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

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

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


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

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

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

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 littermate controls at P18 . Inserts depict high magnification of boxed areas . Scale bars , 50 μm .

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

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

O O O O O O O O O O O O O O O O O O O O O O O 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 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 of Mex3a KO WT intestinal organoids after 5 days of treatment with 50 μM Pioglitazone or vehicle ( DMSO ) . Scale bars , 100 μm .

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

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

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 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

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 of Mex3a KO WT intestinal organoids at day 1 after passaging Pioglitazone - or vehicle ( DMSO ) - treated organoids . 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

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.


C . Coomassie of SDS - PAGE - separated pulldown samples with / without extracts ( C . E ) using GST - tagged FET - NTDs as baits . Sepharose with bound GST was included as control . Background from recombinant baits ( shown by arrowheads ) as well as smaller partial recombinant products is visualized in left panel ( C . E - ) . Several high - molecular are retained by FET - NTDs ( C . E + ) . Black arrowheads indicate bands gel parts analyzed by mass

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 staining of SDS-PAGE-separated pulldown samples with/without cell extracts (C.E) using GST-tagged FET-NTDs as baits. Sepharose with bound GST was included as control. Background from recombinant protein baits (shown by white arrowheads) as well as smaller partial recombinant products is visualized in the left panel (C.E -). Several high-molecular weight proteins are retained by the FET-NTDs (C.E +). Black arrowheads indicate protein bands and gel parts analyzed by mass spectrometry


D . Immunoblot ( IB ) of pulldown samples with FET - NTD baits with / without extracts ( C . E ) . Antibodies against ARID1A , BRG1 BAF60A were used for detection of SWI / SNF components . Input samples diluted 1 : 5 were included as a control .

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

D. Immunoblot analysis (IB) of pulldown samples with FET-NTD baits with/without cell extracts (C.E). Antibodies against ARID1A, BRG1 and BAF60A were used for detection of SWI/SNF components. Input samples diluted 1:5 were included as a control.


E . In situ proximity ligation assays ( PLA ) using antibodies against BRG1 , ARID1A , DDIT3 C - terminal parts of normal FUS show complexes containing FUS / BRG1 , FUS / ARID1A , FUS - DDIT3 / BRG1 FUS - DDIT3 / ARID1A as red spots in nuclei of MLS lines 2645 - 94 402 - 91 . C - terminal parts of FUS are not present in FUS - DDIT3 fusion normal DDIT3 is not expressed in these lines . Merged also include DAPI counterstain in blue . Combinations of antibodies are used to detect interactions ( left panel ) . In control experiments ( right panel ) , one antibody is omitted to evaluate background signals . Scale bars = 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. In situ proximity ligation assays (PLA) using antibodies against BRG1, ARID1A, DDIT3 and C-terminal parts of normal FUS show protein complexes containing FUS/BRG1, FUS/ARID1A, FUS-DDIT3/BRG1 and FUS-DDIT3/ARID1A as red fluorescent spots in nuclei of MLS cell lines 2645-94 and 402-91. C-terminal parts of FUS are not present in the FUS-DDIT3 fusion protein and normal DDIT3 is not expressed in these cell lines. Merged images also include DAPI nuclear counterstain in blue. Combinations of primary antibodies are used to detect interactions (left panel). In control experiments (right panel), one primary antibody is omitted to evaluate the background fluorescent signals. Scale bars = 10 μm.


B . Immunoblot ( IB ) of GST - pulldown samples with deletion mutants of FUS ‑ NTD ‑ GST as baits with / without extract ( C . E ) . ARID1A BRG1 were used as tracers of SWI / SNF binding . Coomassie of recombinant are shown in lower panel . acid numbers are indicated

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblot analysis (IB) of GST-pulldown samples with deletion mutants of FUS‑NTD‑GST as baits with/without cell extract (C.E). ARID1A and BRG1 were used as tracers of SWI/SNF binding. Coomassie staining of recombinant proteins are shown in the lower panel. Amino acid numbers are indicated


C . Immunoblot ( IB ) of FUS - NTD ‑ GST pulldown samples of extracts ( C . E . ) after treatment with RNase or DNase , using antibodies against ARID1A BRG1

O O O O O O O O O O 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. Immunoblot analysis (IB) of FUS-NTD‑GST pulldown samples of cell extracts (C.E.) after treatment with RNase or DNase, using antibodies against ARID1A and BRG1


D . Immunoblot ( IB ) of SWI / SNF components ( ARID1A , BRG1 , BAF170 , BAF155 , BAF60A , SS18 BAF47 ) full - FET ( EWSR1 FUS ) remaining on sepharose - bound FUS - NTD ‑ GST pulldown baits after increasing stringency washes with 100 , 150 , 250 , 500 or 1000 mM NaCl . Note loss of BRG1 at NaCl > 250mM

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

D. Immunoblot analysis (IB) of SWI/SNF components (ARID1A, BRG1, BAF170, BAF155, BAF60A, SS18 and BAF47) and full-length FET proteins (EWSR1 and FUS) remaining on sepharose-bound FUS-NTD‑GST pulldown baits after increasing stringency washes with 100, 150, 250, 500 or 1000 mM NaCl. Note loss of BRG1 at NaCl concentration > 250mM


A . Immunoblot ( IB ) of co - immunoprecipitated with extracted SWI / SNF from MLS 402 ‑ 91 EWS TC - 71 . Antibodies against BRG1 , DDIT3 ( FUS - DDIT3 ) , C - terminal parts of normal FUS EWSR1 , FLI1 ( EWSR1 - FLI1 ) were used for detection . In order to directly quantify fraction of bound non - bound , relative amounts of for each IP ‑ sample were loaded on gel , with consideration taken for dilutions during immunoprecipitation procedure . I : input of extract , B : bound , NB : not bound . One representative immunoblot is shown . Immunoblots from replicates are shown in source data . B . Graphs showing of bound to total ( bound + non ‑ bound ) signal intensities from immunoblots for FUS ‑ DDIT3 , EWSR1 FUS in MLS 402 - 91 EWSR1 - FLI1 , EWSR1 FUS in EWS TC - 71 . Mean ± is shown with replicates indicated by circles ( n = 3 ) . Student ' s t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Original data for quantifications , including p - values , are shown as source 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblot analysis (IB) of proteins co-immunoprecipitated with nuclear extracted SWI/SNF from MLS cell line 402‑91 and EWS cell line TC-71. Antibodies against BRG1, DDIT3 (FUS-DDIT3), the C-terminal parts of normal FUS and EWSR1, and FLI1 (EWSR1-FLI1) were used for detection. In order to directly quantify the fraction of bound and non-bound protein, relative amounts of protein for each IP‑sample were loaded on the gel, with consideration taken for dilutions during the immunoprecipitation procedure. I: input of nuclear extract, B: bound proteins, NB: proteins not bound. One representative immunoblot is shown. Immunoblots from all replicates are shown in the source data. B. Graphs showing the percentage of bound to total (bound + non‑bound) signal intensities from immunoblots for FUS‑DDIT3, EWSR1 and FUS in MLS 402-91 cell line and EWSR1-FLI1, EWSR1 and FUS in EWS TC-71 cell line. Mean ± SEM is shown with individual replicates indicated by circles (n=3). Student's t-test, *p<0.05, **p<0.01, ***p<0.001. Original data for all quantifications, including p-values, are shown as source data.


C . Immunoblot of co - immunoprecipitated with extracted SWI / SNF from model HT1080 with overexpressed FUS - DDIT3 - dsRED , EWSR1 - FLI1 - EGFP or dsRED are shown as representative immunoblots . Antibodies against BRG1 , DDIT3 ( FUS - DDIT3 - dsRED ) , C ‑ terminal parts of normal FUS EWSR1 , EZH2 FLI1 ( EWSR1 - FLI1 - EGFP ) were used for detection . In order to directly quantify fraction of bound non - bound , relative amounts of for each IP ‑ sample were loaded on gel , with consideration taken for dilutions during immunoprecipitation procedure . I : input of extract , B : bound , NB : not bound . Immunoblots from replicates are shown in source data ( with either dsRED - or EGFP ‑ tagged expressed ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblot analysis of proteins co-immunoprecipitated with nuclear extracted SWI/SNF from the model cell line HT1080 with overexpressed FUS-DDIT3-dsRED, EWSR1-FLI1-EGFP or dsRED are shown as representative immunoblots. Antibodies against BRG1, DDIT3 (FUS-DDIT3-dsRED), the C‑terminal parts of normal FUS and EWSR1, EZH2 and FLI1 (EWSR1-FLI1-EGFP) were used for detection. In order to directly quantify the fraction of bound and non-bound protein, relative amounts of protein for each IP‑sample were loaded on the gel, with consideration taken for dilutions during the immunoprecipitation procedure. I: input of nuclear extract, B: bound proteins, NB: proteins not bound. Immunoblots from all replicates are shown in the source data (with either dsRED- or EGFP‑tagged proteins expressed).


D . Graphs showing of bound to total ( bound + non ‑ bound ) signal intensities from immunoblots for fusion ( FUS ‑ DDIT3 or EWSR1 - FLI1 ) , EWSR1 , FUS EZH2 in model HT1080 with overexpressed FUS - DDIT3 - dsRED / - EGFP ( n = 4 , circles ) , EWSR1 - FLI1 - EGFP ( n = 3 , squares ) or dsRED / EGFP ( n = 4 , triangles ) . Mean ± is shown with replicates indicated . Student ' s t - , no significant changes , p > 0 . 05 . Original data for quantifications , including p - values , are shown as source 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

D. Graphs showing the percentage of bound to total (bound + non‑bound) signal intensities from immunoblots for the fusion protein (FUS‑DDIT3 or EWSR1-FLI1), EWSR1, FUS and EZH2 in the model cell line HT1080 with overexpressed FUS-DDIT3-dsRED/-EGFP (n=4, circles), EWSR1-FLI1-EGFP (n=3, squares) or dsRED/EGFP (n=4, triangles). Mean ± SEM is shown with individual replicates indicated. Student's t-test, no significant changes, p>0.05. Original data for all quantifications, including p-values, are shown as source data.


A . Immunoblot ( IB ) of histone modifications in stably transfected HT1080 lines expressing EGFP , FUS ‑ DDIT3 ‑ EGFP or EWSR1 ‑ FLI1 ‑ EGFP . Antibodies against H3K27Ac , H3K27me3 , H3K4me3 histone loading control H4 for detection of histone modifications antibodies against EZH2 loading control GAPDH to evaluate catalytic PRC2 amount . Immunoblot with GFP antibody is shown to verify expression of FET fusion oncoproteins EGFP . One representative immunoblot is shown . Graphs showing amount of ( H3K27me3 , H3K4me3 , H3K27me3 / H3K4me3 , H3K27Ac , EZH2 ) quantified from immunoblots relative each corresponding loading control H4 or GAPDH , normalized to parental HT1080 . Mean ± is shown with replicates indicated by circles from two experiments with stable transfection by squares from two experiments with transient transfection ( 24h 48h , n = 4 . Student ' s t - , ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Original data for quantifications , including p - values , are shown as source 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 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 B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblot analysis (IB) of histone modifications in stably transfected HT1080 cell lines expressing EGFP, FUS‑DDIT3‑EGFP or EWSR1‑FLI1‑EGFP. Antibodies against H3K27Ac, H3K27me3, H3K4me3 and histone loading control H4 for detection of histone modifications and antibodies against EZH2 and loading control GAPDH to evaluate catalytic PRC2 amount. Immunoblot analysis with GFP antibody is shown to verify expression of FET fusion oncoproteins and EGFP. One representative immunoblot is shown. Graphs showing amount of protein (H3K27me3, H3K4me3, ratio H3K27me3/H3K4me3, H3K27Ac, and EZH2) quantified from immunoblots relative each corresponding loading control H4 or GAPDH, normalized to parental HT1080. Mean ± SEM is shown with individual replicates indicated by circles from two experiments with stable transfection and by squares from two experiments with transient transfection (24h and 48h , n=4. Student's t-test, ns= not significant, *p<0.05, **p<0.01, ***p<0.001. Original data for all quantifications, including p-values, are shown as source data.


( A , B ) t - SNE visualization of 20 , 009 human retinal colored by ( A ) annotation of 18 transcriptionally distinct clusters ( C0 - C17 ) ( B ) their distribution in 3 donor retina samples .

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

(A, B) t-SNE visualization of 20,009 human retinal cells colored by (A) annotation of 18 transcriptionally distinct clusters (C0-C17) and (B) their distribution in 3 donor retina samples.


C ) Feature expression showing expression patterns of major retinal class markers across 16 retinal clusters . of each circle depicts of expressing marker within cluster . Brown colour indicates ≥ 10 nTrans ( number of transcripts ) .

O O O O O O O O O O O O O O O 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) Feature expression heatmap showing expression patterns of major retinal class markers across 16 retinal cell clusters. The size of each circle depicts the percentage of cells expressing the marker within the cluster. Brown colour indicates ≥10 nTrans (number of transcripts).


D ) t - SNE plots showing expression of a set of selected marker genes for major retinal classes .

D) t-SNE plots showing expression of a set of selected marker genes for major retinal classes.


E ) Correlation matrix for identified 18 clusters . upper triangle depicts z - value for correlation lower triangle depicts correlation coefficient for gene expression in clusters .

O O O O 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) Correlation matrix for the identified 18 clusters. The upper triangle depicts the z-value for correlation and the lower triangle depicts the correlation coefficient for gene expression in clusters.


F ) of differentially expressed genes used to classify types for each cluster compared to other clusters for 18 retinal clusters . rows correspond to top 10 genes most selectively upregulated in clusters ( p < 0 . 01 , Benjamini - Hochberg correction ) columns show ordered by cluster ( C0 - C17 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heatmap of differentially expressed genes used to classify cell types for each cluster compared to all other clusters for the 18 retinal cell clusters. The rows correspond to top 10 genes most selectively upregulated in individual clusters (p< 0.01, Benjamini-Hochberg correction) and the columns show individual cells ordered by cluster (C0-C17).


A ) Feature expression of a panel of known marker genes for rod photoreceptors across identified 16 retinal clusters . Brown colour indicates ≥ 100 nTrans ( number of transcripts ) .

A) Feature expression heatmap of a panel of known marker genes for rod photoreceptors across the identified 16 retinal cell clusters. Brown colour indicates ≥100 nTrans (number of transcripts).


B ) Representation of three donor retina samples in six rod photoreceptor clusters .

B) Representation of the three donor retina samples in the six rod photoreceptor clusters.


C ) Violin plot showing high or low expression of MALAT1 in rod photoreceptor clusters .

C) Violin plot showing high or low expression levels of MALAT1 in rod photoreceptor clusters.


D ) Distribution of rod photoreceptor populations with high MALAT1 expression ( MALAT1 - hi ) or low MALAT1 expression ( MALAT1 - lo ) in three donor retina samples .

O O O O O O O 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) Distribution of rod photoreceptor populations with high MALAT1 expression (MALAT1-hi) or low MALAT1 expression (MALAT1-lo) in three donor retina samples.


E ) in situ hybridization of human peripheral retina showing heterogeneous of MALAT1 expression in rod photoreceptors located in outer layer ( ONL ) . INL : inner layer ; OPL : outer plexiform layer . Scale bar = 20µm .

O O O O O O O O O O O O O O O O O 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) Fluorescent in situ hybridization analysis of human peripheral retina showing heterogeneous levels of MALAT1 expression in the rod photoreceptors located in the outer nuclear layer (ONL). INL: inner nuclear layer; OPL: outer plexiform layer. Scale bar = 20µm.


A , B ) ( A ) t - SNE visualization of human retinal colored by annotation of 13 transcriptionally distinct clusters ( CCA0 - CCA12 ) ( B ) their distribution in 3 donor retina samples .

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

A, B) (A) t-SNE visualization of human retinal cells colored by annotation of 13 transcriptionally distinct clusters (CCA0-CCA12) and (B) their distribution in 3 donor retina samples.


C ) Feature expression showing expression patterns of 7 rod photoreceptor markers across 12 retinal clusters . of each circle depicts of expressing marker within cluster . Brown colour indicates ≥ 50 nTrans ( number of transcripts ) .

C) Feature expression heatmap showing expression patterns of 7 rod photoreceptor markers across 12 retinal cell clusters. The size of each circle depicts the percentage of cells expressing the marker within the cluster. Brown colour indicates ≥50 nTrans (number of transcripts).


D ) t - SNE plots showing expression of MALAT1 .

D) t-SNE plots showing expression of MALAT1.


E ) Expression pattern of MALAT1 in rod photoreceptor showing MALAT1 - hi ( CCA1 , CCA10 ) MALAT1 - lo ( CCA0 ) subpopulations . x - axis depicts normalized transcript .

O O O O O O O O O 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) Expression pattern of MALAT1 in the rod photoreceptor showing MALAT1-hi (CCA1, CCA10) and MALAT1-lo (CCA0) subpopulations. x-axis depicts normalized transcript levels.


F ) t - SNE plots showing expression of major ribosomal genes .

F) t-SNE plots showing expression of major ribosomal genes.


G ) of differentially expressed genes between two MALAT1 - hi clusters CCA1 CCA10 . rows correspond to top 10 genes most selectively upregulated in clusters ( p < 0 . 01 , Benjamini - Hochberg correction ) columns show ordered in CCA1 CCA10 .

O O O O O O O O O O O O O O O O O O O O O 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) Heatmap of differentially expressed genes between the two MALAT1-hi clusters CCA1 and CCA10. The rows correspond to top 10 genes most selectively upregulated in individual clusters (p< 0.01, Benjamini-Hochberg correction) and the columns show individual cells ordered in CCA1 and CCA10.


in situ hybridization of same donor human peripheral retina at different time points post - mortem ( 7 13 hours , Retina 7 ) , showing decreases in MALAT1 - hi rod subpopulations in outer layer ( ONL ) at later time point . INL : inner layer ; OPL : outer plexiform layer . Scale bar = 20µm . arrows indicated MALAT1 - hi rod photoreceptors .

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

Fluorescent in situ hybridization analysis of the same donor human peripheral retina at different time points post-mortem (7 and 13 hours, Retina 7), showing decreases in MALAT1-hi rod subpopulations in the outer nuclear layer (ONL) at later time point. INL: inner nuclear layer; OPL: outer plexiform layer. Scale bar = 20µm. White arrows indicated MALAT1-hi rod photoreceptors.


A ) Feature expression showing expression of 11 known cone photoreceptor markers across 16 retinal clusters . Brown colour indicates ≥ 10 nTrans ( number of transcripts ) .

O O O O O 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) Feature expression heatmap showing the expression of 11 known cone photoreceptor markers across 16 retinal cell clusters. Brown colour indicates ≥10 nTrans (number of transcripts).


C ) Scatter plots showing expression of OPN1LW / OPN1MW or OPN1SW in cone photoreceptors for population C10 . colour depicts expression level for OPN1LW / OPN1MW in .

C) Scatter plots showing expression of OPN1LW/OPN1MW or OPN1SW in individual cone photoreceptors for population C10. The colour depicts expression level for OPN1LW/OPN1MW in individual cells.


D ) of top 20 differentially expressed genes between L / M cones S cones . colour depicts normalised gene expression ( z - score capped at 2 . 5 ) .

O O O O O O O O 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) Heatmap of top 20 differentially expressed genes between L/M cones and S cones. The colour depicts normalised gene expression (z-score capped at 2.5).


E ) Expression pattern of glial markers in Muller glia ( C9 ) retinal astrocytes ( C16 ) . x - axis depicts normalized transcript .

O O 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) Expression pattern of glial markers in Muller glia (C9) and retinal astrocytes (C16). x-axis depicts normalized transcript levels.


C ) t - SNE of human Müller glia MIO - M1 with retinal types identified in this human neural retina atlas .

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

C) t-SNE analysis of the human Müller glia cell line MIO-M1 with the retinal cell types identified in this human neural retina atlas.


B . Southern blotting confirms successful targeting . Introduction of an additional EcoRI site results in a 4 . 1 kb restriction fragment detectable by Southern for targeted ES ( A174T / + ) but not for parental ( + / + ) .

O O O O O O O O O O O O O O O O O O O O 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. Southern blotting confirms successful targeting. Introduction of an additional EcoRI site results in a 4.1 kb restriction fragment detectable by Southern for targeted ES cells (A174T/+) but not for parental DNA (+/+).


C . Capillary sequencing for Rnaseh2b + / + , Rnaseh2bA174T / + , Rnaseh2bA174T / A174T confirmed presence of introduced missense mutation .

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. Capillary sequencing for Rnaseh2b+/+, Rnaseh2bA174T/+, and Rnaseh2bA174T/A174T DNA confirmed the presence of the introduced missense mutation.


D . Mouse genotyping by multiplex PCR . Top : A 221 bp PCR product is present in wild - type mice ( + / + ) ; Rnaseh2bA174T allele ( also ) give a 460 bp product . Bottom : Position of forward ( x ) reverse primers ( y , z ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Mouse genotyping by multiplex PCR. Top: A 221 bp PCR product is present in wild-type mice (+/+); the Rnaseh2bA174T allele (also) give a 460 bp product. Bottom: Position of forward (x) and reverse primers (y, z).


E . Immunoblotting demonstrates depletion of three RNase H2 subunits in Rnaseh2bA174T / A174T MEFs RNASEH2BA177T / A177T LCLs . Representative of three independent experiments .

E. Immunoblotting demonstrates depletion of all three RNase H2 protein subunits in Rnaseh2bA174T/A174T MEFs and RNASEH2BA177T/A177T LCLs. Representative of three independent experiments.


G , H . RNase H2 enzyme activity is reduced in mouse . ( G ) Enzyme activity for Rnaseh2bA174T / A174T MEFs passage - matched Rnaseh2b + / + controls , against RNase H substrate ( : heteroduplex ) RNase H2 - specific substrate , double stranded with a single embedded ribonucleotide ( DRD : ) . Mean activity for three independent lines , error bars , . Enzymatic activity expressed relative to average value of control MEFs . * * * = p < 0 . 001 , two - t - ( n = 3 Rnaseh2bA174T / A174T n = 3 Rnaseh2b + / + control MEF lines ) . ( H ) RNase H2 activity in LCLs from two independent healthy controls an AGS homozygous for RNASEH2B - A177T mutation . Enzyme activity normalised to average activity of control lines . Three independent experiments , error bars . * * * = p < 0 . 001 versus either control , two - t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, H. RNase H2 enzyme activity is reduced in mouse and patient cells. (G) Enzyme activity for Rnaseh2bA174T/A174T MEFs and passage-matched Rnaseh2b+/+ controls, against RNase H substrate (RNA:DNA heteroduplex) and RNase H2-specific substrate, double stranded DNA with a single embedded ribonucleotide (DRD:DNA). Mean activity for three independent cell lines, error bars, SEM. Enzymatic activity expressed relative to the average value of control MEFs. *** = p<0.001, two-tailed t-test (n=3 Rnaseh2bA174T/A174T and n=3 Rnaseh2b+/+ control MEF lines). (H) RNase H2 activity in LCLs from two independent healthy controls and an AGS patient homozygous for the RNASEH2B-A177T mutation. Enzyme activity normalised to average activity of control lines. Three independent experiments, error bars SEM. *** = p<0.001 versus either control, two-tailed t-test.


Figure 2 . Increased ISG expression in tissues from Rnaseh2bA174T / A174T miceA . Transcript of multiple ISGs are significantly elevated in heart . B . Transcript of a subset of ISGs are significantly increased in kidney . C . No ISG upregulation is evident in brain . ISG transcript determined by RT - qPCR normalised to transcript of housekeeping gene HPRT ( Oas1a was undetectable in brain ) . Each data point represents mean of technical replicates of tissue from a single mouse . n = 9 nine - month old Rnaseh2bA174T / A174T mice , n = 4 - matched control wildtype C57BL / 6Jmice . Horizontal , mean ; error bars , . * = p < 0 . 05 , * * = p < 0 . 01 , two - t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Increased ISG expression in tissues from Rnaseh2bA174T/A174T miceA. Transcript levels of multiple ISGs are significantly elevated in heart.B. Transcript levels of a subset of ISGs are significantly increased in kidney.C. No ISG upregulation is evident in the brain. ISG transcript levels determined by RT-qPCR normalised to transcript levels of the housekeeping gene HPRT (Oas1a was undetectable in brain). Each data point represents the mean of technical replicates of tissue RNA from a single mouse. n=9 nine-month old Rnaseh2bA174T/A174T mice, n=4 age-matched control wildtype C57BL/6Jmice. Horizontal line, mean; error bars, SEM. * = p<0.05, ** = p<0.01, two-tailed t-test.


A . Validation of Rnaseh2b - / - MEF lines . RER activity ( DRD : ) is undetectable in a Rnaseh2b - / - consistent with complete inactivation of Rnaseh2b gene ( Reijns et al , 2012 ) . Mean of three independent experiments ; error bars , , * * * * = p < 0 . 0001 , two - t - . Rnaseh2b - / - Rnaseh2b + / + control MEFs on a C57BL / 6 p53 - / - background .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Validation of Rnaseh2b-/- MEF lines. RER activity (DRD:DNA) is undetectable in a Rnaseh2b-/- line consistent with complete inactivation of the Rnaseh2b gene (Reijns et al, 2012). Mean of three independent experiments; error bars, SEM, **** = p<0.0001, two-tailed t-test. Rnaseh2b-/- and Rnaseh2b+/+ control MEFs on a C57BL/6 p53-/- background.


B . ISG transcript are increased in Rnaseh2b - / - MEFs .

B. ISG transcript levels are increased in Rnaseh2b-/- MEFs.


C . BeadArray transcript ( Illumina ) detected induction of multiple ISGs , including cytokines CXCL10 CCL5 , but not other cytokine transcripts in Rnaseh2b - / - MEFs . Plotted , average fold enrichment versus p - value of significantly upregulated transcripts ( p < 0 . 05 , after multiple testing correction ) comparing two Rnaseh2b - / - MEF lines versus 4 Rnaseh2b + / + MEF lines . 17 out of 29 transcripts are ISGs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. BeadArray transcript analysis (Illumina) detected induction of multiple ISGs, including the cytokines CXCL10 and CCL5, but not other cytokine transcripts in Rnaseh2b-/- MEFs. Plotted, average fold enrichment versus p-value of significantly upregulated transcripts (p<0.05, after multiple testing correction) comparing two Rnaseh2b-/- MEF lines versus 4 Rnaseh2b+/+ MEF lines. 17 out of 29 transcripts are ISGs.


D . CXCL10 CCL5 transcripts ( detected by RT - qPCR ) are significantly elevated .

D. CXCL10 and CCL5 transcripts (detected by RT-qPCR) are significantly elevated.


E . Increased CXCL10 CCL5 ( detected by ELISA ) is secreted by Rnaseh2b - / - MEFs . ( B , D , E ) Mean data from three experiments for six independent Rnaseh2b - / - MEF lines versus four independent Rnaseh2b + / + MEF lines . Error bars , . * = p < 0 . 05 , * * = p < 0 . 01 , Mann - Whitney U .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Increased CXCL10 and CCL5 protein (detected by ELISA) is secreted by Rnaseh2b-/- MEFs. (B, D, E) Mean data from three experiments for six independent Rnaseh2b-/- MEF lines versus four independent Rnaseh2b+/+ MEF lines. Error bars, SEM. * = p<0.05, ** = p<0.01, Mann-Whitney U test.


A . ISG activation cytokine secretion in Rnaseh2b - / - MEFs is markedly impaired by cGAS or STING siRNA depletion . Upper left lower panels : RT - qPCR of IFIT1 , cGAS STING transcripts , 48 h after siRNA targeting luciferase ( control ) , cGAS or STING . Upper right panel : CCL5 is significantly reduced in culture supernatants 48 h after cGAS or STING depletion . of CCL5 ( ELISA ) , normalised to luciferase siRNA control in each experiment . Mean from three independent experiments using one Rnaseh2b - / - MEF ; error bars , . * = p < 0 . 05 , * * = p < 0 . 01 , two - t - for RT - qPCR , one sample t - for CCL5ELISA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. ISG activation and cytokine secretion in Rnaseh2b-/- MEFs is markedly impaired by cGAS or STING siRNA depletion. Upper left and lower panels: RT-qPCR of IFIT1, cGAS and STING transcripts, 48 h after siRNA targeting luciferase (control), cGAS or STING. Upper right panel: CCL5 is significantly reduced in culture supernatants 48 h after cGAS or STING depletion. Concentration of CCL5 (ELISA), normalised to luciferase siRNA control levels in each experiment. Mean from three independent experiments using one Rnaseh2b-/- MEF line; error bars, SEM. * = p<0.05, ** = p<0.01, two-tailed t-test for RT-qPCR, one sample t-test for CCL5ELISA.


B - D . ISG induction cytokine secretion is abolished in Rnaseh2b - / - cGAS - / - MEFs . cGAS was targeted by CRISPR / Cas9 genome editing of a Rnaseh2b - / - MEF to inactivate cGAS / STING signalling . In addition to validation , functional inactivation of cGAS was confirmed in Rnaseh2b - / - cGas - / - CRISPR lines by absence of CCL5 secretion in response to dsDNA ( Appendix Fig S4 ) . CCL5 ( B ) CXCL10 ( C ) production , as well as ISG expression ( D ) was abrogated in Rnaseh2b - / - cGAS - / - clones , assessed by ELISA RT - qPCR respectively . Four independent experiments , n = 2 Rnaseh2b - / - cGAS - / - clones , n = 4 Rnaseh2b - / - cGAS + / + clones , error bars , of each experiment * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , two - t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. ISG induction and cytokine secretion is abolished in Rnaseh2b-/- cGAS-/- MEFs. cGAS was targeted by CRISPR/Cas9 genome editing of a Rnaseh2b-/- MEF line to inactivate cGAS/STING signalling. In addition to sequence validation, functional inactivation of cGAS was confirmed in Rnaseh2b-/- cGas-/- CRISPR lines by the absence of CCL5 secretion in response to dsDNA (Appendix Fig S4). CCL5 (B) and CXCL10 (C) production, as well as ISG expression (D) was abrogated in Rnaseh2b-/- cGAS-/- clones, assessed by ELISA and RT-qPCR respectively. Four independent experiments, n=2 Rnaseh2b-/- cGAS-/- clones, n=4 Rnaseh2b-/- cGAS+/+ clones, error bars, SEM of each experiment * = p<0.05, ** = p<0.01, *** = p<0.001, two-tailed t-test.


E . ISG induction in Rnaseh2bA174T / A174T mice is STING dependent . RT - qPCR of extracted from hearts from Sting + / + Rnaseh2bA174T / A174T ( n = 4 ) Sting - / - Rnaseh2bA174T / A174T ( n = 6 ) three - month old mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. ISG induction in Rnaseh2bA174T/A174T mice is STING dependent. RT-qPCR of RNA extracted from hearts from Sting+/+ Rnaseh2bA174T/A174T (n=4) and Sting-/-Rnaseh2bA174T/A174T (n=6) three-month old mice.


F . Absence of STING does not significantly decrease basal ISG expression . RT - qPCR of extracted from hearts from Sting + / + ( n = 3 ) Sting - / - ( n = 5 ) three - month old mice . ( E , F ) Each data point represents mean of technical triplicates from one mouse . Error bars , . * = p < 0 . 05 , Mann - Whitney U .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Absence of STING does not significantly decrease basal ISG expression. RT-qPCR of RNA extracted from hearts from Sting+/+ (n=3) and Sting-/- (n=5) three-month old mice. (E, F) Each data point represents the mean of technical triplicates from one mouse. Error bars, SEM. * = p<0.05, Mann-Whitney U test.


A . Overexpression of RNase H1 in Rnaseh2b - / - restores RNase H activity against : hybrids to 81 ± 10 % of wildtype , while overexpression of RNase H2B restores enzyme activity for cleavage of both : DRD : substrates ( RER ) . Rnaseh2b - / - MEFs were complemented with Rnaseh1 ( + H1 ) , Rnaseh2b ( + H2B ) or EGFP by retroviral infection . Mean of n = 3 independent experiments ± .

A. Overexpression of RNase H1 in Rnaseh2b-/- cells restores RNase H activity against RNA:DNA hybrids to 81 ± 10% of wildtype levels, while overexpression of RNase H2B restores cellular enzyme activity for cleavage of both RNA:DNA and DRD:DNA substrates (RER). Rnaseh2b-/- MEFs were complemented with Rnaseh1 (+H1), Rnaseh2b (+H2B) or EGFP by retroviral infection. Mean of n=3 independent experiments ± SEM.


B , C . damage is reduced to wildtype by complementation with Rnaseh2b but not Rnaseh1 , measured by 53BP1 foci formation in detergent - extracted fixed . ( B ) Representative ( scale bar , 10 µm ) . ( C ) At least 150 were counted for each in three independent experiments . Mean ± , * * * * = p < 0 . 0001 two - t - .

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

B, C. DNA damage is reduced to wildtype levels by complementation with Rnaseh2b but not Rnaseh1, measured by 53BP1 foci formation in detergent-extracted fixed cells. (B) Representative images (scale bar, 10 µm). (C) At least 150 cells were counted for each cell line in three independent experiments. Mean ± SEM, **** = p<0.0001 two-tailed t-test.


D - F . CCL5 ( D ) CXCL10 production ( E ) , as well as ISG induction ( F ) in Rnaseh2b - / - MEFs are reduced close to wildtype ( Rnaseh2b + / + ) , by complementation with Rnaseh2b but not Rnaseh1 . Mean of n = 6 independent experiments ± for complemented ; n = 3 independent experiments for Rnaseh2b - / - parental Rnaseh2b + / + controls . * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 two - t - indicates significantly reduced expression compared to Rnaseh2b - / - parental .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. CCL5 (D) and CXCL10 production (E), as well as ISG induction (F) in Rnaseh2b-/- MEFs are reduced close to wildtype levels (Rnaseh2b+/+), by complementation with Rnaseh2b but not Rnaseh1. Mean of n=6 independent experiments ± SEM for complemented cells; n=3 independent experiments for Rnaseh2b-/- parental and Rnaseh2b+/+ controls cells. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001 two-tailed t-test indicates significantly reduced expression compared to Rnaseh2b-/- parental cells.


( b ) immunoprecipitation using α - FLAG antibody was performed to confirm pull - down of Sα from Sα - engineered ( FNLDD - Sα - LexA ) CH12F3 - 2A . a , b , c bars indicate primers used for ChIP - qPCR as shown in Fig . 1a ( n = 3 ; mean + sd ; two unpaired Student ' s t - ) .

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

(b) Chromatin immunoprecipitation using α-FLAG antibody was performed to confirm the pull-down of Sα chromatin from the Sα-engineered (FNLDD-Sα-LexA) CH12F3-2A cells. The a, b, and c bars indicate the primers used for ChIP-qPCR as shown in Fig. 1a (n=3; mean + sd; two tailed unpaired Student's t-test).


( e ) Bar graph showing with ≥ 2 - fold enrichment as calculated by heavy to SILAC .

(e) Bar graph showing the proteins with ≥2-fold enrichment as calculated by the heavy to light SILAC ratio.


( f ) Effect of siRNA - mediated KD of histone chaperones remodelers on IgM to IgA switching in CH12F3 - 2A . Data are presented as % IgA switching relative to IgA switching in transfected with control siRNA ( siCONT ) ( n = 2 ; mean + sd ) .

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

(f) Effect of the siRNA-mediated KD of histone chaperones and chromatin remodelers on IgM to IgA switching in CH12F3-2A cells. Data are presented as the % IgA switching relative to the IgA switching in cells transfected with control siRNA (siCONT) (n=2; mean + sd).


( a ) Top : Scheme of IgA - switching in CH12F3 - 2A . After electroporation of siRNAs , were cultured for 24 h , then stimulated by CIT , cultured for another 24 h , subjected to FACS . Bottom : Confirmation of siRNA - mediated KD of SAMHD1 in CH12F3 - 2A .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top: Scheme of the IgA-switching assay in CH12F3-2A cells. After electroporation of siRNAs, cells were cultured for 24 h, and then stimulated by CIT, cultured for another 24 h, and subjected to FACS analysis. Bottom: Confirmation of the siRNA-mediated KD of SAMHD1 in CH12F3-2A cells.


( b ) FACS profile of of CH12F3 - 2A undergoing IgA switching after transfection of indicated siRNAs into CH12F3 - 2A cultured with ( + ) or without ( - ) CIT stimulation for 24 h . number in FACS plot represents of expressing IgA on their surface .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FACS profile of the percentage of CH12F3-2A cells undergoing IgA switching after transfection of the indicated siRNAs into CH12F3-2A cells cultured with (+) or without (-) CIT stimulation for 24 h. The number in the FACS plot represents the percentage of cells expressing IgA on their surface.


( c ) Summary of FACS data obtained from three independent experiments . Data are shown as % IgA switching relative to IgA switching in transfected with control siRNA ( siCONT ) ( n = 3 ; mean + sd ; two unpaired Student ' s t - ) .

(c) Summary of the FACS data obtained from three independent experiments. Data are shown as the % IgA switching relative to the IgA switching in cells transfected with control siRNA (siCONT) (n=3; mean + sd; two tailed unpaired Student's t-test).


( d ) Quantitative RT - PCR of μGLT , αGLT , Aid , Samhd1 expression in indicated samples . data were normalized to Gapdh , represents as mean of two independent experiments with standard deviations . Statistical significance as determined by two - unpaired Student ' s t - is shown . ns = not significant .

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

(d) Quantitative RT-PCR analysis of the μGLT, αGLT, Aid, and Samhd1 expression in the indicated samples. The data were normalized to Gapdh, and represents as the mean of the two independent experiments with standard deviations. Statistical significance as determined by two-tailed unpaired Student's t-test is shown. ns = not significant.


( e ) Confirmation of CRISPR / Cas9 mediated KO of Samhd1 in CH12F3 - 2A . Beta - actin was used as a loading control .

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

(e) Confirmation of the CRISPR/Cas9 mediated KO of Samhd1 in CH12F3-2A cells. Beta-actin was used as a loading control.


( f ) CSR time course of WT Samhd1 KO clones . data are shown as of IgA expressing ( n = 2 ; mean ± sd ; two unpaired Student ' s t - ; * * , p < 0 . 01 ) .

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

(f) CSR time course analysis of WT and Samhd1 KO clones. The data are shown as percentage of IgA expressing cells (n=2; mean ± sd; two tailed unpaired Student's t-test; **, p<0.01).


( g ) Proliferation of WT Samhd1 KO clones after stimulation with CIT ( n = 2 ; mean ± sd ; two unpaired Student ' s t - ; ns = not significant ) .

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

(g) Proliferation of WT and Samhd1 KO clones after stimulation with CIT (n = 2; mean ± sd; two tailed unpaired Student's t-test; ns = not significant).


( h ) of proliferation of WT or Samhd1 KO clones of CH12F3 - 2A . Histograms show CFSE dye dilution derived from indicated samples . Aphidicolin ( 2 μg / mL ) was also used as a positive control .

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

(h) Analysis of the cell proliferation of WT or Samhd1 KO clones of CH12F3-2A. Histograms show the CFSE dye dilution derived from the indicated samples. Aphidicolin (2 μg/mL) was also used as a positive control.


( i ) Schematic representation of IgG1 or IgG3 switching in spleen B . Bottom : Western blot of SAMHD1 expression in spleen B derived from WT or Samhd1 KO mice . Tubulin was used as a loading control .

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

(i) Schematic representation of the IgG1 or IgG3 switching assay in spleen B cells. Bottom: Western blot analysis of the SAMHD1 expression in spleen B cells derived from WT or Samhd1 KO mice. Tubulin was used as a loading control.


( j ) Bar graphs show compilation of IgG1 IgG3 CSR assays from five independent experiments . data are shown as of IgG1 ( left ) or IgG3 ( right ) expressing ( n = 5 ; mean ± sd ; two - unpaired Student ' s t - ) .

(j) Bar graphs show the compilation of IgG1 and IgG3 CSR assays from five independent experiments. The data are shown as percentage of IgG1 (left) or IgG3 (right) expressing cells (n=5; mean ± sd; two-tailed unpaired Student's t-test).


( b ) Southern blot of PCR - amplified fragments with a Myc - specific probe from two independent experiments . CH12F3 - 2A were transfected with indicated siRNAs . ( c ) Frequency of IgH / c - Myc chromosomal translocations derived from two independent experiments ( mean ± sd ; Fisher ' s exact ) .

(b) Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments. CH12F3-2A cells were transfected with the indicated siRNAs. (c) Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test).


Western blot of KD of SAMHD1 or TOP1 by transfection of CH12F3 - 2A by SAMHD1 siRNA or by tetracycline ( 50 nM ) treatment , respectively . Expression of Tubulin is shown as a loading control ( e : left ) . Relative expression of TOP1 SAMHD1 as determined by densitometric of western blot in Fig . 3e ( e : right ) .

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

Western blot analysis of the KD of SAMHD1 or TOP1 by the transfection of CH12F3-2A cells by SAMHD1 siRNA or by tetracycline (50 nM) treatment, respectively. Expression of Tubulin is shown as a loading control (e: left). Relative expression of TOP1 and SAMHD1 as determined by densitometric quantification of western blot images in Fig. 3e (e: right).


Southern blot of PCR - amplified fragments with a Myc - specific probe from two independent experiments ( f ) . Frequency of IgH / c - Myc chromosomal translocations derived from two independent experiments ( mean ± sd ; Fisher ' s exact ) ( g ) .

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

Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments (f). Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test) (g).


( h - j ) of IgH / c - Myc translocation frequency in WT Samhd1 KO CH12F3 - 2A . ( h ) Schematic illustration of experimental design for translocation . ( i ) Southern blot of PCR - amplified fragments with a Myc - specific probe from two independent experiments . ( j ) Frequency of IgH / c - Myc chromosomal translocations derived from two independent experiments ( mean ± sd ; Fisher ' s exact ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-j) Analysis of the IgH/c-Myc translocation frequency in WT and Samhd1 KO CH12F3-2A cells. (h) Schematic illustration of the experimental design for the translocation assay. (i) Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments. (j) Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test).


( k - m ) of IgH / c - Myc translocation frequency in spleen B derived from WT ( Samhd1 + / + ) or Samhd1 KO ( Samhd1 - / - ) mice . ( k ) Schematic illustration of experimental design for translocation . ( l ) Southern blot of PCR - amplified fragments with a Myc - specific probe from two independent experiments . ( m ) Frequency of IgH / c - Myc chromosomal translocations derived from two independent experiments ( mean ± sd ; Fisher ' s exact ) .

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

(k-m) Analysis of the IgH/c-Myc translocation frequency in spleen B cell derived from WT (Samhd1+/+) or Samhd1 KO (Samhd1-/-) mice. (k) Schematic illustration of the experimental design for the translocation assay. (l) Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments. (m) Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test).


( b ) Mutation frequency in 5 ′ Sµ region of WT or Samhd1 KO CH12F3 - 2A . Statistical significance as evaluated by Fisher ' s exact is shown . ns = not significant .

(b) Mutation frequency in the 5′ Sµ region of WT or Samhd1 KO CH12F3-2A cells. Statistical significance as evaluated by Fisher's exact test is shown. ns = not significant.


( d ) LM - PCR of AID - induced double - strand breaks in Sµ region of WT or Samhd1 KO CH12F3 - 2A . Wedges indicate a three - fold increase in amount . Amplification of Gapdh was used as an internal loading control .

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

(d) LM-PCR analysis of the AID-induced DNA double-strand breaks in the Sµ region of WT or Samhd1 KO CH12F3-2A cells. Wedges indicate a three-fold increase in the DNA amount. Amplification of Gapdh was used as an internal loading control.


( e ) Gamma - H2AX - ChIP signal in Sµ region is shown as fraction of immunoprecipitated ( % IP ) normalized to total amount of used for immunoprecipitation ( n = 3 ; mean + sd ; two - unpaired Student ' s t - ; ns = not significant ) .

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

(e) Gamma-H2AX-ChIP signal in the Sµ region is shown as the fraction of immunoprecipitated DNA (% IP) normalized to the total amount of DNA used for the immunoprecipitation (n=3; mean + sd; two-tailed unpaired Student's t-test; ns = not significant).


( f ) Left : Scheme of long - range interactions between Sμ - Sα elements in IgH locus before after AID activation . Right : Representative gel picture of 3C detecting Sμ - Sα interaction in WT or Samhd1 KO CH12F3 - 2A with or without CIT stimulation for 24 h . Gapdh was amplified as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left: Scheme of long-range interactions between Sμ-Sα elements in the IgH locus before and after AID activation. Right: Representative gel picture of the 3C assay detecting the Sμ-Sα interaction in WT or Samhd1 KO CH12F3-2A cells with or without CIT stimulation for 24 h. Gapdh was amplified as a loading control.


( a - d ) of Sμ - Sα recombination junctions from WT or CH12F3 - 2A after transfection with control ( siCONT ) or Ligase 4 ( siLIG4 ) siRNA . genomic isolated from WT or Samhd1 KO from three independent experiments were pooled prior to junction . A total of 62 ( siCONT ) 73 ( siLIG4 ) junctions from WT , 60 ( siCONT ) 60 ( siLIG4 ) junctions from Samhd1 KO were sequenced analyzed . Statistical significance as evaluated by Fisher ' s exact ( b ) or two - unpaired Student ' s t - ( d ) or is shown . Schematic representation of Sμ - Sα junction ( a ) . Summary of characteristics of Sμ - Sα recombination junctions ( b ) . Sequences at Sμ - Sα recombination junctions with insertions ( c ) . Summary of average of insertions at Sμ - Sα recombination junctions ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Analysis of Sμ-Sα recombination junctions from WT or CH12F3-2A cells after transfection with control (siCONT) or Ligase 4 (siLIG4) siRNA. The genomic DNA isolated from WT or Samhd1 KO cells from three independent experiments were pooled prior to junction analysis. A total of 62 (siCONT) and 73 (siLIG4) junctions from WT, and 60 (siCONT) and 60 (siLIG4) junctions from Samhd1 KO cells were sequenced and analyzed. Statistical significance as evaluated by Fisher's exact test (b) or two-tailed unpaired Student's t-test (d) or is shown. Schematic representation of the Sμ-Sα junction analysis (a). Summary of the characteristics of Sμ-Sα recombination junctions (b). Sequences at Sμ-Sα recombination junctions with insertions (c). Summary of the average length of insertions at Sμ-Sα recombination junctions (d).


( e - i ) of CRISPR / Cas9 induced chromosomal translocations between Rosa26 ( Chr6 ) H3f3b ( Chr 11 ) genes . genomic isolated from WT or Samhd1 KO from three independent experiments were pooled prior to chromosomal translocation or junction . Schematic representation of chromosomal translocations ( e ) . Representative of agarose gels after PCR - amplification of Rosa26 / H3f3b translocation from WT or Samhd1 KO CH12F3 - 2A ( f ) . Relative frequency of Rosa26 / H3b3f chromosomal translocations ( g ) . Frequency of junctions containing insertions ( h ) average of insertions ( i ) . Statistical significance as evaluated by two - unpaired Student ' s t - ( g , i ) or Fisher ' s exact ( h ) is shown .

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

(e-i) Analysis of the CRISPR/Cas9 induced chromosomal translocations between the Rosa26 (Chr6) and H3f3b (Chr 11) genes. The genomic DNA isolated from WT or Samhd1 KO cells from three independent experiments were pooled prior to chromosomal translocation or junction analysis. Schematic representation of the chromosomal translocations (e). Representative images of agarose gels after PCR-amplification of the Rosa26/H3f3b translocation from WT or Samhd1 KO CH12F3-2A cells (f). Relative frequency of Rosa26/H3b3f chromosomal translocations (g). Frequency of junctions containing insertions (h) and average length of insertions (i). Statistical significance as evaluated by two-tailed unpaired Student's t-test (g, i) or Fisher's exact test (h) is shown.


( b ) Confirmation of expression of GFP - tagged human SAMHD1 ( WT , DN , DA , TA , RE mutants ) . Histone H3 expression is shown as a loading control .

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

(b) Confirmation of the expression of GFP-tagged human SAMHD1 (WT, and DN, DA, TA, and RE mutants). Histone H3 expression is shown as a loading control.


( c ) CSR complementation in Samhd1 KO . Data are shown as % IgA switching relative to IgA switching in WT transfected with empty vector , are derived from three independent experiments ( mean ± sd ; two - unpaired Student ' s t - ) .

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

(c) CSR complementation assay in Samhd1 KO cells. Data are shown as the % IgA switching relative to the IgA switching in WT cells transfected with empty vector, and are derived from three independent experiments (mean ± sd; two-tailed unpaired Student's t-test).


( e ) Effect of deoxyribonucleoside supplementation individually or in combination on rate of IgM to IgA switching in CH12F3 - 2A . Data are presented as % IgA switching relative to IgA switching in treated with an equivalent volume of solvent in which deoxyribonucleosides were dissolved ( n = 3 ; mean ± sd ; two - unpaired Student ' s t - ; * * = p ≤ 0 . 01 ; * * * = 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

(e) Effect of deoxyribonucleoside supplementation individually or in combination on the rate of IgM to IgA switching in CH12F3-2A cells. Data are presented as % IgA switching relative to the IgA switching in cells treated with an equivalent volume of the solvent in which the deoxyribonucleosides were dissolved (n=3; mean ± sd; two-tailed unpaired Student's t-test; ** = p ≤ 0.01; *** = p ≤ 0.001).


( f ) Effect of deoxyribonucleoside supplementation individually or in combination on frequency of IgH / c - Myc translocations in CH12F3 - 2A ( n = 2 ; mean ± sd ; Fisher ' s exact ; ns = not significant ; * * * = p ≤ 0 . 001 ) . dNTPs used for supplementation either individually or in combination were as follows : 2 ′ - deoxyguanosine ( dG ) , 0 . 15 mM ; 2 ′ - deoxyadenosine ( dA ) , 1 . 0 mM ; 2 ′ - deoxycytidine ( dC ) , 12 mM ; 2 ′ - deoxythymidine ( dT ) , 1 . 6 mM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of deoxyribonucleoside supplementation individually or in combination on the frequency of IgH/c-Myc translocations in CH12F3-2A cells (n = 2; mean ± sd; Fisher's exact test; ns = not significant; *** = p ≤ 0.001). The concentration dNTPs used for supplementation either individually or in combination were as follows: 2′-deoxyguanosine (dG), 0.15 mM; 2′-deoxyadenosine (dA), 1.0 mM; 2′-deoxycytidine (dC), 12 mM; and 2′-deoxythymidine (dT), 1.6 mM.


( b ) FACS profile of of WT Samhd1 KO CH12F3 - 2A undergoing IgA switching following transfection with indicated plasmids . number in FACS plot represents of expressing IgA on their surface . ( c ) Summary of FACS data obtained from two independent experiments . data are shown as of IgA expressing upon CSR induced by blunt ( left ) or staggered ( right ) DSBs ( n = 2 ; mean + sd ; two - unpaired Student ' s t - ) ; ns = not significant .

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

(b) FACS profile of the percentage of WT and Samhd1 KO CH12F3-2A cells undergoing IgA switching following transfection with the indicated plasmids. The number in the FACS plot represents the percentage of cells expressing IgA on their surface. (c) Summary of the FACS data obtained from two independent experiments. The data are shown as percentage of IgA expressing cells upon CSR induced by blunt (left) or staggered (right) DSBs (n = 2; mean + sd; two-tailed unpaired Student's t-test); ns = not significant.


( d ) of resected at Sμ - Sα junctions obtained from joining of DSBs with blunt ends . Statistical significance as evaluated by two - unpaired Student ' s t - is shown . ( e ) Summary of characteristics of Sμ - Sα recombination junctions resulting from joining of DSBs with blunt ends .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Length of resected DNA at Sμ-Sα junctions obtained from the joining of DSBs with blunt ends. Statistical significance as evaluated by two-tailed unpaired Student's t-test is shown. (e) Summary of the characteristics of Sμ-Sα recombination junctions resulting from the joining of DSBs with blunt ends.


( f ) of resected at Sμ - Sα junctions obtained from joining of staggered DSBs . Left : of Sμ Sα overhangs were 35 nt 98 nt , respectively . Right : of Sμ Sα overhangs were 35 nt 187 nt , respectively . ( g ) Summary of characteristics of Sμ - Sα recombination junctions resulting from joining of staggered DSBs . Statistical significance as evaluated by Fisher ' s exact is shown .

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

(f) Length of resected DNA at Sμ-Sα junctions obtained from the joining of staggered DSBs. Left: Length of Sμ and Sα overhangs were 35 nt and 98 nt, respectively. Right: Length of Sμ and Sα overhangs were 35 nt and 187 nt, respectively. (g) Summary of the characteristics of Sμ-Sα recombination junctions resulting from the joining of staggered DSBs. Statistical significance as evaluated by Fisher's exact test is shown.


( h ) Summary of average of insertions at Sμ - Sα recombination junctions . Statistical significance as evaluated by two - unpaired Student ' s t - is 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

(h) Summary of the average length of insertions at Sμ-Sα recombination junctions. Statistical significance as evaluated by two-tailed unpaired Student's t-test is shown.


of effect of TdT expression on frequency of CSR as well as characteristics of Sμ - Sα recombination junctions resulting from joining of DSBs with blunt ends . ( j ) Top : Schematic representation of experimental design to analyze effect of TdT expression on CSR in WT Samhd1 KO . Bottom : Western blot of expression of TdT in WT Samhd1 KO . Tubulin was used as a loading control .

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

Analysis of the effect of TdT expression on the frequency of CSR as well as characteristics of Sμ-Sα recombination junctions resulting from the joining of DSBs with blunt ends. (j) Top: Schematic representation of the experimental design to analyze the effect of TdT expression on CSR in WT and Samhd1 KO cells. Bottom: Western blot analysis of the expression of TdT in WT and Samhd1 KO cells. Tubulin was used as a loading control.


( k ) FACS profile of IgA switching in WT or Samhd1 KO CH12F3 - 2A with or without TdT expression . ( l ) Summary of FACS data obtained from two independent experiments ( n = 2 ; mean + sd ; two - unpaired Student ' s t - ; * = p ≤ 0 . 05 ) .

(k) FACS profile of the percentage IgA switching in WT or Samhd1 KO CH12F3-2A cells with or without TdT expression. (l) Summary of the FACS data obtained from two independent experiments (n = 2; mean + sd; two-tailed unpaired Student's t-test; * = p ≤ 0.05).


( m ) Summary of characteristics of Sμ - Sα recombination junctions . Statistical significance as evaluated by Fisher ' s exact is shown . ns = not significant .

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

(m) Summary of the characteristics of Sμ-Sα recombination junctions. Statistical significance as evaluated by Fisher's exact test is shown. ns = not significant.


C , D . Oxidative stress biochemical markers ( lipid peroxidation , ROS production , GSH ) ( C ) p - ERK expression ( D ) in PD control ( CNTR ) fibroblasts ( n = 6 ) . E , F . Oxidative stress biochemical markers ( lipid peroxidation , ROS production , GSH ) ( E ) p - ERK expression ( F ) in PD control ( CNTR ) myoblasts ( n = 4 ) . In instances indicators of stress were increased in PD , compared to respective control samples . Data information : In each experiment at least biological triplicates were analyzed for each or tissue sample ; each was performed at least in duplicate . Data are presented as mean ± SD . Student ' s t - was applied . Statistically significant comparison p - values are indicated . Fi

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

C, D. Oxidative stress biochemical markers (lipid peroxidation, ROS production, GSH levels) (C) and p-ERK expression (D) in PD and control (CNTR) fibroblasts (n=6). E, F. Oxidative stress biochemical markers (lipid peroxidation, ROS production, GSH levels) (E) and p-ERK expression (F) in PD and control (CNTR) myoblasts (n=4). In all instances indicators of stress were increased in PD, compared to the respective control samples. Data information: In each experiment at least biological triplicates were analyzed for each cell line or tissue sample; each assay was performed at least in duplicate. Data are presented as mean ± SD. Student's t-test was applied. Statistically significant comparison p-values are indicated. Fi


A , B . Western Blot of autophagy marker LC3 ( A ) quantitative analyses ( B ) in untreated control fibroblasts ( CNTR ) in 2 PD fibroblasts lines ( PD1 , PD2 ) . PD were untreated ( NT ) or subjected to different treatments to modulate autophagy ( starvation , STAR ; rapamycin , RAPA ; MK6 - 83 ; bafilomycin , BAFI ) . Data information : Starvation of was performed for 4 hours ; treatments with 20 µM rapamycin or 30 µM MK6 - 83 or 100nM bafilomycin was performed for 24 hours . Data presented as mean ± SD . P values were calculated with ANOVA Šídák ' s multiple comparisons . Statistically significant comparison p - values are indicated .

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

A, B. Western Blot analysis of the autophagy marker LC3 (A) and quantitative analyses (B) in untreated control fibroblasts (CNTR) and in 2 PD fibroblasts cell lines (PD1, PD2). PD cells were untreated (NT) or subjected to different treatments to modulate autophagy (starvation, STAR; rapamycin, RAPA; MK6-83; bafilomycin, BAFI). Data information: Starvation of cells was performed for 4 hours; treatments with 20 µM rapamycin or 30 µM MK6-83 or 100nM bafilomycin was performed for 24 hours. Data presented as mean ± SD. P values were calculated with ANOVA Šídák's multiple comparisons test. Statistically significant comparison p-values are indicated.


C . ROS production , lipid peroxidation , GSH in PD fibroblasts ( n = 3 ) . CNTR ( bars ) , CNTR after treatment ( grey bars ) , PD samples ( black bars ) PD after treatment ( grey bars ) . Physiological or pharmacological enhancement of autophagy resulted in correction of oxidative stress in PD , while bafilomycin treatment further increased stress in CNTR PD . Data information : Starvation of was performed for 4 hours ; treatments with 20 µM rapamycin or 30 µM MK6 - 83 or 100nM bafilomycin was performed for 24 hours . Data presented as mean ± SD . P values were calculated with ANOVA Šídák ' s multiple comparisons . Statistically significant comparison p - values are indicated .

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

C. ROS production, lipid peroxidation, and GSH levels in PD fibroblasts (n=3). CNTR (white bars), CNTR after treatment (dark grey bars), PD samples (black bars) and PD after treatment (light grey bars). Physiological or pharmacological enhancement of autophagy resulted in correction of oxidative stress in PD cells, while bafilomycin treatment further increased stress in CNTR and PD cells. Data information: Starvation of cells was performed for 4 hours; treatments with 20 µM rapamycin or 30 µM MK6-83 or 100nM bafilomycin was performed for 24 hours. Data presented as mean ± SD. P values were calculated with ANOVA Šídák's multiple comparisons test. Statistically significant comparison p-values are indicated.


A , B , C . Correlations between of single stress indicators correction of GAA activity by rhGAA in 6 different PD fibroblasts . ROS production lipid peroxidation inversely correlate with GAA attained in after incubation with rhGAA for 4 hours . of correlation was calculated , coefficient of Pearson is indicated . 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 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

A, B, C. Correlations between the levels of single stress indicators and correction of GAA activity by rhGAA in 6 different PD fibroblasts. ROS production and lipid peroxidation inversely correlate with the GAA levels attained in cells after incubation with rhGAA for 4 hours. The analysis of correlation was calculated, and the coefficient of Pearson is indicated. D


A . viability in control PD fibroblasts ( 3 different lines ) measured by MTT in presence of increased concentrations ( 0 - 100 µM ) of either sodium arsenite ( ARS ) or tert - butyl - peroxide ( TBP ) at different time points . Data presented as a mean ± SD . B . ROS production , lipid peroxidation , GSH in fibroblasts ( 3 different lines ) after 6 hours of treatment with 100 µM ARS or 10 µM TBP . Both oxidative agents induced increases in oxidative stress in control ( CNTR ) in PD . Data presented as a mean ± SD . Significance was calculated by one - way ANOVA followed by Šídák ' s multiple comparisons . C Data information : statistically significant p - values are indicated .

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

A. Cell viability in control and PD fibroblasts (3 different cell lines) measured by MTT assay in the presence of increased concentrations (0-100 µM) of either sodium arsenite (ARS) or tert-butyl-peroxide (TBP) at different time points. Data presented as a mean ± SD. B. ROS production, lipid peroxidation, and GSH levels in fibroblasts (3 different cell lines) after 6 hours of treatment with 100 µM ARS and or 10 µM TBP. Both oxidative agents induced increases in oxidative stress in control (CNTR) and in PD cells. Data presented as a mean ± SD. Significance was calculated by one-way ANOVA followed by Šídák's multiple comparisons test. C Data information: statistically significant p-values are indicated.


A . Effect of antioxidants on ROS production , lipid peroxidation , GSH in PD fibroblasts ( 3 different lines for each treatment , each assayed at least in ducplicate ) . were incubated for 24 hours with : idebenone ( IDE ) 0 . 5 µM ; edaravone ( EDA ) 50 µM ; N - acetylcysteine ( NAC ) 0 . 5 mM ; resveratrol ( RESV ) 30 µM . Mean of control ( CNTR ) values are taken as equal to 100 . results are shown as mean ± SD . Data information : To calculate statistical significance one - way ANOVA was applied for experiments followed by Dunnett ' s . Statistically significant p - values are indicated .

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

A. Effect of antioxidants on ROS production, lipid peroxidation, and GSH levels in PD fibroblasts (3 different cell lines for each treatment, each cell line assayed at least in ducplicate). Cells were incubated for 24 hours with: idebenone (IDE) 0.5 µM; edaravone (EDA) 50 µM; N-acetylcysteine (NAC) 0.5 mM; resveratrol (RESV) 30 µM. Mean of control (CNTR) values are taken as equal to 100. The results are shown as mean ± SD. Data information: To calculate statistical significance one-way ANOVA was applied for all experiments followed by Dunnett's test. Statistically significant p-values are indicated.


E . Confocal immunofluorescence of GAA LAMP2 ; representative fibroblast lines ( PD1 ) . Confocal 63x ; Scale bar 50 µm ; Brightness + 20 % . F . of GAA / LAMP2 colocalization in PD1 . results are expressed as means ± SD of 5 for each condition . G . of GAA / LAMP2 colocalization ; mean of analyses performed in 3 PD fibroblast lines . For each PD , 5 for each condition were quantified . results are expressed as means ± SD of 5 for each condition . Data information : To calculate statistical significance one - way ANOVA was applied for experiments followed by Dunnett ' s . Statistically significant p - values are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal immunofluorescence analysis of GAA and LAMP2; representative fibroblast cell lines (PD1). Confocal 63x images; Scale bar 50 µm; Brightness +20%. F. Percent of GAA/LAMP2 colocalization in PD1. The results are expressed as means ± SD of 5 images for each condition. G. Percent of GAA/LAMP2 colocalization; mean of the analyses performed in 3 PD fibroblast cell lines. For each PD patient, 5 images for each condition were quantified. The results are expressed as means ± SD of 5 images for each condition. Data information: To calculate statistical significance one-way ANOVA was applied for all experiments followed by Dunnett's test. Statistically significant p-values are indicated.


D . Mean of results obtained in control ( two lines in duplicate ) PD fibroblast ( 3 different lines , each tested in duplicate in three different experiments ) . For each , amount of M6PR positive was normalized , taking that observed in not - treated fibroblasts as 1 . Data presented as mean ± SD . Data information : For statistical a one - way ANOVA followed by Šídák ' s post hoc was applied .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Mean of results obtained in control (two cell lines in duplicate) and PD fibroblast (3 different cell lines, each cell line tested in duplicate in three different experiments). For each cell line, the amount of M6PR positive cells was normalized, taking that observed in not-treated fibroblasts as 1. Data presented as mean ± SD. Data information: For statistical analysis a one-way ANOVA followed by Šídák's post hoc test was applied.


C . GAA activity in tissues from Gaa KO mouse after treatment with rhGAA alone ( black bars ) ( n of mice = 10 ) with co - dosing of rhGAA 2 gr / Kg / day NAC ( n of mice = 7 , grey bars ) . Data presented as mean ± SD . Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100 . A Student ' s t - was applied to compare results in each of tissues . D . GAA activity in tissues from Gaa KO mouse after treatment with rhGAA alone ( black bars ) ( n of mice = 4 ) with co - dosing of rhGAA 100 mg / Kg / day IDE ( n of mice = 6 , grey bars ) . Data presented as mean ± SD . Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100 . A Student ' s t - was applied to compare results in each of tissues . E

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

C. GAA activity in tissues from the Gaa KO mouse after treatment with rhGAA alone (black bars) (n of mice = 10) and with co-dosing of rhGAA and 2 gr/Kg/day NAC (n of mice =7, dark grey bars). Data presented as mean ± SD. Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100. A Student's t-test was applied to compare the results in each of the tissues. D. GAA activity in tissues from the Gaa KO mouse after treatment with rhGAA alone (black bars) (n of mice =4) and with co-dosing of rhGAA and 100 mg/Kg/day IDE (n of mice =6, light grey bars). Data presented as mean ± SD. Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100. A Student's t-test was applied to compare the results in each of the tissues. E


E , F . Western Blot analyses of GAA quantitative analyses of enzyme in representative tissues from Gaa KO mouse after treatment with rhGAA alone ( n of mice = 2 ) or in combination with NAC ( n of mice = 3 ) ( E ) or IDE ( n of mice = 3 ) ( F ) . In F Data are presented as mean ± SD .

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

E, F. Western Blot analyses of GAA and quantitative analyses of the enzyme in representative tissues from the Gaa KO mouse after treatment with rhGAA alone (n of mice =2) or in combination with NAC (n of mice =3) (E) or IDE (n of mice =3) (F). In F Data are presented as mean ± SD.


G . Glycogen in tissues from Gaa KO mouse after treatment with rhGAA alone ( n of mice = 3 ) or in combination with NAC ( n of mice = 3 ) . H . Glycogen in tissues from Gaa KO mouse after treatment with rhGAA alone ( n of mice = 4 ) or in combination with IDE ( n of mice = 6 ) . Data information : i data are presented as mean ± SD . Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100 . A Student ' s t - was applied to compare results in each tissue .

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

G. Glycogen assay in tissues from the Gaa KO mouse after treatment with rhGAA alone (n of mice = 3) or in combination with NAC (n of mice = 3). H. Glycogen assay in tissues from the Gaa KO mouse after treatment with rhGAA alone (n of mice =4) or in combination with IDE (n of mice = 6). Data information: i data are presented as mean ± SD. Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100. A Student's t-test was applied to compare the results in each tissue.


Uncoupled ( LEAK ) mitochondrial respiration of soleus ( SOL ) extensor digitorum longus ( EDL ) muscle of wild type ( WT ) vs . Ucp1 - TG ( TG ) mice ( WT n = 9 , TG n = 5 ) .

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

Uncoupled (LEAK) mitochondrial respiration of soleus (SOL) and extensor digitorum longus (EDL) muscle of wild type (WT) vs. Ucp1-TG (TG) mice (WT n=9, TG n=5).


Relative mRNA expression in quadriceps ( Quad ) of ISR components ( n = 8 per genotype ) .

Relative mRNA expression in quadriceps (Quad) of ISR components (n=8 per genotype).


Representative immunoblots of ISR component eIF2α of phospho - eIF2α ( p - eIF2aSer51 ) relative expression in quadriceps skeletal muscle ( WT n = 3 , TG n = 4 ) .

O O O O O O O O O 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 immunoblots of ISR component eIF2α and quantification of phospho-eIF2α (p-eIF2aSer51) relative protein expression in quadriceps skeletal muscle (WT n=3, TG n=4).


Multi - tissue transcriptomic profiling of Gdf15 gene expression . is shown as raw ct expression values ( n = 4 per genotype ) . of Gdf15 mRNA expression in TG mice is shown as fold change compared to WT ( WT n = 5 , TG n = 4 ) .

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

Multi-tissue transcriptomic profiling of Gdf15 gene expression. Heatmap is shown as raw ct expression values (n=4 per genotype). Quantification of Gdf15 mRNA expression in TG mice is shown as fold change compared to WT littermates (WT n=5, TG n=4).


Skeletal muscle ( Quad Gastroc ) GDF15 content normalized to total content ( WT n = 9 , TG n = 18 ) .

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

Skeletal muscle (Quad and Gastroc) GDF15 protein content normalized to total protein content (WT n=9, TG n=18).


Ex vivo secretion of GDF15 from SOL EDL muscle of WT vs . TG mice ( n = 6 per genotype ) after 2h incubation normalized to muscle wet ( mg ) .

Ex vivo secretion of GDF15 from SOL and EDL muscle of WT vs. TG mice (n=6 per genotype) after 2h incubation normalized to muscle wet weight (mg).


Mouse GDF15 plasma in post - absorptive state of WT vs . TG mice at 20wks of ( WT n = 8 , TG n = 9 ; WT n = 5 , TG n = 6 ) .

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

Mouse GDF15 plasma levels in post-absorptive state of male and female WT vs. TG mice at 20wks of age (male WT n=8, TG n=9; female WT n=5, TG n=6).


Mouse GDF15 plasma in post - absorptive state of WT vs . TG mice fed low - fat diet ( LFD ) or high - fat diet ( HFD ) at 24wks of ( LFD WT n = 6 , TG n = 5 ; HFD WT n = 5 , TG n = 5 ) .

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

Mouse GDF15 plasma levels in post-absorptive state of male WT vs. TG mice fed low-fat diet (LFD) or high-fat diet (HFD) at 24wks of age (LFD WT n=6, TG n=5; HFD WT n=5, TG n=5).


Mouse GDF15 plasma in post - absorptive state of young ( 10wks ) vs . old ( 95wks ) WT TG mice ( young WT n = 10 , TG n = 13 ; old WT n = 13 , TG n = 13 ) .

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

Mouse GDF15 plasma levels in post-absorptive state of young (10wks) vs. old (95wks) male WT and TG mice (young WT n=10, TG n=13; old WT n=13, TG n=13).


Gdf15 gene expression of differentiated C2C12 muscle treated with vehicle control ( Ctrl ) or chemical mitochondrial uncoupler ( FCCP , 1µM vs . 5µM ) for 5hrs ( n = 3 biological replicates )

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

Gdf15 gene expression of differentiated C2C12 muscle cells treated with vehicle control (Ctrl) or chemical mitochondrial uncoupler (FCCP, 1µM vs. 5µM) for 5hrs (n=3 biological replicates)


Genotyping PCR panel of Gdf15 HSA - Ucp1 loci shown for wild type ( WT ) , Gdf15 - KO ( KO ) , Ucp1 - TG ( TG ) Ucp1 - TGxGdf15 - KO ( TGxKO ) mice .

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

Genotyping PCR panel of Gdf15 and HSA-Ucp1 loci shown for wild type (WT), Gdf15-KO (KO), Ucp1-TG (TG) and Ucp1-TGxGdf15-KO (TGxKO) mice.


Relative mRNA expression in quadriceps ( Quad ) of Ucp1 Gdf15 in 20wks WT ( n = 8 ) , TG ( n = 7 ) TGxKO ( n = 6 ) mice .

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

Relative mRNA expression in quadriceps (Quad) of Ucp1 and Gdf15 in 20wks male WT (n=8), TG (n=7) and TGxKO (n=6) mice.


Plasma mGDF15 at 20wks of ( WT n = 9 , TG n = 10 , TGxKO n = 9 ) .

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

Plasma mGDF15 levels at 20wks of age (WT n=9, TG n=10, TGxKO n=9).


Voluntary wheel running ( VWR ) activity shown hourly over 24 - hrs at 15 wks of ( n = 4 per genotype ) .

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

Voluntary wheel running (VWR) activity shown hourly over 24-hrs at 15 wks of age (n=4 per genotype).


Grip strength at 10 - 20wks of ( WT n = 15 , TG n = 14 , TGxKO n = 11 ) .

Grip strength at 10 - 20wks of age (WT n=15, TG n=14, TGxKO n=11).


Skeletal muscle mass relative to lean mass of quadriceps ( Quad ) gastrocnemius ( Gastroc ) from mice at 95wk of ( WT n = 8 , TG n = 5 , TGxKO n = 5 ) .

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

Skeletal muscle mass relative to body lean mass of quadriceps (Quad) and gastrocnemius (Gastroc) from mice at 95wk of age (WT n=8, TG n=5, TGxKO n=5).


Representative H & E histological of tibialis anterior ( TA ) muscle ( G ) ( scale bars represent 50 µm ) , cross - sectional ( CSA ) of myofibers ( H ) at 95wks ( WT n = 12 , TG n = 8 , TGxKO n = 8 ) .

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

Representative H&E histological staining of tibialis anterior (TA) muscle (G) (scale bars represent 50 µm), and cross-sectional area (CSA) of myofibers (H) at 95wks (WT n=12, TG n=8, TGxKO n=8).


Representative traces of oxygen consumption rate ( OCR ) during substrate - uncoupler - inhibitor - titration ( SUIT ) protocol for mitochondrial respiratory capacity in permeabilized mouse soleus muscle , PM ( Pyruvate + Malate ; LEAK respiration ) , ADP ( OXPHOS capacity ) , Cyt c ( cytochrome c , integrity of outer mt - ) , G ( glutamate ; ) , S ( succinate ) , U ( Uncoupler , FCCP ) , Rot ( rotenone ) , Ama ( antimycin A ; less than 2 % residual oxygen consumption , ROX ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 traces of oxygen consumption rate (OCR) during substrate-uncoupler-inhibitor-titration (SUIT) protocol for mitochondrial respiratory capacity in permeabilized mouse soleus muscle fibers, PM (Pyruvate+Malate; LEAK respiration), ADP (OXPHOS capacity), Cyt c (cytochrome c, integrity of outer mt-membrane), G (glutamate;), S (succinate), U (Uncoupler, FCCP), Rot (rotenone), Ama (antimycin A; less than 2% residual oxygen consumption, ROX).


Mitochondrial respiratory capacity ( oxygen consumption rate , OCR ) from oxidative soleus ( SOL ) ( J ) glycolytic extensor digitorum longus ( EDL ) muscle ( K ) , NetOXPHOS control of 95wks old mice ( L ) ( n = 5per genotype ) .

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

Mitochondrial respiratory capacity (oxygen consumption rate, OCR) from oxidative soleus (SOL) (J) and glycolytic extensor digitorum longus (EDL) muscle fibers (K), and NetOXPHOS control ratio of 95wks old mice (L) (n=5per genotype).


Quadriceps ( Quad ) relative mRNA expression of ISR components at 95wks of ( WT n = 8 , TG n = 5 , TGxKO n = 5 ) .

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

Quadriceps (Quad) relative mRNA expression of ISR components at 95wks of age (WT n=8, TG n=5, TGxKO n=5).


Representative immunoblots of ISR component eIF2α in quadriceps ( Quad ) muscle of 95wks old mice .

Representative immunoblots of ISR component eIF2α in quadriceps (Quad) muscle of 95wks old mice.


Gastrocnemius enzyme activity of NQO1 GPX in 95wks old mice ( WT n = 8 , TG n = 5 , TGxKO n = 5 ) .

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

Gastrocnemius enzyme activity of NQO1 and GPX in 95wks old male mice (WT n=8, TG n=5, TGxKO n=5).


mass ( A ) , lean mass ( B ) fat mass ( C ) development . Data are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO )

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

Body mass (A), body lean mass (B) and body fat mass (C) development. Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice


mass ( D ) , lean mass ( E ) fat mass ( F ) during aging at 20wks , 45wks 95wks of . Data are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO )

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

Body mass (D), body lean mass (E) and body fat mass (F) during aging at 20wks, 45wks and 95wks of age. Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice


Subcutaneous adipose tissue ( sWAT ) ( G ) epididymal adipose tissue ( eWAT ) ( H ) mass development at 10wks , 20wks , 45wks 95wks of . Data are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice at 10wks ( WT n = 6 , TG n = 7 , TGxKO n = 5 ) , 20wks ( WT n = 10 , TG n = 10 , TGxKO n = 10 ) , 45wks ( WT n = 5 , TG n = 6 , TGxKO n = 7 ) 95wks ( WT n = 8 , TG n = 5 , TGxKO n = 5 ) .

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

Subcutaneous white adipose tissue (sWAT) (G) and epididymal white adipose tissue (eWAT) (H) mass development at 10wks, 20wks, 45wks and 95wks of age. Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 10wks (WT n=6, TG n=7, TGxKO n=5), 20wks (WT n=10, TG n=10, TGxKO n=10), 45wks (WT n=5, TG n=6, TGxKO n=7) and 95wks (WT n=8, TG n=5, TGxKO n=5).


Representative H & E histological of sWAT at 20wks of ( scale bars represent 50 μm ) Data are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice .

O O O O O O O O O O O O O O O O O O O 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 H&E histological staining of sWAT at 20wks of age (scale bars represent 50 μm) Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.


relative mRNA expression profile in sWAT of mice ( WT n = 7 , TG n = 8 , TGxKO n = 8 ) ( B ) . Data are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice .

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

relative mRNA expression profile in sWAT of male mice (WT n=7, TG n=8, TGxKO n=8) (B). Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.


Representative immunoblots ( C ) ( D ) of UCP1 expression in sWAT of mice at 20wks of ( WT n = 4 , TG n = 6 , TGxKO n = 4 ) . Data are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunoblots (C) and quantification (D) of UCP1 protein expression in sWAT of male mice at 20wks of age (WT n=4, TG n=6, TGxKO n=4). Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.


Relative mRNA expression profile in eWAT ( WT n = 7 , TG n = 8 , TGxKO n = 8 ) . Data are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice .

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

Relative mRNA expression profile in eWAT (WT n=7, TG n=8, TGxKO n=8). Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.


Plasma FGF21 from mice at 20wks of ( n = 10 per genotype ) . Plasma Leptin from mice at 20wks of ( n = 5 per genotype ) . Post - absorptive plasma insulin at 20wks of ( WT n = 8 , TG n = 9 , TG n = 9 ) . : Data are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice .

O O O O O O O O O O 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

Plasma FGF21 levels from male mice at 20wks of age (n=10 per genotype). Plasma Leptin levels from male mice at 20wks of age (n=5 per genotype). Post-absorptive plasma insulin levels at 20wks of age (WT n=8, TG n=9, TG n=9). : Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.


Blood glucose ( I ) insulin ( J ) with total under curve ( AUC ) of insulin ( K ) during oral glucose tolerance ( oGTT ) at 17wks of ( WT n = 8 , TG n = 11 , TG n = 11 ) . Data are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice .

O B-MEASURED_VAR O O O B-MEASURED_VAR 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

Blood glucose (I) and insulin levels (J) with total area under the curve (AUC) of insulin (K) during oral glucose tolerance test (oGTT) at 17wks of age (WT n=8, TG n=11, TG n=11). Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.


Physical activity of mice shown hourly over 24 - hrs as day night time . Data shown are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice at 17 - 18wks of ( WT n = 12 , TG n = 11 , TG n = 10 ) shown as means +

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

Physical activity of male mice shown hourly over 24-hrs and as day and night time. Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEM


energy expenditure of mice shown hourly over 24 - hrs as day night time . Data shown are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice at 17 - 18wks of ( WT n = 12 , TG n = 11 , TG n = 10 ) shown as means +

energy expenditure of male mice shown hourly over 24-hrs and as day and night time. Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEM


, total assimilated energy of mice shown hourly over 24 - hrs as day night time . Table showing mean values of assimilated energy ( kJ ) at day / night time per 24 - hrs . Energy balance calculated as delta of assimilated energy ( AE ) energy expenditure ( EE ) energy balance ( J ) per 24 - hrs . Data shown are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice at 17 - 18wks of ( WT n = 12 , TG n = 11 , TG n = 10 ) shown as means +

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

, total assimilated energy of male mice shown hourly over 24-hrs and as day and night time. Table showing mean values of assimilated energy (kJ) at day/night time and per 24-hrs. Energy balance calculated as delta of assimilated energy (AE) and energy expenditure (EE) and energy balance (J) per 24-hrs. Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEM