H . In the same experimental settings , populations of stimulated ( proliferating ) CD4 + T cells were identified cytofluorometrically and included in rectangles . Values ( as % of total population ) are shown in red in each representative plot identify and summed up in a graph as the means ± SD of n = 3 independent experiments . * p < 0 . 05 ; * * * 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

H. In the same experimental settings, populations of stimulated (proliferating) CD4+ T cells were identified cytofluorometrically and included in rectangles. Values (as % of total population) are shown in red in each representative plot identify and summed up in a graph as the means ± SD of n = 3 independent experiments. *p<0.05; ***p<0.001.


( C ) T2 - weighted MR images in coronal plane show abscess formation in the left lower pulmonary lobe ( upper panel ) and in spleen and left axilla ( lower panel ) .

O O O O O O O O O O O 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) T2-weighted MR images in coronal plane show abscess formation in the left lower pulmonary lobe (upper panel) and in spleen and left axilla (lower panel).


( D ) Histological sections of patient biopsies stained with hematoxylin and eosin ( left panel ) or with an actin antibody ( right panel ) . Scale bars : skin : left 1000µm , right 100µm ; lung : left 500µm , right 250µm ; spleen : left 1000µm , right 75µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histological sections of patient biopsies stained with hematoxylin and eosin (left panel) or with an actin antibody (right panel). Scale bars: skin: left 1000µm, right 100µm; lung: left 500µm, right 250µm; spleen: left 1000µm, right 75µm.


( A ) Expression of indicated proteins was determined by western blot in patient - derived and control fibroblasts and B cells and A549 OTULIN WT and KO cells . One representative out of three independent experiments 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

(A) Expression of indicated proteins was determined by western blot in patient-derived and control fibroblasts and B cells and A549 OTULIN WT and KO cells. One representative out of three independent experiments is shown.


( B ) FLAG - tagged tandem ubiquitin binding entity ( TUBE ) assay was performed to pull down linear ubiquitin linkages in patient - derived and control fibroblasts and B cells . One representative out of three independent experiments is shown .

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

(B) FLAG-tagged tandem ubiquitin binding entity (TUBE) assay was performed to pull down linear ubiquitin linkages in patient-derived and control fibroblasts and B cells. One representative out of three independent experiments is shown.


Biochemical characterization of recombinant OTULIN variants by means of differential scanning fluorimetry ( DSF ) A ) DSF measurements with OTULINWT , OTULINM86I or OTULINW167S . Melting temperatures ( Tm ) are calculated from five independent experiments with standard deviations . * , P = 0 . 011 ; * * * * , P = 6 . 64x10 - 18 , unpaired t - test .

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

Biochemical characterization of recombinant OTULIN variants by means of differential scanning fluorimetry (DSF) A) DSF measurements with OTULINWT, OTULINM86I or OTULINW167S. Melting temperatures (Tm) are calculated from five independent experiments with standard deviations. *, P = 0.011; ****, P = 6.64x10-18, unpaired t-test.


Biochemical characterization of recombinant OTULIN variants by means of surface plasmon resonance ( SPR ) measurements ( B ) SPR measurements and steady - state binding curves with calculated dissociation constants ( Kd ) after injection of a concentration series of OTULINC129A , OTULINC129A / M86I or OTULINC129A / W167S to CM5 - immobilized di - ubiquitin chains .

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

Biochemical characterization of recombinant OTULIN variants by means of surface plasmon resonance (SPR) measurements (B) SPR measurements and steady-state binding curves with calculated dissociation constants (Kd) after injection of a concentration series of OTULINC129A, OTULINC129A/M86I or OTULINC129A/W167S to CM5-immobilized di-ubiquitin chains.


( C ) Linear ubiquitin linkages isolated from A549 OTULIN KO cells by M1 TUBE assay were incubated with increasing concentrations of recombinant OTULINWT , OTULINM86I and OTULINW167S or catalytically inactive OTULINC129A as control for 1 hour . Afterwards , samples were subjected to analysis by western blot for the indicated proteins . Images are representative of three independent experiments .

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

(C) Linear ubiquitin linkages isolated from A549 OTULIN KO cells by M1 TUBE assay were incubated with increasing concentrations of recombinant OTULINWT, OTULINM86I and OTULINW167S or catalytically inactive OTULINC129A as control for 1 hour. Afterwards, samples were subjected to analysis by western blot for the indicated proteins. Images are representative of three independent experiments.


( A - C ) A549 OTULIN KO cells were transfected with empty vector or different OTULIN constructs as indicated and analyzed by western blot . ( D ) FLAG - tagged tandem ubiquitin binding entity ( TUBE ) assay was performed in B cells from control , patient , mother and father to pull down linear ubiquitin linkages . One representative ( A - D ) of three independent experiments 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O

(A - C) A549 OTULIN KO cells were transfected with empty vector or different OTULIN constructs as indicated and analyzed by western blot. (D) FLAG-tagged tandem ubiquitin binding entity (TUBE) assay was performed in B cells from control, patient, mother and father to pull down linear ubiquitin linkages. One representative (A - D) of three independent experiments is shown.


Patient - derived and control fibroblasts were used . ( A ) FLAG - tagged tandem ubiquitin binding entity ( TUBE ) assay was performed to pull down linear ubiquitin linkages upon stimulation with 500ng / ml TNF . ( B ) The TNFR1 - SC was isolated using 500ng / ml TAP - TNF . ( C ) Cells were stimulated with 100ng / ml TNF for the indicated times and analyzed by western blot . One representative ( A - C ) of three independent experiments 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 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

Patient-derived and control fibroblasts were used. (A) FLAG-tagged tandem ubiquitin binding entity (TUBE) assay was performed to pull down linear ubiquitin linkages upon stimulation with 500ng/ml TNF. (B) The TNFR1-SC was isolated using 500ng/ml TAP-TNF. (C) Cells were stimulated with 100ng/ml TNF for the indicated times and analyzed by western blot. One representative (A - C) of three independent experiments is shown.


( E ) B cells were incubated with 50µg / ml cycloheximide ( CHX ) for the indicated times and subjected to analysis by western blot . One representative ( left panel ) out of 4 individual experiments analyzed by densitometry ( right panel ) is shown . Data are presented as mean ± SD . * , P = 0 . 02 , unpaired t - test .

O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) B cells were incubated with 50µg/ml cycloheximide (CHX) for the indicated times and subjected to analysis by western blot. One representative (left panel) out of 4 individual experiments analyzed by densitometry (right panel) is shown. Data are presented as mean ± SD. *, P = 0.02, unpaired t-test.


( G ) Fibroblasts were incubated with 50µg / ml cycloheximide ( CHX ) or DMSO and stimulated with TNF as indicated . Viability was measured after 24 hours . Data are presented as mean ± SD of 5 individual experiments performed in three technical replicates ; * * * , P = 0 . 0008 , mixed - effects analysis with Tukey ' s multiple comparisons test .

O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fibroblasts were incubated with 50µg/ml cycloheximide (CHX) or DMSO and stimulated with TNF as indicated. Viability was measured after 24 hours. Data are presented as mean ± SD of 5 individual experiments performed in three technical replicates; ***, P = 0.0008, mixed-effects analysis with Tukey's multiple comparisons test.


Paneth cell quantification on Lgr5 - DTReGFP duodenum ( Duo ) and Ileum ( Ile ) . Left panel : Representative images of Lendrum ' s staining that evidence Paneth cell granules . Arrows show differentiated Paneth cells . Right panel : quantification of the Paneth cells per 10 intervilli regions . Each symbol indicates the value for a given embryo .

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

Paneth cell quantification on Lgr5-DTReGFP duodenum (Duo) and Ileum (Ile). Left panel: Representative images of Lendrum's staining that evidence Paneth cell granules. Arrows show differentiated Paneth cells. Right panel: quantification of the Paneth cells per 10 intervilli regions. Each symbol indicates the value for a given embryo.


Expression analysis by qRT - PCR of the indicated Paneth cell markers in Lgr5 - DTReGFP ileums ( n = 8 WT , 12 HE , 10 KO ) .

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

Expression analysis by qRT-PCR of the indicated Paneth cell markers in Lgr5-DTReGFP ileums (n= 8 WT, 12 HE, 10 KO).


X - gal staining in Axin2Lac / + - Lgr5 - GFP - CreERT2 WT or KO ileums .

X-gal staining in Axin2Lac/+-Lgr5-GFP-CreERT2 WT or KO ileums.


Gene expression analysis by qRT - PCR of stem cell markers in Lgr5 - DTReGFP ileums ( n = 3 WT , 6 HE , 6 KO ) .

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

Gene expression analysis by qRT-PCR of stem cell markers in Lgr5-DTReGFP ileums (n= 3 WT, 6 HE, 6 KO).


FACS quantification of the number of eGFP + ve ( ISC ) cells per small intestine at the indicated developmental stages in Lgr5 HEs and Lgr5 KOs . Each symbol indicates the value for a given embryo . Mean value for each group is depicted on the graph .

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

FACS quantification of the number of eGFP+ve (ISC) cells per small intestine at the indicated developmental stages in Lgr5 HEs and Lgr5 KOs. Each symbol indicates the value for a given embryo. Mean value for each group is depicted on the graph.


Plating efficiency of ex vivo cultured E18 . 5 small intestines quantified 6 days after initial seeding . Each symbol indicates the value for a given embryo .

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

Plating efficiency of ex vivo cultured E18.5 small intestines quantified 6 days after initial seeding. Each symbol indicates the value for a given embryo.


Influence of mouse Rspondin 1 concentration on growth of replated Lgr5 - DTReGFP WT and Lgr5 KO organoids after 5 days of culture .

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

Influence of mouse Rspondin 1 concentration on growth of replated Lgr5-DTReGFP WT and Lgr5 KO organoids after 5 days of culture.


Paneth cell quantification on Lgr5 - DTReGFP ileums . Each symbol indicates the value for a given embryo .

Paneth cell quantification on Lgr5-DTReGFP ileums. Each symbol indicates the value for a given embryo.


Immunofluorescence showing Olfm4 + ve cells in Lgr5 - DTReGFP duodenums . Cell membranes are shown with b - catenin and nuclei were counterstained with DAPI . Quantification of Olfm4 + ve cells per intervilli regions in duodenum and ileum . Each symbol indicates the value for a given embryo .

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

Immunofluorescence showing Olfm4+ve cells in Lgr5-DTReGFP duodenums. Cell membranes are shown with b-catenin and nuclei were counterstained with DAPI. Quantification of Olfm4+ve cells per intervilli regions in duodenum and ileum. Each symbol indicates the value for a given embryo.


Axin2 expression levels detected by RNAscope on Lgr5 - DTReGFP proximal intestine .

Axin2 expression levels detected by RNAscope on Lgr5-DTReGFP proximal intestine.


Gene expression analysis by qRT - PCR of stem cell and differentiation markers in Lgr5 - DTReGFP ileums ( vehicle - treated : 7 WT and 7 KO ; LGK974 - treated : 3 WT and 7 KO ) .

O O O O O 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

Gene expression analysis by qRT-PCR of stem cell and differentiation markers in Lgr5-DTReGFP ileums (vehicle-treated: 7 WT and 7 KO; LGK974-treated: 3 WT and 7 KO).


Representative pictures of lineage tracing in Lgr5 - expressing or Lgr5 cKO intestine after 10 days of chase and quantification of the number of traced clones per recombined surface at PN18 ( ileum ) . Each symbol indicates the value for a given mouse .

O O O O O O O O O O O O O O O O O O O O O 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 pictures of lineage tracing in Lgr5-expressing or Lgr5 cKO intestine after 10 days of chase and quantification of the number of traced clones per recombined surface at PN18 (ileum). Each symbol indicates the value for a given mouse.


Representative immunofluorescence pictures showing Paneth cells ( Plyz + ve ) in traced clones ( RFP + ve ) from control ( Lgr5Cre / + ) and cKO ( Lgr5Cre / flox ) PN18 and adult ileums . Arrow heads point to Plyz + ve cells in traced clones . Quantification of the number of Paneth cell number per recombined RFP + ve crypt on control and cKO ileums in PN18 . Each symbol indicates the value for a given mouse .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunofluorescence pictures showing Paneth cells (Plyz+ve) in traced clones (RFP+ve) from control (Lgr5Cre/+) and cKO (Lgr5Cre/flox) PN18 and adult ileums. Arrow heads point to Plyz+ve cells in traced clones. Quantification of the number of Paneth cell number per recombined RFP+ve crypt on control and cKO ileums in PN18. Each symbol indicates the value for a given mouse.


ISC ( eGFP + ve ) cells were FACS - sorted from Lgr5 - DTReGFP embryos at E16 . 5 and subjected to RNAseq analysis . Heatmap of differentially regulated genes in HE and KO ISCs at E16 . 5 . Biological replicates for RNAseq experiments on ISC Lgr5 - DTReGFP E16 . 5 independent embryonic pools , each obtained from independent litters ( n = 2 HE , 2 KO )

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

ISC (eGFP+ve) cells were FACS-sorted from Lgr5-DTReGFP embryos at E16.5 and subjected to RNAseq analysis. Heatmap of differentially regulated genes in HE and KO ISCs at E16.5. Biological replicates for RNAseq experiments on ISC Lgr5-DTReGFP E16.5 independent embryonic pools, each obtained from independent litters (n= 2 HE, 2 KO)


Compared Hallmarks for upregulated and downregulated genes in the transcriptome of E16 . 5 Lgr5 KOs vs E16 . 5 HEs , adult HEs vs E18 . 5 HE embryos and adult Lgr5 - Cre cKO vs HEs . * Asterisks refer to given pathways for which differentially modulated genes were found upregulated and downregulated . Right panels : GSEA showing enrichment of the epithelial to mesenchymal transition ( EMT ) data set in E16 . 5 Lgr5 KOs vs E16 . 5 HEs , adult HEs vs E18 . 5 HE embryos , and adult Lgr5 cKO vs adult HE modulated genes . NES : Normalized Enrichment Score . Biological replicates for RNAseq experiments on ISC Lgr5 - DTReGFP E16 . 5 independent embryonic pools , each obtained from independent litters ( n = 2 HE , 2 KO ) ; Lgr5 - DTReGFP E18 . 5 individual embryos ( n = 2 HE ) and Lgr5 - DTReGFP adult individual animals ( n = 2 HE ) ; Lgr5 - Cre / flox adult individual animals ( n = 2 HE , 3 cKO ) .

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

Compared Hallmarks for upregulated and downregulated genes in the transcriptome of E16.5 Lgr5 KOs vs E16.5 HEs, adult HEs vs E18.5 HE embryos and adult Lgr5-Cre cKO vs HEs. * Asterisks refer to given pathways for which differentially modulated genes were found upregulated and downregulated. Right panels: GSEA showing enrichment of the epithelial to mesenchymal transition (EMT) data set in E16.5 Lgr5 KOs vs E16.5 HEs, adult HEs vs E18.5 HE embryos, and adult Lgr5 cKO vs adult HE modulated genes. NES: Normalized Enrichment Score. Biological replicates for RNAseq experiments on ISC Lgr5-DTReGFP E16.5 independent embryonic pools, each obtained from independent litters (n= 2 HE, 2 KO); Lgr5-DTReGFP E18.5 individual embryos (n=2 HE) and Lgr5-DTReGFP adult individual animals (n=2 HE); Lgr5-Cre/flox adult individual animals (n= 2 HE, 3 cKO).


Expression levels of genes commonly downregulated in ISCs of Lgr5 KOs vs Lgr5 HEs at E16 . 5 and Lgr5 adult vs E18 . 5 HEs . RPKM : reads per kilobase of transcript per million mapped reads . Below : GeneOntology analysis of downregulated genes identified in the Epithelial Mesenchymal Transition Gene Set ( Hallmark MSigDB collection ) . Biological replicates for RNAseq experiments on ISC Lgr5 - DTReGFP E16 . 5 independent embryonic pools , each obtained from independent litters ( n = 2 HE , 2 KO ) ; Lgr5 - DTReGFP E18 . 5 individual embryos ( n = 2 HE ) and Lgr5 - DTReGFP adult individual animals ( n = 2 HE ) ; Lgr5 - Cre / flox adult individual animals ( n = 2 HE , 3 cKO ) . Data are represented as means ± sem ( C ) .

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

Expression levels of genes commonly downregulated in ISCs of Lgr5 KOs vs Lgr5 HEs at E16.5 and Lgr5 adult vs E18.5 HEs. RPKM: reads per kilobase of transcript per million mapped reads. Below: GeneOntology analysis of downregulated genes identified in the Epithelial Mesenchymal Transition Gene Set (Hallmark MSigDB collection). Biological replicates for RNAseq experiments on ISC Lgr5-DTReGFP E16.5 independent embryonic pools, each obtained from independent litters (n= 2 HE, 2 KO); Lgr5-DTReGFP E18.5 individual embryos (n=2 HE) and Lgr5-DTReGFP adult individual animals (n=2 HE); Lgr5-Cre/flox adult individual animals (n= 2 HE, 3 cKO). Data are represented as means ± sem (C).


Expression profile of Col1a1 , Zeb2 and Cxcl10 genes detected by RNAscope in embryonic and adult intestines . Insets with higher magnification are depicted on the right of each corresponding picture , boundaries between epithelium and mesenchyme are shown with dotted lines and arrows point to epithelial expression .

Expression profile of Col1a1, Zeb2 and Cxcl10 genes detected by RNAscope in embryonic and adult intestines. Insets with higher magnification are depicted on the right of each corresponding picture, boundaries between epithelium and mesenchyme are shown with dotted lines and arrows point to epithelial expression.


Expression of Axin2 , Lgr5 , Lgr4 , Rspo1 , Rspo2 , Rspo3 and Rspo4 genes was analyzed on E15 , E16 . 5 and adult stages by RNAscope . Arrows evidence expressing cells .

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

Expression of Axin2, Lgr5, Lgr4, Rspo1, Rspo2, Rspo3 and Rspo4 genes was analyzed on E15, E16.5 and adult stages by RNAscope. Arrows evidence expressing cells.


Representative pictures showing the influence of mouse Rspondin type and concentration on growth of replated Lgr5 - DTReGFP WT and Lgr5 KO organoids after 4 days of culture . Arrows show alive elements and arrow heads dead elements .

O O O O O O O O O O O O O O O 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 pictures showing the influence of mouse Rspondin type and concentration on growth of replated Lgr5-DTReGFP WT and Lgr5 KO organoids after 4 days of culture. Arrows show alive elements and arrow heads dead elements.


Organoid morphology depends on Rspondin ligand type and concentration . Left panel : growth quantified as the percentage of elements present at day1 surviving at day4 . A mean of 100 elements were analyzed per sample and per condition ( n : 4 WT , 2 KO organoid culture samples , each originating from a given embryo ) . Right panel : organoid complexity measured by branching coefficient . Each symbol indicates the value of a given organoid ( analysis was done on 4 WT and 2 KO organoid culture samples , each originating from a given embryo ) .

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

Organoid morphology depends on Rspondin ligand type and concentration. Left panel: growth quantified as the percentage of elements present at day1 surviving at day4. A mean of 100 elements were analyzed per sample and per condition (n: 4 WT, 2 KO organoid culture samples, each originating from a given embryo). Right panel: organoid complexity measured by branching coefficient. Each symbol indicates the value of a given organoid (analysis was done on 4 WT and 2 KO organoid culture samples, each originating from a given embryo).


Heatmap showing the impact of Rspondin type and concentration ( ng / ml ) on relative Wnt target gene expression ( list from the Sansom APC data set ) in Lgr5 - DTReGFP WT and KO organoids analyzed by RNAseq ( n = 3 WT and 2 organoid culture samples , each originating from a given embryo ) .

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

Heatmap showing the impact of Rspondin type and concentration (ng/ml) on relative Wnt target gene expression (list from the Sansom APC data set) in Lgr5-DTReGFP WT and KO organoids analyzed by RNAseq (n= 3 WT and 2 organoid culture samples, each originating from a given embryo).


Gene expression analysis by qRT - PCR of stem cell markers in Lgr5 - DTREeGFP WT and KO organoids after 5 days of culture ( expression relative to reference genes ) . Each symbol indicates the value for an organoid culture originated from a given embryo .

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

Gene expression analysis by qRT-PCR of stem cell markers in Lgr5-DTREeGFP WT and KO organoids after 5 days of culture (expression relative to reference genes). Each symbol indicates the value for an organoid culture originated from a given embryo.


Immunofluorescence showing Olfm4 + ve cells in Lgr5 - DTReGFP WT and KO organoids cultured in Rspo2 conditions . Epithelium is delineated with b - catenin and nuclei counterstained with DAPI ( Merge ) . Insets : white arrowheads evidence Olfm4 + ve cells concentrated in organoid protrusions . Quantification of Olfm4 + ve area with respect to the total epithelial area . Each symbol indicates the value for an organoid culture originating from a given embryo .

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

Immunofluorescence showing Olfm4+ve cells in Lgr5-DTReGFP WT and KO organoids cultured in Rspo2 conditions. Epithelium is delineated with b-catenin and nuclei counterstained with DAPI (Merge). Insets: white arrowheads evidence Olfm4+ve cells concentrated in organoid protrusions. Quantification of Olfm4+ve area with respect to the total epithelial area. Each symbol indicates the value for an organoid culture originating from a given embryo.


( A ) PI3Kγ is expressed by human hepatocytes and infiltrating immune cells in biopsies from patients with minimal to mild inflammatory activity . Triangles point to immune cells ( clusters ) , including some neutrophils known to express PI3Kγ highly . In the negative control , the primary antibody was replaced by an equal volume buffer . The number of included patients , gender , and diagnosis are summarized

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PI3Kγ is expressed by human hepatocytes and infiltrating immune cells in biopsies from patients with minimal to mild inflammatory activity. Triangles point to immune cells (clusters), including some neutrophils known to express PI3Kγ highly. In the negative control, the primary antibody was replaced by an equal volume buffer. The number of included patients, gender, and diagnosis are summarized


( A ) PI3Kγ is expressed by human hepatocytes and infiltrating immune cells in biopsies from patients with minimal to mild inflammatory activity . Triangles point to immune cells ( clusters ) , including some neutrophils known to express PI3Kγ highly . In the negative control , the primary antibody was replaced by an equal volume buffer . The number of included patients , gender , and diagnosis are

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) PI3Kγ expression in human primary hepatocytes from 20 male ( ♂ ) or female ( ♀ ) donor pools ( HEP , DP20 ) , but not non - parenchymal cells ( NPC ) . Isolated human leukocytes from healthy volunteers ( LEU ) served as positive controls . The characterization of the donor pools is summarized

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PI3Kγ expression in human primary hepatocytes from 20 male (♂) or female (♀) donor pools (HEP, DP20), but not non-parenchymal cells (NPC). Isolated human leukocytes from healthy volunteers (LEU) served as positive controls. The characterization of the donor pools is summarized


( B ) PI3Kγ expression in human primary hepatocytes from 20 male ( ♂ ) or female ( ♀ ) donor pools ( HEP , DP20 ) , but not non - parenchymal cells ( NPC ) . Isolated human leukocytes from healthy volunteers ( LEU ) served as positive controls . The characterization of the donor pools is


( D ) Survival of WT , PI3Kγ null ( left ) and liver - specific PI3Kγ knockout mice ( PI3Kγ floxtg / tg x AlbCre ( tg ) / tg , middle ) or systemic application of the PI3Kγ inhibitor , AS605240 ( right ) in a model of polymicrobial sepsis induced by peritoneal contamination with a human stool suspension . Animal numbers : B6 : 86 ( % male : 52 , % female : 48 ) , B6 - Pi3kcgko / ko : 59 ( % male : 51 , % female 49 ) , B6 - Pi3kcgflox / flox x AlbCre ( tg ) / tg : 25 ( % male : 68 , % female 32 ) , FVB / N Vehicle : 25 ( % male : 44 , % female 56 ) , FVB / N AS : 43 ( % male : 44 , % female 56 )

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

(D) Survival of WT, PI3Kγ null (left) and liver-specific PI3Kγ knockout mice (PI3Kγ floxtg/tg x AlbCre(tg)/tg, middle) or systemic application of the PI3Kγ inhibitor, AS605240 (right) in a model of polymicrobial sepsis induced by peritoneal contamination with a human stool suspension. Animal numbers: B6: 86 (%male: 52, %female: 48), B6-Pi3kcgko/ko: 59 (%male: 51, %female 49), B6-Pi3kcgflox/flox x AlbCre(tg)/tg: 25 (%male: 68, %female 32), FVB/N Vehicle: 25 (%male: 44, %female 56), FVB/N AS: 43 (%male: 44, %female 56)


( C ) Uptake characteristics of T - LipoAS by primary human hepatocytes demonstrate competitive inhibition by cyclosporin A - and energy dependence , confirming active uptake by organic anion transporters ( OATPs ) . The data points are depicted mean ± SD from two batches of primary human hepatocytes in quadruples .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-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) Uptake characteristics of T-LipoAS by primary human hepatocytes demonstrate competitive inhibition by cyclosporin A- and energy dependence, confirming active uptake by organic anion transporters (OATPs). The data points are depicted mean ± SD from two batches of primary human hepatocytes in quadruples.


( F ) T - LipoAS effectively inhibits fMLP - stimulated PI3Kγ , reflected by inhibiting AKT S473 phosphorylation activity in hepatocytes from three different batchtes . The mean pAKT / AKT ratio and standard deviation ( SD ) were calculated for all experiments separately .

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 O O O

(F) T-LipoAS effectively inhibits fMLP-stimulated PI3Kγ, reflected by inhibiting AKT S473 phosphorylation activity in hepatocytes from three different batchtes. The mean pAKT/AKT ratio and standard deviation (SD) were calculated for all experiments separately.


( G ) Episcopic imaging of mice treated with T - LipoAS for 15 or 60 min and relative concentration of the targeting moiety DY - 635 in the different compartments .

O O O O O O O O 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) Episcopic imaging of mice treated with T-LipoAS for 15 or 60 min and relative concentration of the targeting moiety DY-635 in the different compartments.


( B Pro - anti - inflammatory cytokines were analyzed in EDTA - plasma from peritoneal contamination and infection ( PCI ) and sham animals . # p < 0 . 05 against sham , * p < 0 . 05 as indicated ; Kruskal - Wallis ANOVA with controlled false - discovery rates ( Benjamini - Hochberg procedure ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Pro- anti-inflammatory cytokines were analyzed in EDTA-plasma from peritoneal contamination and infection (PCI) and sham animals. #p<0.05 against sham, *p<0.05 as indicated; Kruskal-Wallis ANOVA with controlled false-discovery rates (Benjamini-Hochberg procedure).


C ) anti - inflammatory cytokines were analyzed in EDTA - plasma from peritoneal contamination and infection ( PCI ) and sham animals . # p < 0 . 05 against sham , * p < 0 . 05 as indicated ; Kruskal - Wallis ANOVA with controlled false - discovery rates ( Benjamini - Hochberg procedure ) .

C) anti-inflammatory cytokines were analyzed in EDTA-plasma from peritoneal contamination and infection (PCI) and sham animals. #p<0.05 against sham, *p<0.05 as indicated; Kruskal-Wallis ANOVA with controlled false-discovery rates (Benjamini-Hochberg procedure).


( F , G ) Concentration of ( F ) neutrophils and ( G ) monocytes in 2 mL peritoneal lavage .

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

(F,G) Concentration of (F) neutrophils and (G) monocytes in 2 mL peritoneal lavage.


( A ) Intravital microscopy of elimination of CDFDA , a fluorescent substrate subject to excretory elimination by hepatocytes via Mrp - 2 , allows direct visualization of excretory function in sham or septic ( PCI ) animals treated with vehicle or a hepatocyte - directed inhibitor of PI3Kγ ( T - LipoAS ) .

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

(A) Intravital microscopy of elimination of CDFDA, a fluorescent substrate subject to excretory elimination by hepatocytes via Mrp-2, allows direct visualization of excretory function in sham or septic (PCI) animals treated with vehicle or a hepatocyte-directed inhibitor of PI3Kγ (T-LipoAS).


a - e , Scanning electron microscopy ( SEM ) images of fly eyes expressing DTS7 with or without the indicated HDAC6 transgenes . Lower panels , magnification of ommatidia . a , Normal eyes in DTS7 flies reared at 22 ° C . b , Rough eyes in DTS7 flies reared at 28 ° C . c , d , Degeneration was suppressed by expression of Drosophila HDAC6 ( dHDAC6 ; c ) or human HDAC6 ( hHDAC6 ; d ) , but not a catalytically dead mutant of hHDAC6 ( cat . mut . ; e ) .

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

a-e, Scanning electron microscopy (SEM) images of fly eyes expressing DTS7 with or without the indicated HDAC6 transgenes. Lower panels, magnification of ommatidia. a, Normal eyes in DTS7 flies reared at 22 °C. b, Rough eyes in DTS7 flies reared at 28 °C. c, d, Degeneration was suppressed by expression of Drosophila HDAC6 (dHDAC6; c) or human HDAC6 (hHDAC6; d), but not a catalytically dead mutant of hHDAC6 (cat. mut.; e).


f - j , SEM images of fly eyes expressing AR52 with or without the indicated HDAC6 transgenes . Lower panels , magnification of ommatidia . f , Normal eyes in AR52 flies reared without ( - ) DHT . g , Rough eyes in AR52 flies reared with ( + ) DHT . Degeneration was suppressed by expression of Drosophila HDAC6 ( h ) or human HDAC6 ( i ) , but not a catalytically dead mutant of human HDAC6 ( j ) .

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

f-j, SEM images of fly eyes expressing AR52 with or without the indicated HDAC6 transgenes. Lower panels, magnification of ommatidia. f, Normal eyes in AR52 flies reared without (-) DHT. g, Rough eyes in AR52 flies reared with (+) DHT. Degeneration was suppressed by expression of Drosophila HDAC6 (h) or human HDAC6 (i), but not a catalytically dead mutant of human HDAC6 (j).


k - p , Detection of UPS reporter in imaginal eye discs from third - instar larvae by confocal microscopy . High level fluorescence was found in flies expressing GFP ( k , positive control ) , but fluorescence was barely detectable in control flies expressing CL1 - GFP ( l , negative control ) . CL1 - GFP accumulates in DTS7flies with temperature - dependent proteasome impairment ( compare m to n ) and in AR52flies with ligand - dependent degeneration ( compare o to p ) . The retinal phenotypes of 200 to > 1 , 000 flies of each genotype were examined . Quantitative analyses of eye phenotypes and proteasome impairment are presented in Supplementary Figs S2 and S3 , respectively . ( DHT , dihydrotestosterone . )

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

k-p, Detection of UPS reporter in imaginal eye discs from third-instar larvae by confocal microscopy. High level fluorescence was found in flies expressing GFP (k, positive control), but fluorescence was barely detectable in control flies expressing CL1-GFP (l, negative control). CL1-GFP accumulates in DTS7flies with temperature-dependent proteasome impairment (compare m to n) and in AR52flies with ligand-dependent degeneration (compare o to p). The retinal phenotypes of 200 to >1,000 flies of each genotype were examined. Quantitative analyses of eye phenotypes and proteasome impairment are presented in Supplementary Figs S2 and S3, respectively. (DHT, dihydrotestosterone.)


a - e , Representative examples of autophagic vacuoles detected by TEM in retinal sections used to generate the quantitative data shown in ( f ) . a , An autophagosome ( red arrow ) containing cytoplasmic contents in a photoreceptor neuron from an AR52fly reared on DHT . b , Higher magnification of the autophagosome in a . c , Multiple autophagolysosomes ( red arrows ) containing dense , amorphous material from an AR52fly reared on DHT . d , A juxtanuclear multilamellar body ( red arrow ) from a DTS7fly reared at 28 ° C . e , A multivesicular body ( red arrow ) from a DTS7fly reared at 28 ° C . f , A significant increase in the frequency of neurons with autophagic figures in DTS7 flies reared at 28 ° C compared to those reared at 22 ° C , and in AR52 flies reared on DHT compared to those reared off DHT . Data show mean ± s . d . , n = 59 - 82 neurons in 5 sections per condition . No accumulation of autophagic figures was found in AR12 flies .

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

a-e, Representative examples of autophagic vacuoles detected by TEM in retinal sections used to generate the quantitative data shown in (f). a, An autophagosome (red arrow) containing cytoplasmic contents in a photoreceptor neuron from an AR52fly reared on DHT. b, Higher magnification of the autophagosome in a. c, Multiple autophagolysosomes (red arrows) containing dense, amorphous material from an AR52fly reared on DHT. d, A juxtanuclear multilamellar body (red arrow) from a DTS7fly reared at 28 °C. e, A multivesicular body (red arrow) from a DTS7fly reared at 28 °C. f, A significant increase in the frequency of neurons with autophagic figures in DTS7 flies reared at 28 °C compared to those reared at 22 °C, and in AR52 flies reared on DHT compared to those reared off DHT. Data show mean ± s.d., n = 59-82 neurons in 5 sections per condition. No accumulation of autophagic figures was found in AR12 flies.


g - l , SEM images of fly eyes expressing the indicated transgenes . RNAi knockdown ( KD ) of atg6 and atg12 enhances degeneration in DTS7 flies reared at 28 ° C ( compare h , i to g ) and AR52 flies reared on DHT ( compare k , l to j ) . 200 to > 1 , 000 fly eyes of each genotype were examined . Quantitative analyses of eye phenotypes are presented in Supplementary Fig . S2 . ( N , nucleus ; Rh , rhabdomere . )

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

g-l, SEM images of fly eyes expressing the indicated transgenes. RNAi knockdown (KD) of atg6 and atg12 enhances degeneration in DTS7 flies reared at 28 °C (compare h, i to g) and AR52 flies reared on DHT (compare k, l to j). 200 to >1,000 fly eyes of each genotype were examined. Quantitative analyses of eye phenotypes are presented in Supplementary Fig. S2. (N, nucleus; Rh, rhabdomere.)


a , b , d , Western blots from flies expressing the indicated transgenes . a , Steady state levels of AR52 protein are reduced in flies overexpressing Drosophila HDAC6 , but are elevated in flies in which atg6 or atg12 has been knocked down . b , Western blots showing the temporal profile of AR52 protein monomer and high molecular weight aggregate levels after a brief pulse of expression . AR52 protein became detectable by 2 . 5 h after treatment with RU486 , reached a peak at 10 h , and then slowly decayed . c , A logarithmic plot of AR52 / actin ratios was used to determine the line of best fit by regression analysis ( y = Ae - Kx ) . R2 = 0 . 9117 ( AR52 - DHT ) , R2 = 0 . 7808 ( AR52 + DHT ) , R2 = 0 . 9719 ( HDAC6 + ( AR52 - DHT ) ) , R2 = 0 . 9644 ( HDAC6 + ( AR52 + DHT ) ) . Half - life was determined by the slope of the best fit line with the equation t1 / 2 = 0 . 693 / K . Half - life of AR52 in vivo was reduced ∼ 2 - fold in flies co - expressing Drosophila HDAC6 and did not differ significantly in the presence ( broken lines ) or absence ( solid lines ) of DHT . Plots of the mean AR52 / actin ratios are shown in Supplementary Fig . S12 . d , Flies co - expressing Drosophila HDAC6 showed a nearly identical profile of induced expression as in b , but AR protein decayed at an accelerated rate . Exogenous Drosophila HDAC6 was detected by immunoblot against the V5 epitope .

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

a, b, d, Western blots from flies expressing the indicated transgenes. a, Steady state levels of AR52 protein are reduced in flies overexpressing Drosophila HDAC6, but are elevated in flies in which atg6 or atg12 has been knocked down. b, Western blots showing the temporal profile of AR52 protein monomer and high molecular weight aggregate levels after a brief pulse of expression. AR52 protein became detectable by 2.5 h after treatment with RU486, reached a peak at 10 h, and then slowly decayed. c, A logarithmic plot of AR52/actin ratios was used to determine the line of best fit by regression analysis (y = Ae-Kx). R2 = 0.9117 (AR52 - DHT), R2 = 0.7808 (AR52 + DHT), R2 = 0.9719 (HDAC6 + (AR52 - DHT)), R2 = 0.9644 (HDAC6 + (AR52 + DHT)). Half-life was determined by the slope of the best fit line with the equation t1/2 = 0.693/K. Half-life of AR52 in vivo was reduced ∼2-fold in flies co-expressing Drosophila HDAC6 and did not differ significantly in the presence (broken lines) or absence (solid lines) of DHT. Plots of the mean AR52/actin ratios are shown in Supplementary Fig. S12. d, Flies co-expressing Drosophila HDAC6 showed a nearly identical profile of induced expression as in b, but AR protein decayed at an accelerated rate. Exogenous Drosophila HDAC6 was detected by immunoblot against the V5 epitope.


a - l , SEM images of fly eyes expressing the indicated transgenes . The rough eye phenotypes caused by proteasome mutation ( a ) or by expression of polyQ - expanded AR ( b ) were both suppressed by rearing flies on the TOR inhibitor rapamycin ( c , d ) . e , f , Rapamycin failed to suppress degeneration in an autophagy - deficient background created by knockdown of atg12 , confirming that rescue by rapamycin is autophagy - dependent . g , h , Rapamycin also failed to suppress degeneration when HDAC6 levels were knocked down , demonstrating that suppression via the TOR pathway is HDAC6 - dependent . k , j , HDAC6 failed to suppress degeneration in an autophagy - deficient background , confirming that rescue by HDAC6 is dependent on autophagy ( compare k , l to i , j ) . 200 to > 1 , 000 fly eyes of each genotype were examined . Quantitative analyses of eye phenotypes are presented in Supplementary Fig . S2 .

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

a-l, SEM images of fly eyes expressing the indicated transgenes. The rough eye phenotypes caused by proteasome mutation (a) or by expression of polyQ-expanded AR (b) were both suppressed by rearing flies on the TOR inhibitor rapamycin (c, d). e, f, Rapamycin failed to suppress degeneration in an autophagy-deficient background created by knockdown of atg12, confirming that rescue by rapamycin is autophagy-dependent. g, h, Rapamycin also failed to suppress degeneration when HDAC6 levels were knocked down, demonstrating that suppression via the TOR pathway is HDAC6-dependent. k, j, HDAC6 failed to suppress degeneration in an autophagy-deficient background, confirming that rescue by HDAC6 is dependent on autophagy (compare k, l to i, j). 200 to >1,000 fly eyes of each genotype were examined. Quantitative analyses of eye phenotypes are presented in Supplementary Fig. S2.


B . Survival frequencies observed after DSB induction at TEL6R in WT , dnl4Δ , sir4Δ cells , expressing or not high levels of Sir3p ( oeSir3 and WT respectively ) . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . C . Survival frequencies observed after DSB induction at LYS2 in the indicated strains . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Survival frequencies observed after DSB induction at TEL6R in WT, dnl4Δ, sir4Δ cells, expressing or not high levels of Sir3p (oeSir3 and WT respectively). Error bars indicate survival standard error (SEM) of at least three independent experiments. C. Survival frequencies observed after DSB induction at LYS2 in the indicated strains. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


F . Representative images of Mre11 - YFP foci in response to an I - SceI - induced DSB at LYS2 in WT cells , expressing or not high levels of Sir3p ( oeSir3 and WT respectively ) . Scale bars are 2 μm . G . Quantification of cells with DSB induced Mre11 - YFP foci after DSB induction at LYS2 I - SceI cleavage site in WT , sae2Δ and Sir3 overexpressing ( oeSir3 ) strains . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of Mre11-YFP foci in response to an I-SceI-induced DSB at LYS2 in WT cells, expressing or not high levels of Sir3p (oeSir3 and WT respectively). Scale bars are 2 μm. G. Quantification of cells with DSB induced Mre11-YFP foci after DSB induction at LYS2 I-SceI cleavage site in WT, sae2Δ and Sir3 overexpressing (oeSir3) strains. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


H . Survival frequencies after DSB induction at LYS2 locus , in strains where SIR3 is expressed from its native , pADH1 or pGPD promoters respectively and in which SAE2 is expressed or not from a high copy number 2µ plasmid . Fold increase in Sir3 protein by pAHD1 or pGPD is indicated . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Survival frequencies after DSB induction at LYS2 locus, in strains where SIR3 is expressed from its native, pADH1 or pGPD promoters respectively and in which SAE2 is expressed or not from a high copy number 2µ plasmid. Fold increase in Sir3 protein by pAHD1 or pGPD is indicated. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


A . Representative images of Sir3 - mCherry and Sae2 - GFP signal in WT and SIR3 overexpressing cells . Scale bars are 2 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

A. Representative images of Sir3-mCherry and Sae2-GFP signal in WT and SIR3 overexpressing cells. Scale bars are 2 μm.


B . Sir3 - binding at TEL6R in untagged , WT , sir3Δ cells or in cells overexpressing Sir3 ( oeSir3 ) . Binding is probed by ChIP - qPCR 0 . 2 ( red arrows ) and 1kb ( blue arrows ) from telomeres and at the OGG1 control locus using antibodies against Sae2 - GFP . The mean of three independent biological replicates is shown and error bars correspond to the variation between replicates ( SEM ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Sir3-binding at TEL6R in untagged, WT, sir3Δ cells or in cells overexpressing Sir3 (oeSir3). Binding is probed by ChIP-qPCR 0.2 (red arrows) and 1kb (blue arrows) from telomeres and at the OGG1 control locus using antibodies against Sae2-GFP. The mean of three independent biological replicates is shown and error bars correspond to the variation between replicates (SEM).


C . Co - immunoprecipitation between Sir3 and Sae2 - GFP from cells overexpressing Sir3 , analysed by Western blot with anti - GFP and anti - Sir3 antibodies . D . Co - immunoprecipitation between Sir3 and Sae2 - GFP from WT cells using antibodies against Sae2 - GFP , analysed by Western blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Co-immunoprecipitation between Sir3 and Sae2-GFP from cells overexpressing Sir3, analysed by Western blot with anti-GFP and anti-Sir3 antibodies. D. Co-immunoprecipitation between Sir3 and Sae2-GFP from WT cells using antibodies against Sae2-GFP, analysed by Western blot.


D . Yeast two - hybrid interaction analysis between Sae2C and Sir3SaID domains in WT or sir4Δ cells . Growth on - His + 3AT and blue coloration on X - gal indicate an interaction .

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

D. Yeast two-hybrid interaction analysis between Sae2C and Sir3SaID domains in WT or sir4Δ cells. Growth on -His + 3AT and blue coloration on X-gal indicate an interaction.


B . Representative images of two hybrid assays in the yKD1991 strain testing the interaction of the WT or the mutant SIR3SaID fragment isolated from the screen with SAE2C or SIR4C . C . Representative images of two hybrid assays testing the interaction of the WT or the mutant SIR3SaIDT557I fragment with SAE2C or SIR4C .

B. Representative images of two hybrid assays in the yKD1991 strain testing the interaction of the WT or the mutant SIR3SaID fragment isolated from the screen with SAE2C or SIR4C. C. Representative images of two hybrid assays testing the interaction of the WT or the mutant SIR3SaIDT557I fragment with SAE2C or SIR4C.


A . Survival frequencies after DSB induction at LYS2 locus in WT or sae2Δ strains where the Sir3SaID or Sir3SaIDT557I domains are overexpressed from a GPD promoter at the SIR3 locus . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Survival frequencies after DSB induction at LYS2 locus in WT or sae2Δ strains where the Sir3SaID or Sir3SaIDT557I domains are overexpressed from a GPD promoter at the SIR3 locus. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


B . Representative images of Sae2 - GFP in WT cells and in cells overexpressing either full - length Sir3 or the sir3SaID domain . Scale bars are 2 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

B. Representative images of Sae2-GFP in WT cells and in cells overexpressing either full-length Sir3 or the sir3SaID domain. Scale bars are 2 μm.


F . Representative silver - stained gels of in vitro GST - pulldown of GST or GST - Sir3SaID , GST - sir3 - T557ISaID and Sae2C purified peptides . Control : Sae2C ( 300 ng , lane 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 O O

F. Representative silver-stained gels of in vitro GST-pulldown of GST or GST-Sir3SaID, GST-sir3-T557ISaID and Sae2C purified peptides. Control: Sae2C (300 ng, lane 6).


G . Co - immunoprecipitation between Sae2 - GFP and Sir3 from untagged , Sae2 - GFP WT cells , and Sae2 - GFP cells overexpressing WT Sir3 ( oeSir3 , WT ) , Sae2 - GFP sir3Δ or Sae2 - GFP overexpressing the sir3 - T557I mutant ( oeSir3 , T557I ) using antibodies against Sae2 - GFP , analysed by Western blot with anti - GFP and anti - Sir3 antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Co-immunoprecipitation between Sae2-GFP and Sir3 from untagged, Sae2-GFP WT cells, and Sae2-GFP cells overexpressing WT Sir3 (oeSir3, WT), Sae2-GFP sir3Δ or Sae2-GFP overexpressing the sir3-T557I mutant (oeSir3, T557I) using antibodies against Sae2-GFP, analysed by Western blot with anti-GFP and anti-Sir3 antibodies.


A . Survival frequencies after DSB induction at LYS2 locus in the indicated strains . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

A. Survival frequencies after DSB induction at LYS2 locus in the indicated strains. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


C . Survival frequencies after DSB induction at LYS2 locus in the indicated strains . Insertion of the strong TEF1p promoter upstream of the SIR4 ORF leads to Sir4 overexpression . Insertion of the ADH1p promoter upstream of SIR3 leads to mild Sir3 overexpression . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Survival frequencies after DSB induction at LYS2 locus in the indicated strains. Insertion of the strong TEF1p promoter upstream of the SIR4 ORF leads to Sir4 overexpression. Insertion of the ADH1p promoter upstream of SIR3 leads to mild Sir3 overexpression. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


E . Representative images of two hybrid assays testing the interaction between the full - length Sir3 and full - length Sae2 proteins in WT cells expressing or not high levels of Sir4 ( oeSir4 and WT respectively ) .

O O O O O O O O O O O O O O O O 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. Representative images of two hybrid assays testing the interaction between the full-length Sir3 and full-length Sae2 proteins in WT cells expressing or not high levels of Sir4 (oeSir4 and WT respectively).


A . Relative fold enrichment of Sae2 at 0 . 2 kb from I - SceI site was evaluated by qPCR after ChIP with anti - GFP antibodies . The error bars indicate the variation between at least three biological replicas ( SEM ) . B . Relative fold enrichment of Sae2 at 0 . 2 kb from TEL6R after DSB induction at the LYS2 I - SceI site was evaluated by qPCR after ChIP with anti - GFP antibodies . The error bars indicate the variation between at least three biological replicas ( SEM ) . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

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

A. Relative fold enrichment of Sae2 at 0.2 kb from I-SceI site was evaluated by qPCR after ChIP with anti-GFP antibodies. The error bars indicate the variation between at least three biological replicas (SEM). B. Relative fold enrichment of Sae2 at 0.2 kb from TEL6R after DSB induction at the LYS2 I-SceI site was evaluated by qPCR after ChIP with anti-GFP antibodies. The error bars indicate the variation between at least three biological replicas (SEM). Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


D . Survival frequencies after DSB induction at LYS2 locus in sae2 - E267E mutant cells overexpressing or not full - length Sir3 ( oeSir3 ) . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Survival frequencies after DSB induction at LYS2 locus in sae2-E267E mutant cells overexpressing or not full-length Sir3 (oeSir3). Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


E . Survival curves of mice challenged with SARS - CoV - 2 Wuhan - D614G at 104 PFU , after a primo - infection by BANAL - 236 or Wuhan - 372 viruses at 103 or 104 PFU . Survival of K18 - hACE2 naive mice infected by SARS - CoV - 2 Wuhan - D614G at 104 PFU is presented as a reference ( yellow ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Survival curves of mice challenged with SARS-CoV-2 Wuhan-D614G at 104 PFU, after a primo-infection by BANAL-236 or Wuhan-372 viruses at 103 or 104 PFU. Survival of K18-hACE2 naive mice infected by SARS-CoV-2 Wuhan-D614G at 104 PFU is presented as a reference (yellow).


F . Histopathological analysis , 3 days post - inoculation , of the lung of K18 - hACE2 mice infected with 104 PFU of BANAL - 236 ( a - d ) or SARS - CoV - 2 Wuhan - 372 ( e , f ) viruses . Interstitial pneumonia characterized by interstitial inflammation , often centered on blood vessels ( perivasculitis , black arrows ) or bronchi / bronchioles ( black arrowheads ) , and endothelial cell injury and inflammation ( endothelitis ) . Lesions were globally more severe after Wuhan - 372 infection ( n = 4 ; all of moderate severity ) than after BANAL - 236 infection ( n = 4 ; 3 / 4 of minimal severity ( a - c ) and 1 / 4 of moderate severity ( d ) ) . Scale bar : 100 µ . Low magnification images are provided in Figure S3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Histopathological analysis, 3 days post-inoculation, of the lung of K18-hACE2 mice infected with 104 PFU of BANAL-236 (a-d) or SARS-CoV-2 Wuhan-372 (e,f) viruses. Interstitial pneumonia characterized by interstitial inflammation, often centered on blood vessels (perivasculitis, black arrows) or bronchi/bronchioles (black arrowheads), and endothelial cell injury and inflammation (endothelitis). Lesions were globally more severe after Wuhan-372 infection (n=4; all of moderate severity) than after BANAL-236 infection (n=4; 3/4 of minimal severity (a-c) and 1/4 of moderate severity (d)). Scale bar: 100 µ. Low magnification images are provided in Figure S3.


Neutralizing antibody titer ( expressed in ED50 ) of mice primo - infected at 103 ( left ) or 104 ( right ) PFU by BANAL - 236 ( red ) or SARS - CoV - 2 Wuhan - 372 ( blue ) viruses . The antibody response against the Wuhan - D614G challenge strain of mice primo - infected by BANAL - 236 is presented as a dashed line . PNT means pseudo - neutralization .

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

Neutralizing antibody titer (expressed in ED50) of mice primo-infected at 103 (left) or 104 (right) PFU by BANAL-236 (red) or SARS-CoV-2 Wuhan-372 (blue) viruses. The antibody response against the Wuhan-D614G challenge strain of mice primo-infected by BANAL-236 is presented as a dashed line. PNT means pseudo-neutralization.


B . Effects of infection by BANAL - 236 virus in non - human primates . Animal # MF1 is presented in green and animal # MF2 in purple . Clinical score , body weight variation ( in % ) , CT scores ( compared to Wuhan - 372 ) and neutralizing antibodies are measured from 10 days before infection to 43 days post - infection .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Effects of infection by BANAL-236 virus in non-human primates. Animal #MF1 is presented in green and animal #MF2 in purple. Clinical score, body weight variation (in %), CT scores (compared to Wuhan-372) and neutralizing antibodies are measured from 10 days before infection to 43 days post-infection.


C . Viral load expressed in log10 copies of genomic RNA ( solid line ) or sub - genomic RNA ( dashed line ) per mL of tracheal , nasopharyngeal , BAL and rectal fluids . Lower limit of quantification is presented in red for both genomic ( solid line ) and sub - genomic ( dashed line ) RNA , and limits of detection are presented in green for both genomic ( solid line ) and sub - genomic ( dashed line ) RNA . Results were compared to historical data of SARS - CoV - 2 Wuhan - 372 virus inoculated at 106 or 105 PFU ( presented as the mean virus load + / - 95 % CI expressed in log10 copies of genomic RNA / mL ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Viral load expressed in log10 copies of genomic RNA (solid line) or sub-genomic RNA (dashed line) per mL of tracheal, nasopharyngeal, BAL and rectal fluids. Lower limit of quantification is presented in red for both genomic (solid line) and sub-genomic (dashed line) RNA, and limits of detection are presented in green for both genomic (solid line) and sub-genomic (dashed line) RNA. Results were compared to historical data of SARS-CoV-2 Wuhan-372 virus inoculated at 106 or 105 PFU (presented as the mean virus load +/- 95% CI expressed in log10 copies of genomic RNA/mL).


( A - C ) Results of Pseudoneutralisation ( PNT ) , Luciferase immunoprecipitation ( LIPS ) and LuLISA tests are shown . Pre - pandemic French sera ( black ) were used as negative controls and Laotian sera samples from confirmed SARS - CoV - 2 infection ( green ) were used as cross - reacting positive controls . Laotian sera samples collected in the general population before 2019 ( blue , n = 100 ) or late 2020 ( gray , n = 100 ) or in people exposed to bats ( purple , n = 74 ) were tested for BANAL - 236 ( A ) , BANAL - 52 ( B ) and BANAL - 103 ( C ) antibody responses . The ANOVA non - parametric Kruskal - Wallis test was conducted to compare each sub - population to the reference French population ( * P < 0 . 05 ; * * P < 0 . 005 ; * * * P < 0 . 0005 ; * * * * P < 0 . 0001 ) . The red bars represent the median .

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

(A-C) Results of Pseudoneutralisation (PNT), Luciferase immunoprecipitation (LIPS) and LuLISA tests are shown. Pre-pandemic French sera (black) were used as negative controls and Laotian sera samples from confirmed SARS-CoV-2 infection (green) were used as cross-reacting positive controls. Laotian sera samples collected in the general population before 2019 (blue, n=100) or late 2020 (gray, n=100) or in people exposed to bats (purple, n=74) were tested for BANAL-236 (A), BANAL-52 (B) and BANAL-103 (C) antibody responses. The ANOVA non-parametric Kruskal-Wallis test was conducted to compare each sub-population to the reference French population (* P<0.05; **P<0.005; ***P<0.0005; ****P<0.0001). The red bars represent the median.


( A ) - ( D ) Hepatocytes accumulate apical bulkheads upon bile duct ligation ( BDL ) and MDR2 KO . ( A ) - ( B ) Overview images of ( A ) murine liver tissue after control surgery or bile duct ligation ( BDL ) for 24h ( N = 3 biological replicates ) , or ( B ) control and MDR2 KO mice after 12 weeks ( N = 3 biological replicates ) . Immunofluorescence for the apical membrane marker CD13 ( magenta ) , F - Actin ( green ) and nuclei ( grey ) . Representative images are shown . Scale bar 10 µm . ( C ) - ( D ) High - resolution imaging of individual bile canaliculi in murine liver tissue ( C ) after control and 24h BDL surgery , or ( D ) in control and MDR2 KO mice , stained for F - Actin ( green ) . Representative images are shown . Scale bar 2 µm . Apical bulkheads are highlighted with red arrowheads .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Hepatocytes accumulate apical bulkheads upon bile duct ligation (BDL) and MDR2 KO. (A)-(B) Overview images of (A) murine liver tissue after control surgery or bile duct ligation (BDL) for 24h (N=3 biological replicates), or (B) control and MDR2 KO mice after 12 weeks (N=3 biological replicates). Immunofluorescence for the apical membrane marker CD13 (magenta), F-Actin (green) and nuclei (grey). Representative images are shown. Scale bar 10 µm. (C)-(D) High-resolution imaging of individual bile canaliculi in murine liver tissue (C) after control and 24h BDL surgery, or (D) in control and MDR2 KO mice, stained for F-Actin (green). Representative images are shown. Scale bar 2 µm. Apical bulkheads are highlighted with red arrowheads.


Apical bulkheads in the adult liver resemble those described in the embryonic liver . ( E ) Transmission electron microscopy ( TEM ) longitudinal 80 nm - thin serial section on liver tissue with BDL for 24h shows the ultrastructure characteristic of apical bulkheads . Representative images are shown . Scale bar 1 µm . I - III represent consecutive sections with 460 nm distance showing four apical bulkheads ( B1 - B4 ) sealed by electron - dense tight junctions . Scale bar 1 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

Apical bulkheads in the adult liver resemble those described in the embryonic liver. (E) Transmission electron microscopy (TEM) longitudinal 80 nm-thin serial section on liver tissue with BDL for 24h shows the ultrastructure characteristic of apical bulkheads. Representative images are shown. Scale bar 1 µm. I-III represent consecutive sections with 460 nm distance showing four apical bulkheads (B1-B4) sealed by electron-dense tight junctions. Scale bar 1 µm.


Hepatocytes form aberrantly dilated canaliculi formed by > 3 cells after BDL . ( I ) Transmission electron microscopy ( TEM ) in liver tissue after 24h BDL . Scale bar 10 µm . Bile canaliculi ( BC ) can be severely dilated similar to the size of sinusoids ( S ) and are formed by multiple hepatocytes ( see inset A , scale bar 1 µm ) . Apical bulkheads ( marked with red arrowheads ) are visible . Individual hepatocytes are part of a liver cell rosette with one part of their apical membrane while they form normal canaliculi with apical bulkheads at another pole ( see inset A , cell 3 and 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hepatocytes form aberrantly dilated canaliculi formed by >3 cells after BDL. (I) Transmission electron microscopy (TEM) in liver tissue after 24h BDL. Scale bar 10 µm. Bile canaliculi (BC) can be severely dilated similar to the size of sinusoids (S) and are formed by multiple hepatocytes (see inset A, scale bar 1 µm). Apical bulkheads (marked with red arrowheads) are visible. Individual hepatocytes are part of a liver cell rosette with one part of their apical membrane while they form normal canaliculi with apical bulkheads at another pole (see inset A, cell 3 and 4).


( A ) - ( B ) Hepatocytes accumulate apical bulkheads in the liver tissue of primary sclerosing cholangitis ( PSC ) patients ( N = 3 control patients and N = 4 PSC patients ) . ( A ) Overview images in liver tissue of control and PSC patients . Immunofluoresence for the apical membrane marker BSEP ( magenta ) , F - Actin ( green ) and nuclei ( grey ) . Scale bar 10 µm . ( B ) High - resolution imaging of F - Actin ( green ) showing individual bile canaliculi in control or PSC liver tissue . Red arrowheads mark apical bulkheads . Scale bar 2 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

(A)-(B) Hepatocytes accumulate apical bulkheads in the liver tissue of primary sclerosing cholangitis (PSC) patients (N=3 control patients and N=4 PSC patients). (A) Overview images in liver tissue of control and PSC patients. Immunofluoresence for the apical membrane marker BSEP (magenta), F-Actin (green) and nuclei (grey). Scale bar 10 µm. (B) High-resolution imaging of F-Actin (green) showing individual bile canaliculi in control or PSC liver tissue. Red arrowheads mark apical bulkheads. Scale bar 2 µm.


( C ) Different lumen morphologies of a typical bile canaliculus ( left column ) and bile duct ( middle column ) in control patients and rosette ( right column ) in PSC patients . Immunofluorescence for BSEP ( magenta ) and F - Actin ( green ) . Shows also differences dependent on the imaging plane i . e . transversal ( top row ) or longitudinal ( bottom row ) cut of the lumen . White dashed lines mark the lumen . Scale bar 2 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

(C) Different lumen morphologies of a typical bile canaliculus (left column) and bile duct (middle column) in control patients and rosette (right column) in PSC patients. Immunofluorescence for BSEP (magenta) and F-Actin (green). Shows also differences dependent on the imaging plane i.e. transversal (top row) or longitudinal (bottom row) cut of the lumen. White dashed lines mark the lumen. Scale bar 2 µm.


( F ) Liver cell rosettes in PSC patients are formed by hepatocyte - like cells ( N = 3 control patients and N = 4 PSC patients ) . Immunofluoresence for BSEP ( magenta ) , F - Actin ( green ) and nuclei ( grey ) in healthy parenchyma ( left ) and bile duct ( middle ) in control patients and liver cell rosettes in PSC patients ( right ) . Scale bar 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Liver cell rosettes in PSC patients are formed by hepatocyte-like cells (N=3 control patients and N=4 PSC patients). Immunofluoresence for BSEP (magenta), F-Actin (green) and nuclei (grey) in healthy parenchyma (left) and bile duct (middle) in control patients and liver cell rosettes in PSC patients (right). Scale bar 10 µm.


( A ) - ( D ) Liver cell rosettes in PSC patients do not acquire other markers of bile duct cells or intermediate hepatobiliary cells . Immunofluoresence for different bile duct cell or intermediate hepatobiliary cell type marker ( magenta ) , F - Actin ( green ) and nuclei ( grey ) in healthy parenchyma ( left ) and bile duct ( middle ) in control patients and liver cell rosettes ( right ) in PSC patients ( N = 3 control patients and N = 4 PSC patients ) . Scale bar 10 µm . ( A ) SOX9 , ( B ) Pan - CK , Representative images are shown .

(A)-(D)Liver cell rosettes in PSC patients do not acquire other markers of bile duct cells or intermediate hepatobiliary cells. Immunofluoresence for different bile duct cell or intermediate hepatobiliary cell type marker (magenta), F-Actin (green) and nuclei (grey) in healthy parenchyma (left) and bile duct (middle) in control patients and liver cell rosettes (right) in PSC patients (N=3 control patients and N=4 PSC patients). Scale bar 10 µm. (A) SOX9, (B) Pan-CK, Representative images are shown.


( A ) - ( D ) Liver cell rosettes in PSC patients do not acquire other markers of bile duct cells or intermediate hepatobiliary cells . Immunofluoresence for different bile duct cell or intermediate hepatobiliary cell type marker ( magenta ) , F - Actin ( green ) and nuclei ( grey ) in healthy parenchyma ( left ) and bile duct ( middle ) in control patients and liver cell rosettes ( right ) in PSC patients ( N = 3 control patients and N = 4 PSC patients ) . Scale bar 10 µm . TROP2 CD133 / PROM1 . Representative images are shown .

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

(A)-(D)Liver cell rosettes in PSC patients do not acquire other markers of bile duct cells or intermediate hepatobiliary cells. Immunofluoresence for different bile duct cell or intermediate hepatobiliary cell type marker (magenta), F-Actin (green) and nuclei (grey) in healthy parenchyma (left) and bile duct (middle) in control patients and liver cell rosettes (right) in PSC patients (N=3 control patients and N=4 PSC patients). Scale bar 10 µm. TROP2 CD133/PROM1. Representative images are shown.


3D reconstruction of the bile canaliculi network reveals fundamental changes in the network architecture of PSC patients . ( A ) Top panel : 3D reconstructed bile canaliculi network across full central vein to portal vein ( CV - PV ) axes in control ( left ) and PSC ( right ) patients . Bottom panel : Projection of the 3D reconstruction . Colours correspond to the mean bile canaliculi diameter in µm . Scale bar 100 µm . ( B ) - ( I ) Quantification of bile canaliculi network characteristics from the 3D reconstruction . Represented are mean values with standard deviation of control and PSC patients at different zones across the CV - PV axis . 0 represents the zone surrounding the CV and 10 the area surrounding the PV . N = 3 with 1x axis / patient for control patients and N = 4 with 3x axis / patient for PSC patients . Paired t - test * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . ( B ) bile canaliculi radius [ µm ] , ( C ) volume fraction [ % ] , ( D ) surface area / volume ( µm2 / mm3 ) , ( E ) connectivity ( 1 / # disjointed bile canaliculi ) , ( F ) branches crossing regions [ # / mm2 ] . ( G ) junctions / length [ # /

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

3D reconstruction of the bile canaliculi network reveals fundamental changes in the network architecture of PSC patients. (A) Top panel: 3D reconstructed bile canaliculi network across full central vein to portal vein (CV-PV) axes in control (left) and PSC (right) patients. Bottom panel: Projection of the 3D reconstruction. Colours correspond to the mean bile canaliculi diameter in µm. Scale bar 100 µm. (B)-(I) Quantification of bile canaliculi network characteristics from the 3D reconstruction. Represented are mean values with standard deviation of control and PSC patients at different zones across the CV-PV axis. 0 represents the zone surrounding the CV and 10 the area surrounding the PV. N=3 with 1x axis/patient for control patients and N=4 with 3x axis/patient for PSC patients. Paired t-test *P < 0.05, **P < 0.01, ***P < 0.001. (B) bile canaliculi radius [µm], (C) volume fraction [%], (D) surface area/volume (µm2/mm3), (E) connectivity (1/# disjointed bile canaliculi), (F) branches crossing regions [#/mm2]. (G) junctions/length [#/µm


( A ) Liver cell rosettes occur in early - and late - stage PSC patients and in end - stage alcoholic liver disease ( ALD ) patients . Immunofluorescence of Fibronectin ( magenta ) , F - actin ( green ) and nuclei ( grey ) in early - stage PSC ( N = 3 ) , late - stage PSC ( N = 5 ) and end - stage ALD ( N = 5 ) patients showing liver cell rosettes . Representative images are shown . Scale bar 50 µm . Examples of rosettes are labelled with a red asterisk and examples of sinusoids are labelled with a red arrowhead . ( B ) Similar numbers of liver cell rosettes in early - stage and end - stage PSC patients . Quantification of the total number of rosettes in early - stage PSC ( N = 3 ) , late - stage PSC ( N = 5 ) and end - stage ALD ( N = 5 ) patients . Unpaired t - test * P < 0 . 05 . ( C ) Similar mean diameter of analysed liver cell rosettes in different liver diseases . Quantification of the mean rosette diameter in early - stage PSC ( N = 3 ) , late - stage PSC ( N = 5 ) and end - stage ALD ( N = 5 ) . Unpaired t - test . Data information : Boxplot shows the 25th and 75th percentiles and the central band shows the median value . Whiskers extend to the min and max values .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Liver cell rosettes occur in early- and late-stage PSC patients and in end-stage alcoholic liver disease (ALD) patients. Immunofluorescence of Fibronectin (magenta), F-actin (green) and nuclei (grey) in early-stage PSC (N=3), late-stage PSC (N=5) and end-stage ALD (N=5) patients showing liver cell rosettes. Representative images are shown. Scale bar 50 µm. Examples of rosettes are labelled with a red asterisk and examples of sinusoids are labelled with a red arrowhead. (B) Similar numbers of liver cell rosettes in early-stage and end-stage PSC patients. Quantification of the total number of rosettes in early-stage PSC (N=3), late-stage PSC (N=5) and end-stage ALD (N=5) patients. Unpaired t-test *P < 0.05. (C) Similar mean diameter of analysed liver cell rosettes in different liver diseases. Quantification of the mean rosette diameter in early-stage PSC (N=3), late-stage PSC (N=5) and end-stage ALD (N=5). Unpaired t-test. Data information: Boxplot shows the 25th and 75th percentiles and the central band shows the median value. Whiskers extend to the min and max values.


( C ) - ( D ) DCA treatment induces a cholestatic response in primary hepatocytes . Gene expression analysis of genes involved in bile acid metabolism and transport in primary hepatocytes treated with DMSO or 200 µM DCA for 16h . Represented are mean Cq values normalized to Gapdh expression ( N = 4 biological replicates ) . One sample t - test * P < 0 . 05 , * * * P < 0 . 001 . ( C ) Abcb11 / Bsep , ( D ) Cyp7a1 / Cyp7 . Data information : Boxplots show the 25th and 75th percentiles and the central band shows the median value . Whiskers extend to the min and max values .

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 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

(C) -(D) DCA treatment induces a cholestatic response in primary hepatocytes. Gene expression analysis of genes involved in bile acid metabolism and transport in primary hepatocytes treated with DMSO or 200 µM DCA for 16h. Represented are mean Cq values normalized to Gapdh expression (N=4 biological replicates). One sample t-test *P < 0.05, ***P < 0.001. (C) Abcb11/Bsep, (D) Cyp7a1/Cyp7. Data information: Boxplots show the 25th and 75th percentiles and the central band shows the median value. Whiskers extend to the min and max values.


( I - J ) DCA treatment in 3D hepatocyte organoids also induce canalicular dilation and rosette - like canaliculi and Sox9 expression . Hepatocyte organoids were treated with DMSO or 200 µM DCA for 24h ( N = 3 biological replicates ) . ( I ) Maximum projection of full organoids stained for F - actin ( green ) . Scale bar 50 µm . Inset A and B . Maximum projection of zoomed - in parts of the bile canaliculi network in DMSO - treated ( A ) and DCA - treated ( B ) hepatocyte organoids stained with F - actin ( green ) , nuclei ( white ) and the tight junction protein ZO - 1 ( yellow ) . Scale bar 10 µm . ( J ) High - resolution microscopy of individual bile canaliculi in DMSO - treated and DCA - treated hepatocyte organoids stained for F - actin ( green ) . Red arrows indicate apical bulkheads . Scale bar 2 µm .

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

(I-J) DCA treatment in 3D hepatocyte organoids also induce canalicular dilation and rosette-like canaliculi and Sox9 expression. Hepatocyte organoids were treated with DMSO or 200 µM DCA for 24h (N=3 biological replicates). (I) Maximum projection of full organoids stained for F-actin (green). Scale bar 50 µm. Inset A and B. Maximum projection of zoomed-in parts of the bile canaliculi network in DMSO-treated (A) and DCA-treated (B) hepatocyte organoids stained with F-actin (green), nuclei (white) and the tight junction protein ZO-1 (yellow). Scale bar 10 µm. (J) High-resolution microscopy of individual bile canaliculi in DMSO-treated and DCA-treated hepatocyte organoids stained for F-actin (green). Red arrows indicate apical bulkheads. Scale bar 2 µm.


( K ) Maximum projection of F - actin ( green ) and nuclei ( white ) , and the bile duct cell marker Sox9 ( magenta ) in DMSO - treated and DCA - treated hepatocyte organoids ( N = 3 biological replicates ) . Untreated bile duct cell organoids serve as positive control . Scale bar 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 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

(K) Maximum projection of F-actin (green) and nuclei (white), and the bile duct cell marker Sox9 (magenta) in DMSO-treated and DCA-treated hepatocyte organoids (N=3 biological replicates). Untreated bile duct cell organoids serve as positive control.Scale bar 50 µm.


( A ) Rosette - like canaliculi with reduced number of apical bulkheads can emerge from usual canaliculi with accumulated apical bulkheads . Timelapse imaging with brightfield phase - contrast of primary hepatocytes either untreated ( top row ) , DMSO - treated ( middle row ) or DCA - treated ( bottom row ) . An image was acquired every 15 minutes . Representative images are shown ( N = 3 biological replicates ) . Inserts show magnification of bile canaliculi with red arrows pointing towards apical bulkheads . Red stars mark areas in between apical bulkheads that markedly bulge out . Scale bar 10 µm .

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

(A)Rosette-like canaliculi with reduced number of apical bulkheads can emerge from usual canaliculi with accumulated apical bulkheads. Timelapse imaging with brightfield phase-contrast of primary hepatocytes either untreated (top row), DMSO-treated (middle row) or DCA-treated (bottom row). An image was acquired every 15 minutes. Representative images are shown (N=3 biological replicates). Inserts show magnification of bile canaliculi with red arrows pointing towards apical bulkheads. Red stars mark areas in between apical bulkheads that markedly bulge out. Scale bar 10 µm.


( B ) Pharmacological inhibition of canalicular water influx can prevent canalicular dilation upon DCA treatment . Confocal microscopy of CD13 ( magenta ) , F - actin ( green ) and nuclei ( white ) in primary hepatocytes treated with DMSO and 200 µM DCA with 200 µM phloretin and 1 µM ouabain for 16h . Scale bar 10 µm . Representative images are shown .

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

(B) Pharmacological inhibition of canalicular water influx can prevent canalicular dilation upon DCA treatment. Confocal microscopy of CD13 (magenta), F-actin (green) and nuclei (white) in primary hepatocytes treated with DMSO and 200 µM DCA with 200 µM phloretin and 1 µM ouabain for 16h. Scale bar 10 µm. Representative images are shown.


( C ) Quantification of the mean bile canaliculi volume in µm3of primary hepatocyte treated with DMSO and 200 µM DCA with 200 µM Phloretin and 1 µM Ouabain for 16h ( N = 4 biological replicates ) . Unpaired t - test * * * P < 0 . 001 . Data information : Boxplots show the 25th and 75th percentiles and the central band shows the median value . Whiskers extend to the min and max values .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O 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) Quantification of the mean bile canaliculi volume in µm3of primary hepatocyte treated with DMSO and 200 µM DCA with 200 µM Phloretin and 1 µM Ouabain for 16h (N=4 biological replicates). Unpaired t-test ***P < 0.001. Data information: Boxplots show the 25th and 75th percentiles and the central band shows the median value. Whiskers extend to the min and max values.


( D ) - ( E ) Pharmacological inhibitors do not affect key bile acid signalling pathways . Gene expression analysis of different genes involved in bile acid signalling in primary hepatocytes treated with DMSO or 200 µM DCA with 200 µM Phloretin and 1 µM Ouabain for 16h . Represented are mean Cq values normalized to Gapdh expression and whiskers show min and max values ( N = 5 biological replicates ) . Unpaired t - test . ( D ) Cyp7a1 / Cyp7 , ( E ) Nr0b2 / Shp . Data information : Boxplots show the 25th and 75th percentiles and the central band shows the median value . Whiskers extend to the min and max values .

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

(D)- (E) Pharmacological inhibitors do not affect key bile acid signalling pathways. Gene expression analysis of different genes involved in bile acid signalling in primary hepatocytes treated with DMSO or 200 µM DCA with 200 µM Phloretin and 1 µM Ouabain for 16h. Represented are mean Cq values normalized to Gapdh expression and whiskers show min and max values (N=5 biological replicates). Unpaired t-test. (D) Cyp7a1/Cyp7, (E) Nr0b2/Shp. Data information: Boxplots show the 25th and 75th percentiles and the central band shows the median value. Whiskers extend to the min and max values.


( C ) The 3 . 1 Å 2Fo - Fc map ( 1 . 2 σ ) around the DNA of the final refined ATPγS - MjMR - DNA complex structure .

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

(C) The 3.1 Å 2Fo-Fc map (1.2 σ) around the DNA of the final refined ATPγS-MjMR-DNA complex structure.


( D ) A ribbon representation of the 3 . 1 Å ATPγS - MjMR - DNA structure showing the Mre11 nuclease , capping , and C - terminal three helix - bundle domain ( light pink ) , Rad50 molecules ( green and light blue ) , and the DNA ( yellow and orange ) . ATPγS is shown in spheres . See Movie EV 1 for the movement of the complex .

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

(D) A ribbon representation of the 3.1 Å ATPγS-MjMR-DNA structure showing the Mre11 nuclease, capping, and C-terminal three helix-bundle domain (light pink), Rad50 molecules (green and light blue), and the DNA (yellow and orange). ATPγS is shown in spheres. See Movie EV 1 for the movement of the complex.


( A ) A ribbon diagram showing the overall DNA recognition by the ATPγS - Rad50 dimer .

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

(A) A ribbon diagram showing the overall DNA recognition by the ATPγS-Rad50 dimer.


( B ) Close - up view of the interaction between the internal segment ( major and minor grooves ) of the DNA strand and the edge of lobe I of Rad50A ( green ) . Loop α1 - α2 wedge faces the minor groove between the 10th and 13 ' th phosphates of the template ( orange ) and non - template ( yellow ) strands , respectively . ( C ) Close - up view of the interaction between the DNA strand and the α1 - α2 loop in an orientation different from Fig 2B .

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

(B) Close-up view of the interaction between the internal segment (major and minor grooves) of the DNA strand and the edge of lobe I of Rad50A (green). Loop α1-α2 wedge faces the minor groove between the 10th and 13' th phosphates of the template (orange) and non-template (yellow) strands, respectively. (C) Close-up view of the interaction between the DNA strand and the α1-α2 loop in an orientation different from Fig 2B.


( B , C ) DNA binding analysis for the Interactions between WT or four MjMRcd mutants and a closed circular dsDNA ( φx174RFII ) in the absence ( B ) or presence ( C ) of AMP - PNP / Mg2 + . Each protein sample was incubated with φx174RFII dsDNA ( 3 . 5 nM ) for 30 min on 4 ° C . The following molar ratio of protein : DNA is used in Fig 3B - 3E ; lane 2 , 50 : 1 ; lane 3 , 100 : 1 ; lane 4 , 200 : 1 ; lane 5 , 500 : 1 ; lane 6 , 1000 : 1 ; lane 7 , 2000 : 1 . We note that some MRcd variants may form a second form of the complex with DNA .

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

(B, C) DNA binding analysis for the Interactions between WT or four MjMRcd mutants and a closed circular dsDNA (φx174RFII) in the absence (B) or presence (C) of AMP-PNP/Mg2+. Each protein sample was incubated with φx174RFII dsDNA (3.5 nM) for 30 min on 4°C. The following molar ratio of protein: DNA is used in Fig 3B-3E; lane 2, 50:1; lane 3, 100:1; lane 4, 200:1; lane 5, 500:1; lane 6, 1000:1; lane 7, 2000:1. We note that some MRcd variants may form a second form of the complex with DNA.