( c ) Cultured ventral midbrain dopaminergic neurons from Ctr and from WT - LRRK2 and G / S - LRRK2 transgenic mice , transduced with the same reporter as in b and co - stained with antibody to tyrosine hydroxylase ( TH ) . Nuclei are highlighted with DAPI ( 4 ′ , 6 - diamidino - 2 - phenylindole ) in blue . Left : representative images . Right : average number of puncta per cell in > 50 TH - positive cells . Scale bars , 10 μm . All values are mean + s . e . m . ; differences with Ctr ( * ) and between WT LRRK2 and G / S LRRK2 ( § ) were significant at P 0 . 05 ; unpaired t - test in a and ANOVA and Bonferroni test in b , c .

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(c) Cultured ventral midbrain dopaminergic neurons from Ctr and from WT-LRRK2 and G/S-LRRK2 transgenic mice, transduced with the same reporter as in b and co-stained with antibody to tyrosine hydroxylase (TH). Nuclei are highlighted with DAPI (4′,6-diamidino-2-phenylindole) in blue. Left: representative images. Right: average number of puncta per cell in >50 TH-positive cells. Scale bars, 10 μm. All values are mean + s.e.m.; differences with Ctr (*) and between WT LRRK2 and G/S LRRK2 (§) were significant at P 0.05; unpaired t-test in a and ANOVA and Bonferroni test in b,c.


( a ) Immunoblots of rat liver lysosomes incubated with WT or G / S LRRK2 alone or with 1 mM GTP and / or GAPDH . Duplicate samples are shown . Bottom : quantification of lysosome - bound LRRK2 expressed as a multiple of the amount bound without additions ( n = 6 independent experiments ) .

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(a) Immunoblots of rat liver lysosomes incubated with WT or G/S LRRK2 alone or with 1 mM GTP and/or GAPDH. Duplicate samples are shown. Bottom: quantification of lysosome-bound LRRK2 expressed as a multiple of the amount bound without additions (n = 6 independent experiments).


( b ) Effect of kinase inhibitor on WT , G / S , or the kinase - dead mutant D / A LRRK2 incubated with rat liver lysosomes in presence or absence of GAPDH ( 5 μg ) . Right : quantification expressed as a multiple of the association without additions ( n = 3 - 5 independent experiments ) .

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(b) Effect of kinase inhibitor on WT, G/S, or the kinase-dead mutant D/A LRRK2 incubated with rat liver lysosomes in presence or absence of GAPDH (5 μg). Right: quantification expressed as a multiple of the association without additions (n = 3-5 independent experiments).


( c , d ) Transient transfection of HEK293 cells with the myc - or GFP - tagged LRRK2 constructs indicated at top left in c . Full , full length ; M , KFERQ - like motifs ( M1 - M8 ) . ( c ) Binding to GST - hsc70 . Left , representative immunoblots ; input lanes , one - tenth of input material . NT , non - transfected cells . Immunoblot for hsc70 is shown as loading control . Top right : quantification of LRRK2 bound to hsc70 . Where indicated , samples were also incubated with RNase A to compete the KFERQ - mediated binding of hsc70 ( n = 3 or 4 independent experiments ) . ( d ) Coimmunoprecipitation ( IP ) of hsc70 with anti - myc or anti - GFP in the same cells . Inputs ( Inp ) , one - quarter of starting volume .

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(c,d) Transient transfection of HEK293 cells with the myc- or GFP-tagged LRRK2 constructs indicated at top left in c. Full, full length; M, KFERQ-like motifs (M1-M8). (c) Binding to GST-hsc70. Left, representative immunoblots; input lanes, one-tenth of input material. NT, non-transfected cells. Immunoblot for hsc70 is shown as loading control. Top right: quantification of LRRK2 bound to hsc70. Where indicated, samples were also incubated with RNase A to compete the KFERQ-mediated binding of hsc70 (n = 3 or 4 independent experiments). (d) Coimmunoprecipitation (IP) of hsc70 with anti-myc or anti-GFP in the same cells. Inputs (Inp), one-quarter of starting volume.


( e ) Binding or LRRK2 to rat liver lysosomes treated with the indicated concentrations of trypsin . LAMP - 1 is shown to confirm the integrity of the lysosomes .

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(e) Binding or LRRK2 to rat liver lysosomes treated with the indicated concentrations of trypsin. LAMP-1 is shown to confirm the integrity of the lysosomes.


( f ) Co - IP of LAMP - 2A with anti - LRRK2 in lysosomes incubated with WT or G / S LRRK2 . Inp , one - quarter of starting sample ; IgG , immunoglobulin G . Values : LAMP - 2A recovered in the IP ( IP L2A ) corrected for the amount of LRRK2 pulled down and expressed as a multiple of WT value .

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(f) Co-IP of LAMP-2A with anti-LRRK2 in lysosomes incubated with WT or G/S LRRK2. Inp, one-quarter of starting sample; IgG, immunoglobulin G. Values: LAMP-2A recovered in the IP (IP L2A) corrected for the amount of LRRK2 pulled down and expressed as a multiple of WT value.


( g , h ) Duplicate LAMP - 2A immunoblots of rat liver lysosomes incubated alone ( − ) or with WT or G / S LRRK2 ( g ) or with lysosomes from control ( Ctr ) or from WT - LRRK2 or G / S - LRRK2 transgenic mice ( h ) and subjected to blue native electrophoresis . Arrow , 700 - kDa multimeric complex . All values are mean + s . e . m . ; differences compared to none or untreated ( * ) or to WT or full - length protein ( § ) were significant at P 0 . 05 ; unpaired t - test in a and ANOVA and Bonferroni test in b , c . Full - length blots and gels in Supplementary Figure 12 .

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(g,h) Duplicate LAMP-2A immunoblots of rat liver lysosomes incubated alone (−) or with WT or G/S LRRK2 (g) or with lysosomes from control (Ctr) or from WT-LRRK2 or G/S-LRRK2 transgenic mice (h) and subjected to blue native electrophoresis. Arrow, 700-kDa multimeric complex. All values are mean + s.e.m.; differences compared to none or untreated (*) or to WT or full-length protein (§) were significant at P 0.05; unpaired t-test in a and ANOVA and Bonferroni test in b,c. Full-length blots and gels in Supplementary Figure 12.


( a ) Immunofluorescence for LAMP - 2A and LAMP - 1 of sections from the dorsal motor nuclei of the vagus nerves of brains from two different unaffected control ( Ctr ) individuals and two individuals with Parkinson ' s disease with the G / S LRRK2 mutation . Left : images from representative fields . Right : number of fluorescent puncta per cell . Black dots , values in individual cells ; red lines , mean .

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(a) Immunofluorescence for LAMP-2A and LAMP-1 of sections from the dorsal motor nuclei of the vagus nerves of brains from two different unaffected control (Ctr) individuals and two individuals with Parkinson's disease with the G/S LRRK2 mutation. Left: images from representative fields. Right: number of fluorescent puncta per cell. Black dots, values in individual cells; red lines, mean.


( b ) Immunohistochemistry for LAMP - 2A in the same samples .

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(b) Immunohistochemistry for LAMP-2A in the same samples.


( c ) Immunofluorescence for LAMP - 2A ( red ) and LRRK2 ( green ) in the same samples . Merged images from the two fluorophore channels are shown ( for colocalization quantification , see Supplementary Fig . 7b ) . Scale bars , 10 μm . Values are mean + s . e . m . ; * P 0 . 05 , ANOVA and Bonferroni test .

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(c) Immunofluorescence for LAMP-2A (red) and LRRK2 (green) in the same samples. Merged images from the two fluorophore channels are shown (for colocalization quantification, see Supplementary Fig. 7b). Scale bars, 10 μm. Values are mean + s.e.m.; *P 0.05, ANOVA and Bonferroni test.


( a ) Top : LRRK2 immunoblot of rat liver lysosomes incubated with WT , G / S , D / A ( kinase - dead ) , I / T or R / C mutant LRRK2 , alone or in the presence of protease inhibitors ( PI ) . Input lanes , one - quarter of input material . Bottom : quantification of binding and uptake ( calculated as in Fig . 2b ; n = 4 or 5 independent experiments ) .

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(a) Top: LRRK2 immunoblot of rat liver lysosomes incubated with WT, G/S, D/A (kinase-dead), I/T or R/C mutant LRRK2, alone or in the presence of protease inhibitors (PI). Input lanes, one-quarter of input material. Bottom: quantification of binding and uptake (calculated as in Fig. 2b; n = 4 or 5 independent experiments).


( b ) Left : immunoblots of lysosomes incubated as in a , alone or in the presence of increasing concentrations of RNase A ( left ) or GAPDH ( right ) . Right : quantification of LRRK2 bound , as a multiple of the binding when incubated with lysosomes alone ( n = 3 independent experiments ) .

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(b) Left: immunoblots of lysosomes incubated as in a, alone or in the presence of increasing concentrations of RNase A (left) or GAPDH (right). Right: quantification of LRRK2 bound, as a multiple of the binding when incubated with lysosomes alone (n = 3 independent experiments).


( c ) Top : coimmunoprecipitation ( co - IP ) of LAMP - 2A ( L2A ) with anti - LRRK2 from lysosomes incubated with LRRK2proteins as in a . Co - immunoprecipitated fractions were immunoblotted for LAMP - 2A and LRRK2 . Input : immunoblot for LAMP - 2A before the affinity purification . Bottom : amount of LAMP - 2A coimmunoprecipitated , expressed as a multiple of the value with WT LRRK2 ( n = 3 or 4 independent experiments ) .

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(c) Top: coimmunoprecipitation (co-IP) of LAMP-2A (L2A) with anti-LRRK2 from lysosomes incubated with LRRK2proteins as in a. Co-immunoprecipitated fractions were immunoblotted for LAMP-2A and LRRK2. Input: immunoblot for LAMP-2A before the affinity purification. Bottom: amount of LAMP-2A coimmunoprecipitated, expressed as a multiple of the value with WT LRRK2 (n = 3 or 4 independent experiments).


( d ) Co - IP of LAMP - 2A with anti - GFP in tet - on HEK293 cells expressing GFP - LRRK2 proteins and pulsed for 24 h with doxycycline . Inputs ( Inp ) , one - quarter of material added .

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(d) Co-IP of LAMP-2A with anti-GFP in tet-on HEK293 cells expressing GFP-LRRK2 proteins and pulsed for 24 h with doxycycline. Inputs (Inp), one-quarter of material added.


( e ) The same cells as in d and control cells transduced with KFERQ - PA - mCherry1 . Left : representative images . Nuclei are highlighted with DAPI in blue . Right : Quantification after photoactivation and 16 h of serum deprivation ( n > 150 cells ) . All values are mean + s . e . m . Differences with WT ( * ) or with the indicated mutants ( § ) were significant at P 0 . 05 ; unpaired t - test in a - c and ANOVA and Bonferroni test in e . Scale bars , 10 μm . Full - length blots and gels in Supplementary Figure 12 .

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(e) The same cells as in d and control cells transduced with KFERQ-PA-mCherry1. Left: representative images. Nuclei are highlighted with DAPI in blue. Right: Quantification after photoactivation and 16 h of serum deprivation (n > 150 cells). All values are mean + s.e.m. Differences with WT (*) or with the indicated mutants (§) were significant at P 0.05; unpaired t-test in a-c and ANOVA and Bonferroni test in e. Scale bars, 10 μm. Full-length blots and gels in Supplementary Figure 12.


( a ) α - syn immunoblot of rat liver lysosomes incubated with WT or mutant ( A53T ) α - syn alone ( − ) or with WT LRRK2 or G / S LRRK2 . Inp , input ; Mon , monomers . Right : quantification of α - syn oligomers , expressed as a multiple of amounts in samples without LRRK2 ( n = 4 or 5 independent experiments ) .

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(a) α-syn immunoblot of rat liver lysosomes incubated with WT or mutant (A53T) α-syn alone (−) or with WT LRRK2 or G/S LRRK2. Inp, input; Mon, monomers. Right: quantification of α-syn oligomers, expressed as a multiple of amounts in samples without LRRK2 (n = 4 or 5 independent experiments).


( b - d ) Degradation of radiolabeled proteins ( b ) , binding of GST - α - syn ( c ) and levels of endogenous α - syn ( d ) in intact brain lysosomes ( Lysos ) from non - transgenic ( Ctr ) and G / S LRRK2 transgenic mice . Bottom panel in c shows lower exposure to visualize monomers of α - syn . Homog , homogenates ; olig , oligomers ; I , input ( n = 3 independent experiments ) .

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(b-d) Degradation of radiolabeled proteins (b), binding of GST-α-syn (c) and levels of endogenous α-syn (d) in intact brain lysosomes (Lysos) from non-transgenic (Ctr) and G/S LRRK2 transgenic mice. Bottom panel in c shows lower exposure to visualize monomers of α-syn. Homog, homogenates; olig, oligomers; I, input (n = 3 independent experiments).


( e ) Ventral midbraindopaminergic neuronal cultures from control ( Ctr ) or WT - LRRK2 or G / S - LRRK2 transgenic mice untreated or treated with RNAi for LAMP - 2A ( L2A ( − ) ) . ( f ) Colocalization between α - syn and total LAMP - 2 from cells in e ( n > 50 tyrosine hydroxylase ( TH ) - positive neurons ) . Total LAMP - 2 is not reduced in L2A knockdown cells because LAMP - 2B is upregulated .

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(e) Ventral midbraindopaminergic neuronal cultures from control (Ctr) or WT-LRRK2 or G/S-LRRK2 transgenic mice untreated or treated with RNAi for LAMP-2A (L2A(−)). (f) Colocalization between α-syn and total LAMP-2 from cells in e (n > 50 tyrosine hydroxylase (TH)-positive neurons). Total LAMP-2 is not reduced in L2A knockdown cells because LAMP-2B is upregulated.


( g ) iPSC lines from controls and from patients with G / S LRRK2 - mutation Parkinson ' s disease , differentiated for 30 d and immunostained for LAMP - 2A , α - syn and TH . Merged channels ( left ) and magnified green and red channels from boxed region ( right ) . Arrows , colocalization LAMP - 2A and α - syn . ( h ) Percentage of LAMP - 2A + puncta colocalizing with α - syn in TH + cells ( n > 50 ) for two control and two LRRK2 iPSC lines treated as in g .

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(g) iPSC lines from controls and from patients with G/S LRRK2-mutation Parkinson's disease, differentiated for 30 d and immunostained for LAMP-2A, α-syn and TH. Merged channels (left) and magnified green and red channels from boxed region (right). Arrows, colocalization LAMP-2A and α-syn. (h) Percentage of LAMP-2A+ puncta colocalizing with α-syn in TH+ cells (n > 50) for two control and two LRRK2 iPSC lines treated as in g.


( i , j ) iPSC lines as in g , differentiated for 30 or 75 d and untreated or treated with RNAi for LAMP - 2A . ( i ) Immunofluorescence for LAMP - 2A , α - syn and GFP in LAMP - 2A - RNAi iPSC lines differentiated for 30 d . Bottom : higher magnifications of boxed areas . ( j ) Percentage of TH + cells showing cytoplasmic accumulation of α - syn . n ≥ 2 experiments using two control and two LRRK2 iPSC lines ( n > 70 and n > 150 TH + cells in Ctr and LRRK2 , respectively ) . Scale bars , 10 μm . All values are mean + s . e . m . Differences from WT or control ( * ) or between control or knockdown cells ( § ) are significant at P 0 . 05 ; unpaired t - test in a , h and ANOVA and Bonferroni test in f , j . Full - length blots and gels in Supplementary Figure 12 .

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(i,j) iPSC lines as in g, differentiated for 30 or 75 d and untreated or treated with RNAi for LAMP-2A. (i) Immunofluorescence for LAMP-2A, α-syn and GFP in LAMP-2A-RNAi iPSC lines differentiated for 30 d. Bottom: higher magnifications of boxed areas. (j) Percentage of TH+ cells showing cytoplasmic accumulation of α-syn. n ≥ 2 experiments using two control and two LRRK2 iPSC lines (n > 70 and n > 150 TH+ cells in Ctr and LRRK2, respectively). Scale bars, 10 μm. All values are mean + s.e.m. Differences from WT or control (*) or between control or knockdown cells (§) are significant at P 0.05; unpaired t-test in a,h and ANOVA and Bonferroni test in f,j. Full-length blots and gels in Supplementary Figure 12.


A . Whole mount in situ hybridization ( WISH ) of evi1at 20 ( left ) and 32 ( middle ) hpf . evi1expression is visible in various structures of the brain , neuronal structures , the posterior pronephric duct ( ppnd ) and the branchial arches ( ba ) , as well as in the VDA ( ventral dorsal aorta ) region . Additionally , evi1co - localizes with the endothelial marker flk1 ( right ) .

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A. Whole mount in situ hybridization (WISH) of evi1at 20 (left) and 32 (middle) hpf. evi1expression is visible in various structures of the brain, neuronal structures, the posterior pronephric duct (ppnd) and the branchial arches (ba), as well as in the VDA (ventral dorsal aorta) region. Additionally, evi1co-localizes with the endothelial marker flk1 (right).


B , C . WISH of runx1 / c - myb ; in HSPCs ( B ) , mpo in neutrophils ( left ) and l - plastin ( right ) in monocytes / macrophages ( C ) at 36 hpf in control ( upper ) and evi1 MO ( lower ) injected embryos . D - G . WISH of globinin erythrocytes of 6 dpf old embryos ( D ) , of rag - 1in the thymus of 5 dpf embryos ( E ; red asterisk ) , of cd41at 52 hpf ( F ) , and gata2bat 32 hpf ( G ) for both control ( upper ) and evi1 morpholino ( lower ) injected embryos . H . Quantitation of results is shown for each gene , displaying the percentages of embryos with normal vs . changed expression in each condition . A Fisher ´ s exact test was applied to calculate statistical significance . P < 0 . 001 ( * * * ) .

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B, C. WISH of runx1/c-myb ;in HSPCs (B), mpo in neutrophils (left) and l-plastin (right) in monocytes/macrophages (C) at 36 hpf in control (upper) and evi1 MO (lower) injected embryos.D-G. WISH of globinin erythrocytes of 6 dpf old embryos (D), of rag-1in the thymus of 5 dpf embryos (E; red asterisk), of cd41at 52 hpf (F), and gata2bat 32 hpf (G) for both control (upper) and evi1 morpholino (lower) injected embryos.H. Quantitation of results is shown for each gene, displaying the percentages of embryos with normal vs. changed expression in each condition. A Fisher´s exact test was applied to calculate statistical significance. P < 0.001 (***).


I . WISH of runx1 / c - myb in HSPCs of uninjected control and UAS : mEvi1 plasmid DNA injected Tg ( - 1 . 5hsp70l : Gal4 ) embryos with heat - shock induction performed at 14 hpf . J . WISH of runx1 / c - myb in HSPCs of uninjected control and UAS : mEvi1 mRNA in Tg ( fli . 1 : Gal4FF ; UAS : RFP ) embryos , leading to endothelial - specific evi1 overexpression . K . Graph displays quantitation of results from I and J using the same method as above .

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I. WISH of runx1/c-myb in HSPCs of uninjected control and UAS:mEvi1 plasmid DNA injected Tg(-1.5hsp70l:Gal4) embryos with heat-shock induction performed at 14 hpf.J. WISH of runx1/c-myb in HSPCs of uninjected control and UAS:mEvi1 mRNA in Tg(fli.1:Gal4FF;UAS:RFP) embryos, leading to endothelial-specific evi1 overexpression.K. Graph displays quantitation of results from I and J using the same method as above.


A . WISH of notch1b ( upper ) and dll4 ( lower ) in both control ( left ) and evi1 MO injected embryos ( right ) .

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A. WISH of notch1b(upper) and dll4(lower) in both control (left) and evi1 MO injected embryos (right).


B . WISH of notch1b in uninjected ( left ) and UAS : mEvi1 plasmid DNA injected ( right ) Tg ( fli . 1 : Gal4FF ; UAS : RFP ) embryos resulting in endothelial - specific evi1 induction .

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B. WISH of notch1b in uninjected (left) and UAS:mEvi1 plasmid DNA injected (right) Tg(fli.1:Gal4FF;UAS:RFP) embryos resulting in endothelial-specific evi1 induction.


A . Confocal time - lapse live imaging was performed in Tg ( c - myb : GFP ; kdrl : mKate - CAAX ) embryos from 28 to 42 hpf ( Movie EV 1 ) . Shown are three representative time - points in which the transformation from hemogenic endothelial cells to the hematopoietic fate is visible , indicated by the white arrowheads . For each time - point merged images are shown . White arrowheads denote double - positive cells . B . Cell counts of total numbers of kdrl + cmyb1 + cells " in view " , dividing and respectively emerging HSPCs . n = 10 movies from n = 3 biological replicates were analyzed . A non - parametric Mann - Whitney test was used to test for statistical significance and error bars are shown as ± s . d .

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A. Confocal time-lapse live imaging was performed in Tg(c-myb:GFP;kdrl:mKate-CAAX) embryos from 28 to 42 hpf (Movie EV 1). Shown are three representative time-points in which the transformation from hemogenic endothelial cells to the hematopoietic fate is visible, indicated by the white arrowheads. For each time-point merged images are shown. White arrowheads denote double-positive cells.B. Cell counts of total numbers of kdrl+cmyb1+ cells "in view", dividing and respectively emerging HSPCs. n=10 movies from n=3 biological replicates were analyzed. A non-parametric Mann-Whitney test was used to test for statistical significance and error bars are shown as ± s.d.


C . Gene expression analysis of endothelial ( kdrl , flt1 , dab2 , cdh5 ) and blood specific genes ( c - myb , CD45 , CD41 , runx1 ) in sorted kdrl + c - myb + cells from 34 hpf Tg ( c - myb : GFP ; kdrl : mKate - CAAX ) embryos . Cells were isolated from 15 - 25 pooled embryos per sample . Three biological experiments were performed with one representative shown . Error bars indicate s . d . of three technical replicates for each representative experiment .

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C. Gene expression analysis of endothelial (kdrl, flt1, dab2, cdh5) and blood specific genes (c-myb, CD45, CD41, runx1) in sorted kdrl+c-myb+ cells from 34 hpf Tg(c-myb:GFP;kdrl:mKate-CAAX) embryos. Cells were isolated from 15-25 pooled embryos per sample. Three biological experiments were performed with one representative shown. Error bars indicate s.d.
of three technical replicates for each representative experiment.


A . WISH for runx1 / c - mybin 36 hpf evi1 morphant ( left ) and evi1 morphant transgenic Tg ( 5xUAS - E1b : 6xMYC - notch1a ; - 1 . 5hsp70l : Gal4 ) embryos ( right ) with global heat - shock ( HS ) induction at 20 hpf . B . WISH for runx1 / c - myb in 36 hpf evi1 morphant ( left ) and evi1 morphant transgenic Tg ( 5xUAS - E1b : 6xMYC - notch1a ; cdh5 : gal4ff ) embryos with endothelial - specific NICD induction ( right ) . C . WISH for runx1 / c - myb in 36 hpf evi1 morphant ( left ) and evi1 morphant transgenic Tg ( Hsp70l : vegfaa ; myl7 : eGFP ) embryos ( right ) with global heat - shock induction at 20 hpf . D . WISH for runx1 / c - mybin 32 hpf evi1 MO injected embryos ( left ) and embryos injected with both evi1 MO and capped gata2 mRNA ( right ) . E . Quantitation of all rescue experiments . Shown are the percentages of embryos displaying normal or changed runx1 / c - myb expression . A Fisher ´ s exact test was applied to calculate statistical significance . ( n . s . , not significant ; p < 0 . 001 ( * * * ) ; p < 0 . 01 ( * * ) ) .

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A. WISH for runx1/c-mybin 36 hpf evi1 morphant (left) and evi1 morphant transgenic Tg(5xUAS-E1b:6xMYC-notch1a;-1.5hsp70l:Gal4) embryos (right) with global heat-shock (HS) induction at 20 hpf.B. WISH for runx1/c-myb in 36 hpf evi1 morphant (left) and evi1 morphant transgenic Tg(5xUAS-E1b:6xMYC-notch1a;cdh5:gal4ff) embryos with endothelial-specific NICD induction (right).C. WISH for runx1/c-myb in 36 hpf evi1 morphant (left) and evi1 morphant transgenic Tg(Hsp70l:vegfaa;myl7:eGFP) embryos (right) with global heat-shock induction at 20 hpf.D. WISH for runx1/c-mybin 32 hpf evi1 MO injected embryos (left) and embryos injected with both evi1 MO and capped gata2 mRNA (right).E. Quantitation of all rescue experiments. Shown are the percentages of embryos displaying normal or changed runx1/c-myb expression. A Fisher´s exact test was applied to calculate statistical significance. (n.s., not significant; p < 0.001 (***); p<0.01 (**)).


A . Confocal time - lapse live imaging was performed in Tg ( c - myb : GFP ; fli . 1 : UAS ; Gal4 : RFP ) evi1 morphant embryos ( top , Movie EV 2 ) or in Tg ( c - myb : GFP ; fli . 1 : UAS ; Gal4 : RFP ) crossed to Tg ( 5xUAS - E1b : 6xMYC - notch1a ) embryos prior to MO injection ( bottom , Movie EV 3 ) from 28 to 42 hpf . Shown are three representative time - points , in which the transformation from hemogenic endothelial cells to the hematopoietic fate is visible . For each time - point merged images are shown . NICD induction controlled by endothelial fli . 1 ( lower panel ) can rescue the evi1 morphant phenotype and increases the number of HSPCs cells emerging from the VDA ( arrows ) . B . Cell counts of emerging HSPCs in control and evi1 morphant fish with or without endothelial NICD induction . At least n = 5 movies from n = 3 biological replicates were analyzed . A non - parametric Mann - Whitney test was used to test for statistical significance and error bars are shown as ± s . d . ( n . s . , not significant , p < 0 . 05 ( * ) ) .

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A. Confocal time-lapse live imaging was performed in Tg(c-myb:GFP;fli.1:UAS;Gal4:RFP) evi1 morphant embryos (top, Movie EV 2) or in Tg(c-myb:GFP;fli.1:UAS;Gal4:RFP) crossed to Tg(5xUAS-E1b:6xMYC-notch1a) embryos prior to MO injection (bottom, Movie EV 3) from 28 to 42 hpf. Shown are three representative time-points, in which the transformation from hemogenic endothelial cells to the hematopoietic fate is visible. For each time-point merged images are shown. NICD induction controlled by endothelial fli.1 (lower panel) can rescue the evi1 morphant phenotype and increases the number of HSPCs cells emerging from the VDA (arrows).B. Cell counts of emerging HSPCs in control and evi1 morphant fish with or without endothelial NICD induction. At least n=5 movies from n=3 biological replicates were analyzed. A non-parametric Mann-Whitney test was used to test for statistical significance and error bars are shown as ± s.d. (n.s., not significant, p<0.05 (*)).


A . WISH of dll4 ( upper ) and notch1b ( lower ) in control injected embryos ( left ) , evi1 morphants ( middle ) and evi1 morphant transgenic Tg ( Hsp70l : vegfaa ; myl7 : eGFP ) embryos at 32 hpf after heat - shock induction at 20 hpf ( right ) .

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A. WISH of dll4(upper) and notch1b(lower) in control injected embryos (left), evi1 morphants (middle) and evi1 morphant transgenic Tg(Hsp70l:vegfaa;myl7:eGFP) embryos at 32 hpf after heat-shock induction at 20 hpf (right).


B , C . WISH of runx1 / c - myb ( B ) or notch1b ( C ) after treatment with DMSO ( left ) , the VEGF receptor inhibitor SU5461 ( middle ) and respectively SU5461 after injection of UAS : mEvi1 ( inducing endothelial evi1 expression ) in Tg ( fli . 1 : Gal4FF ; UAS : RFP ) embryos ( right ) .

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B, C. WISH of runx1/c-myb(B) or notch1b(C) after treatment with DMSO (left), the VEGF receptor inhibitor SU5461 (middle) and respectively SU5461 after injection of UAS:mEvi1 (inducing endothelial evi1 expression) in Tg(fli.1:Gal4FF;UAS:RFP) embryos (right).


A . Expression of runx1 / c - myb in the VDA after treatment with DMSO vehicle control ( left ) , the PI3K / AKT inhibitor Wortmannin ( WM , middle ) and after joined treatment with WM and enforced NICD expression via HS at 14 hpf in Tg ( 5xUAS - E1b : 6xMYC - notch1a ; - 1 . 5hsp70l : Gal4 ) embryos ( right ) . Graph displays quantitation of results . Shown are the percentages of embryos with normal or decreased runx1 / c - mybexpression for each condition .

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A. Expression of runx1/c-myb in the VDA after treatment with DMSO vehicle control (left), the PI3K/AKT inhibitor Wortmannin (WM, middle) and after joined treatment with WM and enforced NICD expression via HS at 14 hpf in Tg(5xUAS-E1b:6xMYC-notch1a;-1.5hsp70l:Gal4) embryos (right). Graph displays quantitation of results. Shown are the percentages of embryos with normal or decreased runx1/c-mybexpression for each condition.


B . Expression of notch1b after treatment with DMSO vehicle control ( left ) or Wortmannin ( WM , middle ) . Graph displays quantitation of results . Shown are the percentages of embryos with normal or decreased notch1b expression for each condition .

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B. Expression of notch1b after treatment with DMSO vehicle control (left) or Wortmannin (WM, middle). Graph displays quantitation of results. Shown are the percentages of embryos with normal or decreased notch1b expression for each condition.


C . WISH for runx1 / c - myb in control injected embryos ( upper left ) , evi1 morphants ( upper right ) or respectively evi1 MO with myr - AKT injected embryos with HS at 14 hpf ( lower right ) and evi1 MO with UAS - myr - AKT injected transgenic Tg ( fli . 1 : Gal4FFubs3 ; UAS : RFP ) rk embryos . D . Quantitation of results from ( C ) . Shown are the percentages of embryos with normal or decreased runx1 / c - myb expression for each condition .

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C. WISH for runx1/c-myb in control injected embryos (upper left), evi1 morphants (upper right) or respectively evi1 MO with myr-AKT injected embryos with HS at 14 hpf (lower right) and evi1 MO with UAS-myr-AKT injected transgenic Tg(fli.1:Gal4FFubs3; UAS:RFP)rk embryos.D. Quantitation of results from (C). Shown are the percentages of embryos with normal or decreased runx1/c-myb expression for each condition.


E . WISH for notch1b in evi1 Morpholino injected ( left ) and evi1 plus UAS : myr - AKT injected transgenic Tg ( fli . 1 : Gal4FFubs3 ; UAS : RFP ) rk embryos ( right ) . Graph displays quantitation of results . Shown are the percentages of embryos with normal or decreased notch1b expression for each condition .

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E. WISH for notch1b in evi1 Morpholino injected (left) and evi1 plus UAS:myr-AKT injected transgenic Tg(fli.1:Gal4FFubs3; UAS:RFP)rk embryos (right). Graph displays quantitation of results. Shown are the percentages of embryos with normal or decreased notch1b expression for each condition.


( B ) Kaplan - Meier survival curve based on n = 17 144DG11 - treated and n = 9 vehicle - treated animals . 144DG11 significantly increased survival ( log - rank test p - value < 0 . 000692 ) .

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(B) Kaplan-Meier survival curve based on n=17 144DG11-treated and n=9 vehicle-treated animals. 144DG11 significantly increased survival (log-rank test p-value<0.000692).


( M ) average duration in movement in an open field as a function of time after treating Gbeys / ys mice with vehicle ( n = 8 ) or 144DG11 ( n = 9 ) at 6 months of age ( at onset ) . Data information : Two way ANOVA with repeated measures show that , throughout the period , treatment values were not different than vehicle ( p < 0 . 15 ) .

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(M) average duration in movement in an open field as a function of time after treating Gbeys/ys mice with vehicle (n=8) or 144DG11 (n=9) at 6 months of age (at onset). Data information: Two way ANOVA with repeated measures show that, throughout the period, treatment values were not different than vehicle (p<0.15).


( A ) Left panel , Mice , treated as indicated , were sacrificed and the indicated tissues were collected and stained for PG ( arrows ) with PAS following diastase treatment . Scale bars , 200 µm ( liver , muscle ) , 50 µm ( cortex , heart , peripheral nerve ) . Right panel , PAS staining was quantified based on analysis of 4 sections from each tissue in n = 3 wt , n = 7 Gbeys / ys vehicle - treated , and n = 9 144DG11 - treated mice .

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(A) Left panel, Mice, treated as indicated, were sacrificed and the indicated tissues were collected and stained for PG (arrows) with PAS following diastase treatment. Scale bars, 200 µm (liver, muscle), 50 µm (cortex, heart, peripheral nerve). Right panel, PAS staining was quantified based on analysis of 4 sections from each tissue in n=3 wt, n=7 Gbeys/ys vehicle-treated, and n=9 144DG11-treated mice.


( C ) 144DG11 Pharmacokinetics . Gbeys / ys mice injected with 144DG1 were sacrificed 30 , 60 , 90 , and 210 min post injection and the indicated tissues were removed , as well as 200 uL of serum drawn . Graph shows means ( + / - SEM ) of 144DG11 levels in the different tissues determined by LC - MS / MS Results obtained from n = 3 mice at each time point .

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(C) 144DG11 Pharmacokinetics. Gbeys/ys mice injected with 144DG1 were sacrificed 30, 60, 90, and 210 min post injection and the indicated tissues were removed, as well as 200 uL of serum drawn. Graph shows means (+/- SEM) of 144DG11 levels in the different tissues determined by LC-MS/MS Results obtained from n = 3 mice at each time point.


A - F Mice were monitored over a 24 hr period . Effective mass was calculated by ANCOVA Data are mean ± SEM from nine month old mice ( n = 11 , wt vehicle - treated , n = 6 Gbeys / ys vehicle - treated , and n = 7 Gbeys / ys 144DG11 - treated ) . Vehicle - treated Gbeys / ys mice demonstrate lower respiratory quotient ( in the light ) ( A ) , total energy expenditure ( TEE ) ( B ) , and fat oxidation ( C ) compared to wt controls . 144DG11 treatment increased these parameters ( for fat oxidation only in the dark and total time ) . Carbohydrate oxidation and ambulatory activity , not significantly affected by the diseased state , were increased by 144DG11 even beyond wt control levels ( D and E ) ( note , while 144DG11 increased carbohydrate oxidation in the light ( D ) , p was only < 0 . 06 ) . 144DG11 also reversed the decrease in meal size and water sip volume observed in Gbeys / ys mice as compared to wt control ( F ) . Data information : * p < 0 . 05 v wt controls , # p < 0 . 05 v Gbeys / ys vehicle treated mice . Statistical differences were determined by two - tailed t - tests .

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A-F Mice were monitored over a 24 hr period. Effective mass was calculated by ANCOVA Data are mean±SEM from nine month old mice (n=11, wt vehicle-treated, n=6 Gbeys/ys vehicle-treated, and n=7 Gbeys/ys 144DG11-treated). Vehicle-treated Gbeys/ys mice demonstrate lower respiratory quotient (in the light) (A), total energy expenditure (TEE) (B), and fat oxidation (C) compared to wt controls. 144DG11 treatment increased these parameters (for fat oxidation only in the dark and total time). Carbohydrate oxidation and ambulatory activity, not significantly affected by the diseased state, were increased by 144DG11 even beyond wt control levels (D and E) (note, while 144DG11 increased carbohydrate oxidation in the light (D), p was only <0.06). 144DG11 also reversed the decrease in meal size and water sip volume observed in Gbeys/ys mice as compared to wt control (F). Data information: *p<0.05 v wt controls, #p<0.05 v Gbeys/ys vehicle treated mice. Statistical differences were determined by two-tailed t-tests.


( G ) Blood metabolic panel based on n = 3 , 9 . 5 month old mice treated as indicated . Blood glucose was increased and blood triglycerides decreased in Gbeys / ys cells by 144DG11 ( p < 0 . 05 ) .

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(G) Blood metabolic panel based on n=3, 9.5 month old mice treated as indicated. Blood glucose was increased and blood triglycerides decreased in Gbeys/ys cells by 144DG11 (p<0.05).


( A ) PAS staining for total glycogen in skin fibroblasts from different APBD patients . Staining fluorescence was quantified by InCell2200 ( see Methods ) . Means ( + / - SEM ) are statistically different from each other ( p < 0 . 0001 , One Way ANOVA ) . n = 3 technical replicates from each patient indicated at the x - axis were used .

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(A) PAS staining for total glycogen in skin fibroblasts from different APBD patients. Staining fluorescence was quantified by InCell2200 (see Methods). Means (+/- SEM) are statistically different from each other (p<0.0001, One Way ANOVA). n=3 technical replicates from each patient indicated at the x-axis were used.


( B ) PAS staining for total glycogen in APBD87 fibroblasts glucose - starved for 48 h ( left ) , or glucose starved and then replenished for the last 24 h to induce glycogen burden ( right ) . Image acquisition performed by Nikon Eclipse Ti2 microscope using a 40x PlanFluor objective and CY3 filter . Scale bar , 100 µm .

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(B) PAS staining for total glycogen in APBD87 fibroblasts glucose-starved for 48 h (left), or glucose starved and then replenished for the last 24 h to induce glycogen burden (right). Image acquisition performed by Nikon Eclipse Ti2 microscope using a 40x PlanFluor objective and CY3 filter. Scale bar, 100 µm.


( D ) Glycolytic ( red ) and mitochondrial ( blue ) ATP production determined by Agilent ' s Seahorse machine and real - time ATP rate assay kit . HC and APBD patient fibroblasts were serum / glucose - starved for 48 h and then full medium was replenished for 24 h without ( untreated ) , or with ( chronic ) 50 µM 144DG11 . Acute , 50 µM 144DG11 was added on assay for 20 min after 24 h of serum / glucose replenishment . Readings were normalized to cell number as determined by Crystal Violet staining . Shown are mean and SEM values based on n = 3 - 6 technical repeats . For all experiments ( HC , p < 0 . 01 ; APBD1 , p < 0 . 02 ; APBD2 , p < 0 . 09 ; APBD3 , p < 0 . 11 ; One Way ANOVA with Sidak ' s post - hoc correction for multiple comparisons ) glycolytic ATP production was increased , as compared to untreated control , by acute , but not chronic , supplementation of 144DG11 . Mitochondrial ATP production was increased only by acute supplementation of 144DG11 to APBD2 fibroblasts ( p < 0 . 06 ) .

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(D) Glycolytic (red) and mitochondrial (blue) ATP production determined by Agilent's Seahorse machine and real-time ATP rate assay kit. HC and APBD patient fibroblasts were serum/glucose-starved for 48 h and then full medium was replenished for 24 h without (untreated), or with (chronic) 50 µM 144DG11. Acute, 50 µM 144DG11 was added on assay for 20 min after 24 h of serum/glucose replenishment. Readings were normalized to cell number as determined by Crystal Violet staining. Shown are mean and SEM values based on n=3-6 technical repeats. For all experiments (HC, p<0.01; APBD1, p<0.02; APBD2, p<0.09; APBD3, p<0.11; One Way ANOVA with Sidak's post-hoc correction for multiple comparisons) glycolytic ATP production was increased, as compared to untreated control, by acute, but not chronic, supplementation of 144DG11. Mitochondrial ATP production was increased only by acute supplementation of 144DG11 to APBD2 fibroblasts (p<0.06).


( C ) Cellular thermal shift assay ( CETSA ) of different targets of the 144DG11hetero - assembly ( B ) . Only LAMP1 was significantly protected by 144DG11 from heat - mediated denaturation ( see right shift in its Tm in the lower panel ) suggesting its specific interaction with 144DG11 . n = 3 biological replicates . Error bars represent S . D .

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(C) Cellular thermal shift assay (CETSA) of different targets of the 144DG11hetero-assembly (B). Only LAMP1 was significantly protected by 144DG11 from heat-mediated denaturation (see right shift in its Tm in the lower panel) suggesting its specific interaction with 144DG11. n=3 biological replicates. Error bars represent S.D.


( A ) Left panel , Autophagic flux , determined by the extent of lysosomal inhibitors - dependent increase in the ratio of lipidated to non - lipidated LC3 ( LC3II / LC3I ) , is increased by 144DG11 ( 50µM , 24h ) . Right panel , 144DG11 - mediated increase in autophagic flux , demonstrated by enhanced degradation of the autophagy substrate p62 . Shown are representative immunoblots ( grouped from different gels ) and densitometric quantifications + / - s . d . ( for LC3II / LC3I , n = 4 biological replicates , * p < 0 . 0023 ; for p62 , n = 5 biological replicates , * p < 0 . 0099 ; two - tailed t - test ) .

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(A) Left panel, Autophagic flux, determined by the extent of lysosomal inhibitors-dependent increase in the ratio of lipidated to non-lipidated LC3 (LC3II/LC3I), is increased by 144DG11 (50µM, 24h). Right panel, 144DG11-mediated increase in autophagic flux, demonstrated by enhanced degradation of the autophagy substrate p62. Shown are representative immunoblots (grouped from different gels) and densitometric quantifications +/-s.d. (for LC3II/LC3I, n=4 biological replicates, *p<0.0023; for p62, n=5 biological replicates, *p<0.0099; two-tailed t-test).


( B ) Left panel , TEM images of liver tissue from vehicle or 144DG11 - treated Gbeys / ys mice . High - magnification ( right , scale bars = 200 nm ) and low - magnification ( left , scale bars = 1 µm ) images show higher levels of glycogen / polyglucosan in lysosomes and cytosol , respectively . Right panel , quantification ( + / - s . d ) of lysosomal glycogen particles ( n = 3 biological replicates , * p < 0 . 03 , two - tailed t - test ) . G , Glycogen / polyglucosan ; L , Lysosomes ; M , Mitochondria .

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(B) Left panel, TEM images of liver tissue from vehicle or 144DG11-treated Gbeys/ys mice. High-magnification (right, scale bars=200 nm) and low-magnification (left, scale bars=1 µm) images show higher levels of glycogen/polyglucosan in lysosomes and cytosol, respectively. Right panel, quantification (+/- s.d) of lysosomal glycogen particles (n=3 biological replicates, *p<0.03, two-tailed t-test). G, Glycogen/polyglucosan; L, Lysosomes; M, Mitochondria.


( C ) 144DG11 reduces LC3 and p62 in mouse liver , but not muscle ( n = 3 biological replicates , * p < 0 . 04 for LC3 , * * p < 0 . 0007 for p62 ; two - tailed t - test , SEM ) . Scale bars , 10

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(C) 144DG11 reduces LC3 and p62 in mouse liver, but not muscle (n=3 biological replicates, *p<0.04 for LC3, **p<0.0007 for p62; two-tailed t-test, SEM). Scale bars, 10 µm


( E ) LAMP1 - KD and 144DG11 treatment cause lysosomal acidification . Upper panel , flow cytometry results showing that 144DG11 slightly increased acidification ( yellow to blue median fluorescence ratio ( Y / B ) ) in control , GFP - transduced , APBD fibroblasts ( Y / B ( GFP / 144DG11 ) > Y / B ( GFP ) , p < 0 . 12 ) , but significantly acidified LAMP1 - KD , GFP - shLAMP1 - transduced , APBD fibroblasts ( Y / B ( LAMP1 - KD / 144DG11 ) > ( Y / B ( LAMP1 - KD ) , p < 0 . 03 ) . LAMP1 - KD itself led to the most significant acidification ( Y / B ( LAMP1 - KD ) > Y / B ( GFP ) , p < 0 . 007 ) . n = 3 , two - tailed t - tests . Middle panel Lysosensor staining of the corresponding cells . Yellow fluorescence intensity correlates with acidification . Lower panel , PAS ( glycogen ) staining of the corresponding cells . Scale bars , 50 µm .

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(E) LAMP1-KD and 144DG11 treatment cause lysosomal acidification. Upper panel, flow cytometry results showing that 144DG11 slightly increased acidification (yellow to blue median fluorescence ratio (Y/B)) in control, GFP-transduced, APBD fibroblasts (Y/B(GFP/144DG11)>Y/B(GFP), p<0.12), but significantly acidified LAMP1-KD, GFP-shLAMP1-transduced, APBD fibroblasts (Y/B(LAMP1-KD/144DG11)>(Y/B(LAMP1-KD), p<0.03). LAMP1-KD itself led to the most significant acidification (Y/B (LAMP1-KD)>Y/B(GFP), p<0.007). n=3, two-tailed t-tests. Middle panel Lysosensor staining of the corresponding cells. Yellow fluorescence intensity correlates with acidification. Lower panel, PAS (glycogen) staining of the corresponding cells. Scale bars, 50 µm.


( G ) 144DG11 reduces lysosomal ( LAMP1 positive ) area in liver , but not muscle , of Gbeys / ys mice ( n = 3 biological replicates , * p < 0 . 01 , two - tailed t - test , SEM ) . Scale bars , 5 µm for liver ( upper left panel ) , and 10 µm for muscle ( lower left panel ) . Right panel shows quantification of the left panel .

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(G) 144DG11 reduces lysosomal (LAMP1 positive) area in liver, but not muscle, of Gbeys/ys mice (n=3 biological replicates, *p<0.01, two-tailed t-test , SEM).Scale bars, 5 µm for liver (upper left panel), and 10 µm for muscle (lower left panel). Right panel shows quantification of the left panel.


( B ) Morphological characterization of lysosomes based on lysotracker staining of n = 4 HC and n = 4 ( biological replicates ) APBD patient skin fibroblasts starved for 48 h and treated or not with 50 uM 144DG11 for 24 h . Values are deviations from untreated HC . 144DG11 has reduced lysotracker mean intensity , integrated intensity ( DXA ) , and area in both HC and APBD fibroblasts ( * * * p < 0 . 0001 , One - Way ANOVA with Sidak ' s multi - comparison post - hoc test ) .

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(B) Morphological characterization of lysosomes based on lysotracker staining of n=4 HC and n=4 (biological replicates) APBD patient skin fibroblasts starved for 48 h and treated or not with 50 uM 144DG11 for 24 h. Values are deviations from untreated HC. 144DG11 has reduced lysotracker mean intensity, integrated intensity (DXA), and area in both HC and APBD fibroblasts (***p<0.0001, One-Way ANOVA with Sidak's multi-comparison post-hoc test).


( C ) Functional ( mitochondrial membrane potential ( TMRE parameters ) ) and biomass ( mitotracker ) characterization of mitochondria in HC and APBD cells treated and analyzed as in ( B ) . Mean ( * * * , p < 0 . 0001 ) and integrated ( * * * , p < 0 . 0006 ) TMRE intensities , and mitochondrial biomass ( * * * , p < 0 . 0001 ) , were reduced in APBD v HC . 144DG11 increased TMRE mean ( * * , p < 0 . 009 , * * , p < 0 . 006 ) and integrated ( * , p < 0 . 01 , * * , p < 0 . 001 ) intensities in APBD and HC cells , respectively and mitotracker intensity in APBD cells only ( * * * , p < 0 . 0001 ) . Data represent n = 3 biological replicates . Statistical differences analyzed by One - way ANOVA with multiple comparisons .

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(C) Functional (mitochondrial membrane potential (TMRE parameters)) and biomass (mitotracker) characterization of mitochondria in HC and APBD cells treated and analyzed as in (B). Mean (***, p<0.0001) and integrated (***, p<0.0006) TMRE intensities, and mitochondrial biomass (***, p<0.0001), were reduced in APBD v HC. 144DG11 increased TMRE mean (**, p<0.009, **, p<0.006) and integrated (*, p<0.01, **, p<0.001) intensities in APBD and HC cells, respectively and mitotracker intensity in APBD cells only (***, p<0.0001). Data represent n=3 biological replicates. Statistical differences analyzed by One-way ANOVA with multiple comparisons.


A Agarose gel image illustrating PCR amplicons of ltaS ( LBA0447 ) , slpB , and slpX deletions in NCK2187 .

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A Agarose gel image illustrating PCR amplicons of ltaS (LBA0447), slpB, and slpX deletions in NCK2187.


B SDS - PAGE gel of 5 M LiCl ‐ purified S ‐ layer fractions from the parental strains , NCK56 and NCK1909 , NCK2030 ( LTA + , SlpA + , SlpB − , SlpX − ) , and NCK2187 ( LTA − , SlpA + , SlpB − , SlpX − ) .

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B SDS-PAGE gel of 5 M LiCl‐purified S‐layer fractions from the parental strains, NCK56 and NCK1909, NCK2030 (LTA+, SlpA+, SlpB−, SlpX−), and NCK2187 (LTA−, SlpA+, SlpB−, SlpX−).


C B6 mice were orally gavaged with 109 CFU erythromycin ‐ resistant NCK56 or NCK2187 . Fecal pellets were collected daily and tested for the presence of erythromycin ‐ resistant strains . n = 3 mice / group . Data are representative of five independent experiments and are shown as mean ± SEM .

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C B6 mice were orally gavaged with 109 CFU erythromycin‐resistant NCK56 or NCK2187. Fecal pellets were collected daily and tested for the presence of erythromycin‐resistant strains. n = 3 mice/group. Data are representative of five independent experiments and are shown as mean ± SEM.


D ColonicLPL cells were co ‐ cultured with NCK56 or NCK2187 ( 1 : 1 ) , and secreted cytokines were measured in the supernatants . Data are shown as mean ± SEM and are representative of two experiments performed in triplicate . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

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D ColonicLPL cells were co‐cultured with NCK56 or NCK2187 (1:1), and secreted cytokines were measured in the supernatants. Data are shown as mean ± SEM and are representative of two experiments performed in triplicate. *P 0.05, **P 0.01, ***P 0.001.


A B6 mice were orally gavaged with 109CFU NCK56 ( blue ) or NCK2187 ( green ) on days 0 , 3 , 6 , and 9 , or left untreated , and immune responses in the colon analyzed at day 14 by flow cytometry .

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A B6 mice were orally gavaged with 109CFU NCK56 (blue) or NCK2187 (green) on days 0, 3, 6, and 9, or left untreated, and immune responses in the colon analyzed at day 14 by flow cytometry.


B , C B6 FoxP3 ‐ GFP mice were treated and evaluated as in ( A ) . Regulatory cytokine production in FoxP3 ‐ GFP + ( green ‐ dotted bars ) versus FoxP3 ‐ GFP − ( white bars ) cells was measured by intracellular staining and FACS analyses ( C ) .

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B, C B6 FoxP3‐GFP mice were treated and evaluated as in (A). Regulatory cytokine production in FoxP3‐GFP+ (green‐dotted bars) versus FoxP3‐GFP− (white bars) cells was measured by intracellular staining and FACS analyses (C).


A B6 Rag1 − / − mice were injected with 106CD4 + CD45RBhiT cells and then orally gavaged with NCK56 ( red ) , NCK2187 ( green ) , or SlpA ( blue ) , 1 and 3 days after transfer , and subsequently once a week for four consecutive weeks , or left untreated ( magenta ) . A group of mice was co ‐ transferred with CD4 + CD25 + T cells as a positive control for protection ( Tregs ; gray ) . Colitis severity was determined in part by weight loss , diarrhea scores , and FOB . See Supplementary Tables S1 , S2 and S3 for statistical analyses results .

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A B6 Rag1−/−mice were injected with 106CD4+CD45RBhiT cells and then orally gavaged with NCK56 (red), NCK2187 (green), or SlpA (blue), 1 and 3 days after transfer, and subsequently once a week for four consecutive weeks, or left untreated (magenta). A group of mice was co‐transferred with CD4+CD25+T cells as a positive control for protection (Tregs; gray). Colitis severity was determined in part by weight loss, diarrhea scores, and FOB. See Supplementary Tables S1, S2 and S3 for statistical analyses results.


B , C Colitis scores based on histopathology and gross morphology of the colons of mice treated as in ( A ) were also used as measures of disease . Scale bar = 50 μm .

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B, C Colitis scores based on histopathology and gross morphology of the colons of mice treated as in (A) were also used as measures of disease. Scale bar = 50 μm.


D Circulating levels of proinflammatory cytokines were measured in the sera of the mice transferred and treated as in ( A ) , or sham adoptive transferred ( white bars ) .

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D Circulating levels of proinflammatory cytokines were measured in the sera of the mice transferred and treated as in (A), or sham adoptive transferred (white bars).


Colitis was induced in B6 Rag1 − / − mice as described in Fig . A , B Colonic expression of tight junction ‐ associated genes Cldn3 and Ocln , determined by RT - PCR relative to 18S rRNA ( A ) , were used as measures of epithelial barrier integrity . Sham adoptive transferred B6 Rag1 − / − mice ( white bars ) were used as baseline controls in some cases . n = 5 mice / group . Data represent three individual experiments and are shown as mean ± SEM . * P 0 . 05 , * * P 0 . 01 . Black asterisks compare NCK2187 to PBS ‐ treated adoptively transferred mice , and red asterisks to NCK56 ‐ treated mice .

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Colitis was induced in B6 Rag1−/−mice as described in Fig .A, B Colonic expression of tight junction‐associated genes Cldn3 and Ocln, determined by RT-PCR relative to 18S rRNA (A), were used as measures of epithelial barrier integrity. Sham adoptive transferred B6 Rag1−/−mice (white bars) were used as baseline controls in some cases. n = 5 mice/group. Data represent three individual experiments and are shown as mean ± SEM. *P 0.05, **P 0.01. Black asterisks compare NCK2187 to PBS‐treated adoptively transferred mice, and red asterisks to NCK56‐treated mice.


Colitis was induced in B6 Rag1 − / − mice as described in Fig . A , B Passive transepithelial absorption of FITC ‐ dextran ( B ) , was used as measures of epithelial barrier integrity . Sham adoptive transferred B6 Rag1 − / − mice ( white bars ) were used as baseline controls in some cases . n = 5 mice / group . Data represent three individual experiments and are shown as mean ± SEM . * P 0 . 05 , * * P 0 . 01 . Black asterisks compare NCK2187 to PBS ‐ treated adoptively transferred mice , and red asterisks to NCK56 ‐ treated mice .

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Colitis was induced in B6 Rag1−/−mice as described in Fig .A, B Passive transepithelial absorption of FITC‐dextran (B), was used as measures of epithelial barrier integrity. Sham adoptive transferred B6 Rag1−/−mice (white bars) were used as baseline controls in some cases. n = 5 mice/group. Data represent three individual experiments and are shown as mean ± SEM. *P 0.05, **P 0.01. Black asterisks compare NCK2187 to PBS‐treated adoptively transferred mice, and red asterisks to NCK56‐treated mice.


A B6 mice were orally gavaged with 109CFU NCK56 or NCK2187 , and the colonic gene expression of C ‐ type lectin receptors were measured by RT - PCR . Each box represents an individual mouse ; n = 4 . Data represent three individual experiments .

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A B6 mice were orally gavaged with 109CFU NCK56 or NCK2187, and the colonic gene expression of C‐type lectin receptors were measured by RT-PCR. Each box represents an individual mouse; n = 4. Data represent three individual experiments.


B Binding of SlpA to various hFc fusion proteins was analyzed by flow cytometry . Gray tinted line = SlpA ‐ coated beads only ; orange = SlpA ‐ coated beads + secondary antibody ; green = SlpA ‐ coated beads + control fusion protein ; blue = SlpA ‐ coated beads + SIGNR1 ‐ hFc ; red = SlpA ‐ coated beads + SIGNR3 ‐ hFc . Binding assay results were confirmed five independent times .

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B Binding of SlpA to various hFc fusion proteins was analyzed by flow cytometry. Gray tinted line = SlpA‐coated beads only; orange = SlpA‐coated beads + secondary antibody; green = SlpA‐coated beads + control fusion protein; blue = SlpA‐coated beads + SIGNR1‐hFc; red = SlpA‐coated beads + SIGNR3‐hFc. Binding assay results were confirmed five independent times.


C Binding of SlpA to SIGNR3 , but not SIGNR1 expressed by CHO ‐ S cells . Gray tinted line = untransfected CHO ‐ S cells ; blue = untransfected CHO ‐ S cells + labeled SlpA ; green = SIGNR1 ‐ transfected CHO ‐ S cells + labeled SlpA ; red = SIGNR3 ‐ transfected CHO ‐ S cells + labeled SlpA . Binding assays in CHO ‐ S cells were performed three individual times .

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C Binding of SlpA to SIGNR3, but not SIGNR1 expressed by CHO‐S cells. Gray tinted line = untransfected CHO‐S cells; blue = untransfected CHO‐S cells + labeled SlpA; green = SIGNR1‐transfected CHO‐S cells + labeled SlpA; red = SIGNR3‐transfected CHO‐S cells + labeled SlpA. Binding assays in CHO‐S cells were performed three individual times.


D Binding of SlpA to recombinant human DC ‐ SIGN / CD209 ‐ Fc chimera was analyzed by flow cytometry . Gray tinted line = SlpA ‐ coated beads only ; orange = SlpA ‐ coated beads + secondary antibody ; green = SlpA ‐ coated beads + control fusion protein ; red = SlpA ‐ coated beads + DC ‐ SIGN ‐ Fc . Binding assays were performed and confirmed three times .

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D Binding of SlpA to recombinant human DC‐SIGN/CD209‐Fc chimera was analyzed by flow cytometry. Gray tinted line = SlpA‐coated beads only; orange = SlpA‐coated beads + secondary antibody; green = SlpA‐coated beads + control fusion protein; red = SlpA‐coated beads + DC‐SIGN‐Fc. Binding assays were performed and confirmed three times.


E Binding of SlpA to DC ‐ SIGN expressed by CHO ‐ S cells . Gray tinted line = untransfected CHO ‐ S cells ; blue = untransfected CHO ‐ S cells + labeled SlpA ; red = DC ‐ SIGN ‐ transfected CHO ‐ S cells + labeled SlpA . Binding assays in CHO ‐ S cells were performed three individual times .

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

E Binding of SlpA to DC‐SIGN expressed by CHO‐S cells. Gray tinted line = untransfected CHO‐S cells; blue = untransfected CHO‐S cells + labeled SlpA; red = DC‐SIGN‐transfected CHO‐S cells + labeled SlpA. Binding assays in CHO‐S cells were performed three individual times.


F , G IL ‐ 1β production by colonic DCs of naïve WT B6 GF or B6 Signr3 − / − ( KO ) mice treated with NCK56 ( blue ) or NCK2187 ( green ) four times , or left untreated ( black ) , as determined by flow cytometry . n = 5 mice / group . Data represent four individual experiments and are shown as mean ± SEM . * P 0 . 05 . Black asterisks compare NCK2187 to untreated ( PBS ) mice , and red asterisks to NCK56 ‐ treated mice .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G IL‐1β production by colonic DCs of naïve WT B6 GF or B6 Signr3−/− (KO) mice treated with NCK56 (blue) or NCK2187 (green) four times, or left untreated (black), as determined by flow cytometry. n = 5 mice/group. Data represent four individual experiments and are shown as mean ± SEM. *P 0.05. Black asterisks compare NCK2187 to untreated (PBS) mice, and red asterisks to NCK56‐treated mice.


H Frequency of colonic FoxP3 + Tregs in KO mice treated with NCK56 or NCK2187 was measured by flow cytometry . n = 5 mice / group . Data represent four individual experiments and are shown as mean ± SEM .

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H Frequency of colonic FoxP3+ Tregs in KO mice treated with NCK56 or NCK2187 was measured by flow cytometry. n = 5 mice/group. Data represent four individual experiments and are shown as mean ± SEM.


WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . A Colitis severity was determined in part by weight loss . n = 5 mice / group .

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

WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.A Colitis severity was determined in part by weight loss. n = 5 mice/group.


WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . B , C Colitis scores based on histopathology and gross morphology of the colons were also used as measures of disease . Scale bar = 50 μm . n = 5 mice / group . Empty bars = WT ; lined bars = KO ; white bars = untreated ; purple bars = DSS ; red bars = DSS + NCK56 ; green bars = DSS + NCK2187 ; blue bars = DSS + SlpA .

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

WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.B, C Colitis scores based on histopathology and gross morphology of the colons were also used as measures of disease. Scale bar = 50 μm. n = 5 mice/group. Empty bars = WT; lined bars = KO; white bars = untreated; purple bars = DSS; red bars = DSS + NCK56; green bars = DSS + NCK2187; blue bars = DSS + SlpA.


WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . D Colonoscopies were performed in the different groups with a Multi ‐ Purpose Rigid ™ Telescope attached to a TELE PACK X on day 10 .

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

WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.D Colonoscopies were performed in the different groups with a Multi‐Purpose Rigid™ Telescope attached to a TELE PACK X on day 10.


WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . E Mean relative colonic expression of tight junction ‐ associated genes in WT mice . n = 5 mice / group .

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

WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.E Mean relative colonic expression of tight junction‐associated genes in WT mice. n = 5 mice/group.


WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . F Fecalalbumin levels in WT mice as a measure of intestinal permeability . n = 5 mice / group .

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

WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.F Fecalalbumin levels in WT mice as a measure of intestinal permeability. n = 5 mice/group.


Signr3 + / + ( WT ) or Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 1 and − 3 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times , and immunity was analyzed by flow cytometry at day 10 . A Representative plots indicate the frequency of neutrophils in the colons of untreated or DSS ‐ treated WT ( left ) and KO mice ( right ) . Empty bars = WT ; lined bars = KO ; white bars = untreated ; purple bars = DSS ; red bars = DSS + NCK56 ; green bars = DSS + NCK2187 ; blue bars = DSS + SlpA .

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

Signr3+/+ (WT) or Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −1 and −3, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times, and immunity was analyzed by flow cytometry at day 10.A Representative plots indicate the frequency of neutrophils in the colons of untreated or DSS‐treated WT (left) and KO mice (right). Empty bars = WT; lined bars = KO; white bars = untreated; purple bars = DSS; red bars = DSS + NCK56; green bars = DSS + NCK2187; blue bars = DSS + SlpA.


Signr3 + / + ( WT ) or Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 1 and − 3 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times , and immunity was analyzed by flow cytometry at day 10 . B , C Colonic DCs and macrophages ( MΦs ) were analyzed by flow cytometry for the production of IL ‐ 1β ( B ) , and colonic FoxP3 + Tregs were evaluated for co ‐ expression of RORγt + ( C ) . n = 5 mice / group . Gray tinted line = isotype control ; black = untreated ; purple = DSS ; red = DSS + NCK56 ; green = DSS + NCK2187 ; blue = DSS + SlpA .

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Signr3+/+ (WT) or Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −1 and −3, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times, and immunity was analyzed by flow cytometry at day 10.B, C Colonic DCs and macrophages (MΦs) were analyzed by flow cytometry for the production of IL‐1β (B), and colonic FoxP3+ Tregs were evaluated for co‐expression of RORγt+ (C). n = 5 mice/group. Gray tinted line = isotype control; black = untreated; purple = DSS; red = DSS + NCK56; green = DSS + NCK2187; blue = DSS + SlpA.


The violin plot of ACE2 gene expression of all major cell types .

The violin plot of ACE2 gene expression of all major cell types.


Cell type annotations of all four patients by the tSNE plot . Hepatocyte - cholangiocytes cluster includes potential stem - like cells capable of differentiating into both cell types . B ) tSNE plot colored by

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Cell type annotations of all four patients by the tSNE plot. Hepatocyte-cholangiocytes cluster includes potential stem-like cells capable of differentiating into both cell types. B) tSNE plot colored by patients


Heatmap of marker genes of 16 cell types identified . The red box highlights the cell type specific expression of ACE2 .

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Heatmap of marker genes of 16 cell types identified. The red box highlights the cell type specific expression of ACE2.


The violin plot of ACE2 gene expression across all cell types .

The violin plot of ACE2 gene expression across all cell types.


A , B NAD and nicotinamide ( NAM ) levels in heart of WT ( n = 5 , n = 6 , respectively ) , mdx ( n = 5 , n = 5 , respectively ) and mdx / CD38 - / - mice ( n = 6 , n = 5 , respectively ) . C , D NAD and NAM levels in diaphragm of WT ( n = 5 , n = 6 , respectively ) , mdx ( n = 4 , n = 5 , respectively ) and mdx / CD38 - / - mice ( n = 6 , n = 5 , respectively ) . Data information : Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests , significance was assessed using : ANOVA followed by Fisher ' s LSD test ; B : Kruskal - Wallis followed by Dunn ' s test ; D : Kruskal - Wallis followed by Mann - Whitney tests ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

A,B NAD and nicotinamide (NAM) levels in heart of WT (n=5, n=6, respectively), mdx (n=5, n=5, respectively) and mdx/CD38-/- mice (n=6, n=5, respectively). C,D NAD and NAM levels in diaphragm of WT (n=5, n=6, respectively), mdx (n=4, n=5, respectively) and mdx/CD38-/- mice (n=6, n=5, respectively). Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test; B: Kruskal-Wallis followed by Dunn's test; D: Kruskal-Wallis followed by Mann-Whitney tests; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


E , F levels of ADP - ribose ( ADPR ) ( E ) , cyclic ADP - ribose ( cADPR ) ( F ) expressed as nmol / mg protein in heart of WT ( n = 6 ) , mdx ( n = 5 ) and mdx / CD38 - / - mice ( n = 5 , n = 5 , n = 6 respectively ) . Data information : Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests , significance was assessed using : : ANOVA followed by Fisher ' s LSD test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

E,F levels of ADP-ribose (ADPR) (E), cyclic ADP-ribose (cADPR) (F) expressed as nmol/mg protein in heart of WT (n=6), mdx (n=5) and mdx/CD38-/- mice (n=5, n=5, n=6 respectively). Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: : ANOVA followed by Fisher's LSD test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


( G ) qPCR analysis of mRNA levels of Sterile alpha and Toll / interleukin - 1 receptor motif - containing 1 ( SARM1 ) in heart of WT ( n = 6 ) , mdx ( n = 5 ) and mdx / CD38 - / - mice ( n = 5 , n = 5 , n = 6 respectively ) . Data information : Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests , significance was assessed using : ANOVA followed by Fisher ' s LSD test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(G) qPCR analysis of mRNA levels of Sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1) in heart of WT (n=6), mdx (n=5) and mdx/CD38-/- mice (n=5, n=5, n=6 respectively). Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


H , I Levels of ADPR ( H ) , cADPR ( I ) expressed as nmol / mg protein in diaphragm of WT ( n = 6 ) , mdx ( n = 5 ) and mdx / CD38 - / - ( n = 5 ) mice . Data information : Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests , significance was assessed using : ANOVA followed by Fisher ' s LSD test ; ; I : Welch ' s ANOVA followed by Welch ' s t - tests Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

H,I Levels of ADPR (H), cADPR (I) expressed as nmol/mg protein in diaphragm of WT (n=6), mdx (n=5) and mdx/CD38-/- (n=5) mice. Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test; ; I: Welch's ANOVA followed by Welch's t-tests Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


( J ) qPCR analysis mRNA levels of SARM1 in diaphragm of WT ( n = 6 ) , mdx ( n = 5 ) and mdx / CD38 - / - ( n = 5 ) mice . Data information : Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests , significance was assessed using : J : ANOVA Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(J) qPCR analysis mRNA levels of SARM1 in diaphragm of WT (n=6), mdx (n=5) and mdx/CD38-/- (n=5) mice. Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: J: ANOVA Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


K , L Western blot analysis of CD38 protein expression in heart ( K ) and diaphragm ( L ) of WT ( n = 5 , n = 6 , respectively ) and mdx ( n = 5 ) mice . Vinculin is used as housekeeping protein control and the dot plots show the ratio of CD38 / vinculin . Data information : Each dot of the graphs represents a value / mouse . After normality and variance comparison tests , significance was assessed using : unpaired Student ' s t - test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

K, L Western blot analysis of CD38 protein expression in heart (K) and diaphragm (L) of WT (n=5, n=6, respectively) and mdx (n=5) mice. Vinculin is used as housekeeping protein control and the dot plots show the ratio of CD38/vinculin. Data information: Each dot of the graphs represents a value/mouse. After normality and variance comparison tests, significance was assessed using: unpaired Student's t-test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


M , N qPCR analysis of CD38 mRNA levels in heart ( M ) and diaphragm ( N ) of WT ( n = 6 ) and mdx ( n = 5 ) mice . Data information : Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests , significance was assessed using : M : unpaired Student ' s t - test ; N : unpaired Welch ' s t - test . Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

M, N qPCR analysis of CD38 mRNA levels in heart (M) and diaphragm (N) of WT (n=6) and mdx (n=5) mice. Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: M: unpaired Student's t-test; N: unpaired Welch's t-test. Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


A Cardiac function evaluated by echocardiography in mdx / CD38 - / - mice : the dot plots show the left ventricular ( LV ) diastolic ( A1 ) and systolic ( A2 ) inner diameters and LV ejection ( A3 ) fraction in 7 - month - old WT ( n = 5 ) , mdx ( n = 6 ) and mdx / CD38 - / - ( n = 10 ) mice . Data information : : Each dot of the graphs represents a mouse . in duplicate After normality and variance comparison tests , significance was assessed using : A1 ANOVA followed by Fisher ' s LSD test ; A2 , A3 Kruskal - Wallis followed by Dunn ' s test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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 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

A Cardiac function evaluated by echocardiography in mdx/CD38-/- mice: the dot plots show the left ventricular (LV) diastolic (A1) and systolic (A2) inner diameters and LV ejection (A3) fraction in 7-month-old WT (n=5), mdx (n=6) and mdx/CD38-/- (n=10) mice. Data information: : Each dot of the graphs represents a mouse. in duplicate After normality and variance comparison tests, significance was assessed using:A1 ANOVA followed by Fisher's LSD test; A2,A3 Kruskal-Wallis followed by Dunn's test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


B Plasma levels of cardiac stress biomarkers : brain natriuretic peptide ( BNP ) ( B1 ) and cardiac troponin I ( cTnI ) ( B2 ) in 7 - month - old WT ( n = 7 ) , mdx ( n = 6 ) and mdx / CD38 - / - ( n = 11 ) mice . Data information : : Each dot of the graphs represents a mouse . After normality and variance comparison tests , significance was assessed using ANOVA followed by Fisher ' s LSD test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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B Plasma levels of cardiac stress biomarkers: brain natriuretic peptide (BNP) (B1) and cardiac troponin I (cTnI) (B2) in 7-month-old WT (n=7), mdx (n=6) and mdx/CD38-/- (n=11) mice. Data information: : Each dot of the graphs represents a mouse. After normality and variance comparison tests, significance was assessed using ANOVA followed by Fisher's LSD test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


C Cropped images revealed the collagen ( blue ) stained by Masson ' s trichrome staining in the heart of WT , mdx , and mdx / CD38 - / - mice . Dot plot showing the quantification of heart collagen staining area ( % total area ) in 7 - month - old WT , mdx , and mdx / CD38 - / - mice ( n = 4 per group ) . Scale bars : 200 µm . Data information : : Each dot of the graphs represents a mouse . in triplicate After normality and variance comparison tests , significance was assessed using ANOVA followed by Fisher ' s LSD test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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C Cropped images revealed the collagen (blue) stained by Masson's trichrome staining in the heart of WT, mdx, and mdx/CD38-/- mice. Dot plot showing the quantification of heart collagen staining area (% total area) in 7-month-old WT, mdx, and mdx/CD38-/- mice (n=4 per group). Scale bars: 200 µm. Data information: : Each dot of the graphs represents a mouse. in triplicate After normality and variance comparison tests, significance was assessed using ANOVA followed by Fisher's LSD test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.