using DHE→Eth conversion of wild-type (WT) and Δspe1 cells with and without supplementation of low doses of spermidine. Data represent means ± s.e.m. (n = 4; *P 0.001).


( e ) Quantification ( FACS analysis ) of phosphatidylserine externalization and loss of membrane integrity using annexin V / PI co - staining ( at day 5 ) and of DNA fragmentation using TUNEL staining ( at day 3 ) of chronologically ageing wild - type ( WT ) and Δspe1 cells with or without supplementation of 0 . 1 mM spermidine as indicated . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 001 ) .

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(e) Quantification (FACS analysis) of phosphatidylserine externalization and loss of membrane integrity using annexin V/PI co-staining (at day 5) and of DNA fragmentation using TUNEL staining (at day 3) of chronologically ageing wild-type (WT) and Δspe1 cells with or without supplementation of 0.1 mM spermidine as indicated. Data represent means ± s.e.m. (n = 3; *P 0.001).


( a ) Fluorescence microscopy of DHE → Eth conversion and annexin V ( green ) / PI ( red ) co - staining of wild - type cells at day 18 of the chronological ageing experiment shown in Fig . 1b . Scale bars , 10 μm . ( b , c ) Quantification of DHE → Eth conversion ( b ) and annexin V / PI co - staining ( c ) using FACS analysis performed at indicated time - points of the chronological ageing experiment shown in Fig . 1b . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 001 ) .

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(a) Fluorescence microscopy of DHE→Eth conversion and annexin V (green)/PI (red) co-staining of wild-type cells at day 18 of the chronological ageing experiment shown in Fig. 1b. Scale bars, 10 μm. (b, c) Quantification of DHE→Eth conversion (b) and annexin V/PI co-staining (c) using FACS analysis performed at indicated time-points of the chronological ageing experiment shown in Fig. 1b. Data represent means ± s.e.m. (n = 3; *P 0.001).


( d ) Electron microscopy of young log - phase cells ( day 0 ) and of 20 - day - old wild - type cells aged without ( control ) or with spermidine ( 4 mM ) . Representative cells are shown ( for an overview see Supplementary Information , Fig . S3c ) .

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(d) Electron microscopy of young log-phase cells (day 0) and of 20-day-old wild-type cells aged without (control) or with spermidine (4 mM). Representative cells are shown (for an overview see Supplementary Information, Fig. S3c).


( e ) Fluorescence microscopy of chronologically aged wild - type cells ( day 3 and 14 ) expressing an EGFP - tagged version of the yeast HMGB1 homologue ( Nhp6A - EGFP ) with or without ( control ) addition of 4 mM spermidine . Scale bars , 5 μm .

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(e) Fluorescence microscopy of chronologically aged wild-type cells (day 3 and 14) expressing an EGFP-tagged version of the yeast HMGB1 homologue (Nhp6A-EGFP) with or without (control) addition of 4 mM spermidine. Scale bars, 5 μm.


( a ) Relative acetylation ( normalized to controls at each day ) of indicated histone H3 Lys residues determined by quantification of immunoblot analysis . A representative blot using site specific antibodies is shown in b . Data represent means ± s . e . m . ( n = 3 ) . Serial dilutions of protein extracts were applied in western blots to verify linearity before quantification ( examples are shown in Supplementary Information , Fig . S4 ) . For calculation details see section on Methods . P values indicate the result of a two - factor ANOVA corrected by Bonferroni post hoc test . ( b ) Immunoblot of whole - cell extracts of wild - type cells chronologically aged to designated time - points with ( + ) or without ( − ) spermidine application . Blots were probed with antibodies against total histone H3 or H3 acetylation sites at the indicated Lys residues . Full scans of blots are available in Supplementary Information , Fig . S5 .

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(a) Relative acetylation (normalized to controls at each day) of indicated histone H3 Lys residues determined by quantification of immunoblot analysis. A representative blot using site specific antibodies is shown in b. Data represent means ± s.e.m. (n = 3). Serial dilutions of protein extracts were applied in western blots to verify linearity before quantification (examples are shown in Supplementary Information, Fig. S4). For calculation details see section on Methods. P values indicate the result of a two-factor ANOVA corrected by Bonferroni post hoc test. (b) Immunoblot of whole-cell extracts of wild-type cells chronologically aged to designated time-points with (+) or without (−) spermidine application. Blots were probed with antibodies against total histone H3 or H3 acetylation sites at the indicated Lys residues. Full scans of blots are available in Supplementary Information, Fig. S5.


( c ) Relative acetylation ( normalized to wild - type controls at each day ) of indicated histone H3 Lys residues of Δspe1 cells ( open bars ) , compared with wild - type cells ( closed bars ) during chronological ageing . Data represent means ± s . e . m . ( n = 3 ) . P values indicate the result of a two - factor ANOVA corrected by Bonferroni post hoc test .

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(c) Relative acetylation (normalized to wild-type controls at each day) of indicated histone H3 Lys residues of Δspe1 cells (open bars), compared with wild-type cells (closed bars) during chronological ageing. Data represent means ± s.e.m. (n = 3). P values indicate the result of a two-factor ANOVA corrected by Bonferroni post hoc test.


( d ) Relative acetylation ( normalized to day 1 ) of histone H3 Lys 18 residue of chronologically ageing wild - type cells . Data represent means ± s . e . m . ( n = 3 ) .

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(d) Relative acetylation (normalized to day 1) of histone H3 Lys 18 residue of chronologically ageing wild-type cells. Data represent means ± s.e.m. (n = 3).


( e ) Relative acetylation ( normalized to controls ) of histone H3 Lys 14 and 18 residues of cultured human PBMC after 6 days of incubation with or without 20 nM spermidine . Data represent quantification of two independent experiments performed with cells obtained from two different donors .

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(e) Relative acetylation (normalized to controls) of histone H3 Lys 14 and 18 residues of cultured human PBMC after 6 days of incubation with or without 20 nM spermidine. Data represent quantification of two independent experiments performed with cells obtained from two different donors.


( f ) Immunoblot analysis of liver cell extracts ( applied in serial dilutions ) obtained from mice fed with 3 mM spermidine for 200 days and respective control mice of the same age . Blots were probed with antibodies recognizing total histone H3 ( C - terminal epitope ) or specific for acetylated lysine 18 residue ( N - terminal epitope ) . Full scans of blots are available in Supplementary Information , Fig . S5 .

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(f) Immunoblot analysis of liver cell extracts (applied in serial dilutions) obtained from mice fed with 3 mM spermidine for 200 days and respective control mice of the same age. Blots were probed with antibodies recognizing total histone H3 (C-terminal epitope) or specific for acetylated lysine 18 residue (N-terminal epitope). Full scans of blots are available in Supplementary Information, Fig. S5.


( a ) Relative acetylation of histone H3 Lys 9 and 14 residues determined by quantification of immunoblot analysis performed at day 20 of the ageing experiment shown in panel b . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 05 ) . ( b ) Chronological ageing of wild - type ( ▪ , o ) and Δiki3Δsas3 ( ▴ , ▿ ) with ( open symbols ) or without ( closed symbols ) addition of spermidine ( 4 mM ) at day 1 . Data represent means ± s . e . m . ( n = 4 ) . ( c ) Quantification ( FACS analysis ) of phosphatidylserine externalization and loss of membrane integrity ( annexinV / PI costaining ) as well as ROS production ( DHE - > Eth conversion ) performed at day 20 of the experiment shown in b . Data represent means ± s . e . m . ( n = 4 ; * P 0 . 001 ) .

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

(a) Relative acetylation of histone H3 Lys 9 and 14 residues determined by quantification of immunoblot analysis performed at day 20 of the ageing experiment shown in panel b. Data represent means ± s.e.m. (n = 3; *P 0.05). (b) Chronological ageing of wild-type (▪, o) and Δiki3Δsas3 (▴, ▿) with (open symbols) or without (closed symbols) addition of spermidine (4 mM) at day 1. Data represent means ± s.e.m. (n = 4). (c) Quantification (FACS analysis) of phosphatidylserine externalization and loss of membrane integrity (annexinV/PI costaining) as well as ROS production (DHE->Eth conversion) performed at day 20 of the experiment shown in b. Data represent means ± s.e.m. (n = 4; *P 0.001).


( d ) Relative inhibition of histone acetyltransferase activity ( HAT - activity ) by spermidine determined by an in vitro HAT - activity assay of yeast nuclear extracts of wild - type cells . Data represent means ± s . e . m . of three independent experiments ( * P = 0 . 024 ) .

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(d) Relative inhibition of histone acetyltransferase activity (HAT-activity) by spermidine determined by an in vitro HAT-activity assay of yeast nuclear extracts of wild-type cells. Data represent means ± s.e.m. of three independent experiments (*P = 0.024).


( g ) Relative change of ATG7 , ATG11 and ATG15 mRNA levels by spermidine supplementation ( normalised to controls ) after ten days of chronological ageing as determined by reverse transcriptase real - time PCR . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 05 and * * P 0 . 01 ) .

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(g) Relative change of ATG7, ATG11 and ATG15 mRNA levels by spermidine supplementation (normalised to controls) after ten days of chronological ageing as determined by reverse transcriptase real-time PCR. Data represent means ± s.e.m. (n = 3; *P 0.05 and **P 0.01).


( h ) Relative histone H3 Lys 18 acetylation of indicated promoter regions normalized to the acetylation of ATG7 promoter ( pATG7 ) determined by chromatin immunoprecipitation with H3 - K18Ac specific antibody and subsequent quantification of precipitated promoter DNA using quantitative real time PCR . Signals specific for indicated promoter regions were initially compared with non - immunoprecipitated samples and afterwards normalized to the pATG7 signals . Data represent means ± s . e . m . ( n = 3 ) . The P value was calculated using a two - factor ANOVA with promoter and condition ( treated and non - treated ) as independent factors .

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(h) Relative histone H3 Lys 18 acetylation of indicated promoter regions normalized to the acetylation of ATG7 promoter (pATG7) determined by chromatin immunoprecipitation with H3-K18Ac specific antibody and subsequent quantification of precipitated promoter DNA using quantitative real time PCR. Signals specific for indicated promoter regions were initially compared with non-immunoprecipitated samples and afterwards normalized to the pATG7 signals. Data represent means ± s.e.m. (n = 3). The P value was calculated using a two-factor ANOVA with promoter and condition (treated and non-treated) as independent factors.


( a ) Fluorescence microscopy of wild - type yeast cells expressing an EGFP - Atg8p fusion protein with or without ( control ) treatment of 4 mM spermidine for 48 h . White arrows indicate vacuolar localization of EGFP - Atg8p indicative of autophagy . Scale bars , 5 μm .

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(a) Fluorescence microscopy of wild-type yeast cells expressing an EGFP-Atg8p fusion protein with or without (control) treatment of 4 mM spermidine for 48 h. White arrows indicate vacuolar localization of EGFP-Atg8p indicative of autophagy. Scale bars, 5 μm.


( b ) Relative alkaline phosphatase activity ( ALP activity ) indicative of autophagy during chronological ageing of pho8ΔC60 yeast with ( open bars ) or without ( closed bars ) application of spermidine ( 4 mM ) . Data represent means ± s . e . m . ( n = 3 * ; P 0 . 01 and * * P 0 . 001 ) .

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(b) Relative alkaline phosphatase activity (ALP activity) indicative of autophagy during chronological ageing of pho8ΔC60 yeast with (open bars) or without (closed bars) application of spermidine (4 mM). Data represent means ± s.e.m. (n = 3 *; P 0.01 and **P 0.001).


( c ) Relative alkaline phosphatase activity ( ALP activity ) indicative of autophagy during chronological ageing of wild - type ( closed bars ) and Δiki3Δsas3 ( open bars ) cells . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 01 and * * P 0 . 001 ) .

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(c) Relative alkaline phosphatase activity (ALP activity) indicative of autophagy during chronological ageing of wild-type (closed bars) and Δiki3Δsas3 (open bars) cells. Data represent means ± s.e.m. (n = 3; *P 0.01 and **P 0.001).


( d ) Chronological ageing of wild - type ( ▪ , o ) and Δatg7 ( ▴ , ▿ ) with ( open symbols ) or without ( closed symbols ) addition of spermidine ( 4 mM ) at day 1 . Data represent means ± s . e . m . ( n = 4 ) .

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(d) Chronological ageing of wild-type (▪, o) and Δatg7 (▴, ▿) with (open symbols) or without (closed symbols) addition of spermidine (4 mM) at day 1. Data represent means ± s.e.m. (n = 4).


( e ) Chronological ageing on water of wild - type ( ▪ , o ) and Δatg7 ( ▴ , ▿ ) with ( open symbols ) or without ( closed symbols ) addition of spermidine ( 8 mM ) at day 0 . Data represent means ± s . e . m . ( n = 4 ) .

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(e) Chronological ageing on water of wild-type (▪, o) and Δatg7 (▴, ▿) with (open symbols) or without (closed symbols) addition of spermidine (8 mM) at day 0. Data represent means ± s.e.m. (n = 4).


( f ) Quantification ( fluorescence reader ) of ROS production ( DHE → Eth conversion ) by wild - type and Δatg7 cells with and without supplementation of spermidine ( 8 mM ) , obtained from the ageing experiment shown in panel e . Data represent means ± s . e . m . ( n = 4 ; * P 0 . 07 and * * P 0 . 01 ) .

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(f) Quantification (fluorescence reader) of ROS production (DHE→Eth conversion) by wild-type and Δatg7 cells with and without supplementation of spermidine (8 mM), obtained from the ageing experiment shown in panel e. Data represent means ± s.e.m. (n = 4; *P 0.07 and **P 0.01).


( a ) Fluorescence microscopy of Hoechst - counterstained HeLa cells transiently transfected with LC3 - GFP subjected to 100 μM spermidine for 6 h . Representative pictures are shown . ( b ) Percentage of adherent cells exhibiting a clear LC3 - GFP relocalization into cytoplasmic vacuoles . Numbers were determined using micrographs of Hoechst - counterstained HeLa cells as representatively shown in a . Data represent means ± s . d . ( n = 3 ) .

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(a) Fluorescence microscopy of Hoechst-counterstained HeLa cells transiently transfected with LC3-GFP subjected to 100 μM spermidine for 6 h. Representative pictures are shown. (b) Percentage of adherent cells exhibiting a clear LC3-GFP relocalization into cytoplasmic vacuoles. Numbers were determined using micrographs of Hoechst-counterstained HeLa cells as representatively shown in a. Data represent means ± s.d. (n = 3).


( c ) LysoTracker Red staining of vacuoles indicative of autophagy in oesophagus tissue from flies fed with 1 mM spermidine for two days , compared with controls ( without spermidine ) . Nuclei were visualized by Hoechst staining . Scale bars , 10 μm .

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(c) LysoTracker Red staining of vacuoles indicative of autophagy in oesophagus tissue from flies fed with 1 mM spermidine for two days, compared with controls (without spermidine). Nuclei were visualized by Hoechst staining. Scale bars, 10 μm.


( d ) Quantification of autophagic vesicles per nucleus in LysoTracker Red stained muscle tissue of female flies fed with supplementation of 1 mM spermidine or with 10 % glucose ( starved ) for 48 h , compared with normal food ( control ) . Data represent means ± s . e . m . of at least 20 flies for each group ( * P 0 . 01 ) .

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(d) Quantification of autophagic vesicles per nucleus in LysoTracker Red stained muscle tissue of female flies fed with supplementation of 1 mM spermidine or with 10% glucose (starved) for 48 h, compared with normal food (control). Data represent means ± s.e.m. of at least 20 flies for each group (*P 0.01).


( e , f ) Survival of Drosophila during ageing without ( control ) and with supplementation of food at various concentrations of spermidine ( as indicated ) . Autophagy - deficient flies ( f ) homozygous mutant for Atg7 ( Δatg7 ) were compared with flies capable of autophagy ( e ) and heterozygous for Atg7 ( control ) . For details of strains and additional wild - type controls , see Supplementary Information Methods and Fig . S6h , i .

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(e, f) Survival of Drosophila during ageing without (control) and with supplementation of food at various concentrations of spermidine (as indicated). Autophagy-deficient flies (f) homozygous mutant for Atg7 (Δatg7) were compared with flies capable of autophagy (e) and heterozygous for Atg7 (control). For details of strains and additional wild-type controls, see Supplementary Information Methods and Fig. S6h, i.


( g ) Fluorescence microscopy of C . elegans transgenic embryos expressing a full - length plgg - 1DsRED : : LGG - 1 fusion protein indicative of autophagic activity . Shown are two representative pictures of embryos untreated ( control ) or treated with spermidine ( 0 . 2 mM ) supplementation of food . ( h ) Quantification of autophagic activity through measurement of DsRED : : LGG - 1 pixel intensity from images of wild - type animals shown in g , and bec - 1 RNAi knockdown animals ( bec - 1 RNAi ) . Data represent means ± s . e . m . ( n = 3 ) with at least 25 images processed for each trial .

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(g) Fluorescence microscopy of C. elegans transgenic embryos expressing a full-length plgg-1DsRED::LGG-1 fusion protein indicative of autophagic activity. Shown are two representative pictures of embryos untreated (control) or treated with spermidine (0.2 mM) supplementation of food. (h) Quantification of autophagic activity through measurement of DsRED::LGG-1 pixel intensity from images of wild-type animals shown in g, and bec-1 RNAi knockdown animals (bec-1 RNAi). Data represent means ± s.e.m. (n = 3) with at least 25 images processed for each trial.


( i ) Survival of C . elegans during ageing with and without ( control ) supplementation of food ( UV - killed E . coli ) with spermidine ( 0 . 2 mM ) . Wild - type ( N2 ) animals were compared with bec - 1 RNAi animals deficient in autophagy induction . For mean life spans see Supplementary Information , Table S3 .

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(i) Survival of C. elegans during ageing with and without (control) supplementation of food (UV-killed E. coli) with spermidine (0.2 mM). Wild-type (N2) animals were compared with bec-1 RNAi animals deficient in autophagy induction. For mean life spans see Supplementary Information, Table S3.


A , B MARS ( A ) and MARS2 ( B ) mRNA expression levels were measured by real - time PCR in blood samples with 2 and 3 - 4 copies . Relative expression levels were normalized to GAPDH levels , and data were quantified relative to the mRNA expression levels of 2 copy samples . Data are presented as the mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Unpaired Student ' s t

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A, B MARS (A) and MARS2 (B) mRNA expression levels were measured by real-time PCR in blood samples with 2 and 3-4 copies. Relative expression levels were normalized to GAPDH levels, and data were quantified relative to the mRNA expression levels of 2 copy samples. Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Unpaired Student's t test


C , D Immunoblotting analysis was performed to detect MARS protein levels in samples with 2 and 3 - 4 copies . Protein - level intensity shown in ( D ) was quantified using ImageJ and divided by the protein - level intensity of actin . The results were then graphed relative to samples with 2 copies . E , F MARS2 protein expression levels were detected ( E ) and quantified ( F ) . Actin was used as a loading control . Data are presented as the mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Unpaired Student ' s t test was

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C, D Immunoblotting analysis was performed to detect MARS protein levels in samples with 2 and 3-4 copies. Protein-level intensity shown in (D) was quantified using ImageJ and divided by the protein-level intensity of actin. The results were then graphed relative to samples with 2 copies. E, F MARS2 protein expression levels were detected (E) and quantified (F). Actin was used as a loading control. Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Unpaired Student's t test was used


G Cell proliferation was assessed by Ki67 ( red ) immunofluorescent staining in mice neural tube sections at E10 . MARS ( green ) was co - stained . Scale bar : 150 μm .

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G Cell proliferation was assessed by Ki67 (red) immunofluorescent staining in mice neural tube sections at E10. MARS (green) was co-stained. Scale bar: 150 μm.


H Double immunofluorescent staining for MARS ( green ) and Ki67 ( red ) in developing heart sections at E10 . Scale bar : 150 μm .

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H Double immunofluorescent staining for MARS (green) and Ki67 (red) in developing heart sections at E10. Scale bar: 150 μm.


I MARS expression was detected in E80 rhesus monkey brain sections ( n = 3 ) by IHC . Local large magnification is shown as framed . Red arrow indicates germinar layer , blue indicates immature neurons . Scale bar : 1mm .

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I MARS expression was detected in E80 rhesus monkey brain sections (n=3) by IHC. Local large magnification is shown as framed. Red arrow indicates germinar layer, blue indicates immature neurons. Scale bar: 1mm.


MARS protein levels in embryonic chorion ( C ) cells and their corresponding maternal deciduae ( D ) cells were determined by western blot . Each D / C lane pair was quantified relative to D as shown at the bottom .

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MARS protein levels in embryonic chorion (C) cells and their corresponding maternal deciduae (D) cells were determined by western blot. Each D/C lane pair was quantified relative to D as shown at the bottom.


MARS protein levels in embryonic chorion ( C ) cells and their corresponding maternal deciduae ( D ) cells were determined by western blot . The mean MARS levels in all D and C groups were quantified by ImageJ and shown in ( K ) ( n = 6 ) . Data are presented as the mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Wilcoxon matched pairs test was

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

MARS protein levels in embryonic chorion (C) cells and their corresponding maternal deciduae (D) cells were determined by western blot. The mean MARS levels in all D and C groups were quantified by ImageJ and shown in (K) (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was used


L MARS2 mRNA expression in embryonic chorion ( C ) and maternal deciduae ( D ) were detected by qPCR ( n = 6 ) . Data are presented as the mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Wilcoxon matched pairs test was

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L MARS2 mRNA expression in embryonic chorion (C) and maternal deciduae (D) were detected by qPCR (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was used


M MARS protein quantification in the brains of foetal rats ( F ) and their corresponding mothers ( M ) from experiment ( O ) ( n = 6 ) . Data are presented as the mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Wilcoxon matched pairs test was

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M MARS protein quantification in the brains of foetal rats (F) and their corresponding mothers (M) from experiment (O) (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was used


N MARS mRNA levels ( n = 6 ) in the brains of foetal rats ( F ) and their corresponding mothers ( M ) were determined by qPCR ( n = 6 ) . Data are presented as the mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Wilcoxon matched pairs test was

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N MARS mRNA levels (n=6) in the brains of foetal rats (F) and their corresponding mothers (M) were determined by qPCR (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was used


O MARS protein levels in foetal rats ( F ) and their corresponding mothers ( M ) were determined by western blot . Each M / F lane pair was quantified relative to M as shown at the bottom .

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O MARS protein levels in foetal rats (F) and their corresponding mothers (M) were determined by western blot. Each M/F lane pair was quantified relative to M as shown at the bottom.


P Representative result of MARS expression was detected by IHC in tumor tissues ( T ) and adjacent noncancer tissues ( N ) of UCC samples . Scale bar : 50 μm .

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P Representative result of MARS expression was detected by IHC in tumor tissues (T) and adjacent noncancer tissues (N) of UCC samples. Scale bar: 50 μm.


Q Ki67 and MARS expression was analysed by IHC in HCC samples . Scale bar : 20 μm .

Q Ki67 and MARS expression was analysed by IHC in HCC samples. Scale bar: 20 μm.


B Cell growth was determined in the absence and presence of 0 . 5 mM homocysteine or 0 . 5 mM methionine in the culture media of NE4C cells transfected with pcDNA3 . 1 vector or pcDNA3 . 1 - Flag - Bcl - 2 plasmids ( n = 3 ) . 5μg plasmids were transfected in each 1 × 106 cells . The cell index responds to changes in cell number and cell adhesion . Cleaved caspase 3 ( c - caspase3 ) levels were detected to determine apoptosis levels ( right ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test .

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B Cell growth was determined in the absence and presence of 0.5 mM homocysteine or 0.5 mM methionine in the culture media of NE4C cells transfected with pcDNA3.1 vector or pcDNA3.1-Flag-Bcl-2 plasmids (n=3). 5μg plasmids were transfected in each 1×106 cells. The cell index responds to changes in cell number and cell adhesion. Cleaved caspase 3 (c-caspase3) levels were detected to determine apoptosis levels (right). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test.


C , D Apoptotic cells were detected by flow cytometry . The apoptotic rates of untreated , MARS ( C ) - , and MARS2 ( D ) - over - expressing NE4C cells when cultured in the absence and presence of 20 μM homocysteine were normalized to the untreated NE4C cells ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

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C, D Apoptotic cells were detected by flow cytometry. The apoptotic rates of untreated, MARS (C)-, and MARS2 (D)-over-expressing NE4C cells when cultured in the absence and presence of 20 μM homocysteine were normalized to the untreated NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.


E Cells were treated with different methionine concentrations as indicated . Apoptotic rates were detected by flow cytometry and normalized to those of the untreated cells ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

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E Cells were treated with different methionine concentrations as indicated. Apoptotic rates were detected by flow cytometry and normalized to those of the untreated cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.


F Apoptotic rates were determined in the absence and presence of 20 μM homocysteine in the culture media of control and MARS / MARS2 knockdown NE4C cells ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

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F Apoptotic rates were determined in the absence and presence of 20 μM homocysteine in the culture media of control and MARS/MARS2 knockdown NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.


HTL and ROS ( superoxide ) levels were compared ( n = 4 ) between NE4C cells transfected with the empty vector , MARS ( A ) plasmids . Data were normalized against that of vector - transfected cells . HTL levels were determined by LC - MS , and ROS levels were determined by DHE staining assay .

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HTL and ROS (superoxide) levels were compared (n=4) between NE4C cells transfected with the empty vector, MARS (A) plasmids. Data were normalized against that of vector-transfected cells. HTL levels were determined by LC-MS, and ROS levels were determined by DHE staining assay.


HTL and ROS ( superoxide ) levels were compared ( n = 4 ) between NE4C cells transfected with the empty vector or MARS2 ( B ) plasmids . Data were normalized against that of vector - transfected cells . HTL levels were determined by LC - MS , and ROS levels were determined by DHE staining assay .

HTL and ROS (superoxide) levels were compared (n=4) between NE4C cells transfected with the empty vector or MARS2 (B) plasmids. Data were normalized against that of vector-transfected cells. HTL levels were determined by LC-MS, and ROS levels were determined by DHE staining assay.


C MARS was knocked down by small hairpin RNAs in NE4C cells . MARS knockdown efficiencies were confirmed by western blot . HTL and superoxide levels were quantified ( n = 4 ) relative to vector - transfected NE4C cells . Cells were cultured in 20 μM Hcy - containing media .

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C MARS was knocked down by small hairpin RNAs in NE4C cells. MARS knockdown efficiencies were confirmed by western blot. HTL and superoxide levels were quantified (n=4) relative to vector-transfected NE4C cells. Cells were cultured in 20 μM Hcy-containing media.


D Relative superoxide levels were determined by DHE staining assay and compared ( n = 4 ) between NE4C cells with and without MARS knockdown by shRNA . Cells were cultured in 20 μM Hcy - containing media . MARS2 knockdown efficiencies were confirmed by western blot .

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D Relative superoxide levels were determined by DHE staining assay and compared (n=4) between NE4C cells with and without MARS knockdown by shRNA. Cells were cultured in 20 μM Hcy-containing media. MARS2 knockdown efficiencies were confirmed by western blot.


E NE4C cells with and without MARS knockdown were treated with either Hcy ( 20 μM ) or Met ( 75 μM ) , and cellular superoxide levels were detected 6 h after the start of the respective treatment and quantified relative to the untreated NE4C cells ( n = 4 ) .

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E NE4C cells with and without MARS knockdown were treated with either Hcy (20 μM) or Met (75 μM), and cellular superoxide levels were detected 6 h after the start of the respective treatment and quantified relative to the untreated NE4C cells (n=4).


F The cellular ROS ( superoxide ) levels were determined ( n = 4 ) by DHE staining of NE4C cells treated with various HTL concentrations for 4 h . The ROS levels were normalized to those of untreated NE4C cells .

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F The cellular ROS (superoxide) levels were determined (n=4) by DHE staining of NE4C cells treated with various HTL concentrations for 4 h. The ROS levels were normalized to those of untreated NE4C cells.


G Superoxide levels in response to Hcy treatment ( 20 μM , 4 h ) in MARS - expressing and control cells were determined relative to the untreated NE4C cells .

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G Superoxide levels in response to Hcy treatment (20 μM, 4 h) in MARS-expressing and control cells were determined relative to the untreated NE4C cells.


H NE4C cells were cultured in DMEM supplemented with different HTL levels as indicated . N - Hcy levels in HTL - treated and control cells were detected by western blot .

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H NE4C cells were cultured in DMEM supplemented with different HTL levels as indicated. N-Hcy levels in HTL-treated and control cells were detected by western blot.


I Protein N - Hcy levels in response to MARS knockdown ( by shRNA ) and over - expression were detected by western blot .

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I Protein N-Hcy levels in response to MARS knockdown (by shRNA) and over-expression were detected by western blot.