C Percentage of immunosuppression of CTV - stained PBMC co - cultured with MitoTneg or MitoTpos cells after 5 - 7 days of CD4 + naïve cell activation with differentiation media ( ratio MitoT cells : PBMC ) ( n = 3 biological replicates , with different donors of PBMCs ) . Graph shows mean ± SEM and statistical analysis by Student ' s t - test .

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

C Percentage of immunosuppression of CTV-stained PBMC co-cultured with MitoTneg or MitoTpos cells after 5-7 days of CD4+ naïve cell activation with differentiation media (ratio MitoT cells:PBMC) (n=3 biological replicates, with different donors of PBMCs). Graph shows mean ± SEM and statistical analysis by Student's t-test.


B Representative FACS plots of mitoception ( % of MitoTracker + ) in CD4 + or CD8 + T cells from mitocepted PBMC compared to control PBMC , before intravenous injection .

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B Representative FACS plots of mitoception (% of MitoTracker+) in CD4+ or CD8+T cells from mitocepted PBMC compared to control PBMC, before intravenous injection.


C Percent of mouse weight after GVHD induction with IV injection of mitocepted PBMC or control PBMC cells ( n = 3 , with a total of 25 mice per group ) . Control group in grey , were untreated mice ( n = 6 mice ) . * p < 0 . 05 by Mann Whitney t - test .

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C Percent of mouse weight after GVHD induction with IV injection of mitocepted PBMC or control PBMC cells (n=3, with a total of 25 mice per group). Control group in grey, were untreated mice (n=6 mice). * p<0.05 by Mann Whitney t-test.


D Kaplan Meier plot showing percent survival of GVHD mice after transplantation of an equal amount ( 12x106 cells ) of mitocepted PBMC or control PBMC cells ( n = 3 , with a total of 25 mice per group ) ( p = 0 . 0218 by log - rank test ) .

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D Kaplan Meier plot showing percent survival of GVHD mice after transplantation of an equal amount (12x106 cells) of mitocepted PBMC or control PBMC cells (n=3, with a total of 25 mice per group) (p=0.0218 by log-rank test).


E FACS analysis of human Treg ( CD4 + CD25 + FoxP3 + ) cell engraftment in mouse spleen following transplantation of PBMC - MitoT or control PBMC cells ( n = 3 , with a total of 25 and 27 mice per group , respectively ) .

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E FACS analysis of human Treg (CD4+CD25+FoxP3+) cell engraftment in mouse spleen following transplantation of PBMC-MitoT or control PBMC cells (n=3, with a total of 25 and 27 mice per group, respectively).


F FACS analysis of human pro - inflammatory T helper1 ( CD4 + IFNγ + , left panel ) and T cytotoxic ( CD8 + IFNγ + , right panel ) cell engraftment in mouse spleen following transplantation of mitocepted or control PBMC cells ( n = 3 , with a total of 25 and 27 mice per group , 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 O O O O O O O O O O O O O O O O O O O O O O O

F FACS analysis of human pro-inflammatory T helper1 (CD4+IFNγ+, left panel) and T cytotoxic (CD8+IFNγ+, right panel) cell engraftment in mouse spleen following transplantation of mitocepted or control PBMC cells (n=3, with a total of 25 and 27 mice per group, respectively).


G Representative histopathologic images of GVHD in liver and small intestine sections obtained from PBMC - MitoT , PBMC and non - transplanted control samples , stained with Masson ' s trichrome staining . Asterisk showed increased periportal collagen deposits in the liver and increased piknotic cells in the small intestine crypts . Arrowheads represents a reduced number of Paneth cells .

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G Representative histopathologic images of GVHD in liver and small intestine sections obtained from PBMC-MitoT, PBMC and non-transplanted control samples, stained with Masson's trichrome staining. Asterisk showed increased periportal collagen deposits in the liver and increased piknotic cells in the small intestine crypts. Arrowheads represents a reduced number of Paneth cells.


H - I Semiquantitative scoring system for pathologic damage in the liver and small intestine of mitocepted PBMC ( n = 8 independent mice ) or control PBMC transplanted mice ( n = 5 independent mice ) .

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H-I Semiquantitative scoring system for pathologic damage in the liver and small intestine of mitocepted PBMC (n=8 independent mice) or control PBMC transplanted mice (n=5 independent mice).


J Quantification of Paneth cells per crypt , from at least 50 crypts per PBMC - MitoT ( n = 8 mice ) , PBMC transplanted ( n = 5 mice ) or non - transplanted control ( n = 3 ) 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

J Quantification of Paneth cells per crypt, from at least 50 crypts per PBMC-MitoT (n=8 mice), PBMC transplanted (n=5 mice) or non-transplanted control (n=3) mouse.


Representative histopathologic images of GVHD in lung sections obtained from PBMC - MitoT , PBMC and non - transplanted control , stained with Masson ' s trichrome staining .

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Representative histopathologic images of GVHD in lung sections obtained from PBMC-MitoT, PBMC and non-transplanted control, stained with Masson's trichrome staining.


L Semiquantitative scoring system for pathologic damage in lung of mitocepted PBMC ( n = 8 independent mice ) or control PBMC transplanted mice ( n = 5 independent mice ) .

L Semiquantitative scoring system for pathologic damage in lung of mitocepted PBMC (n=8 independent mice) or control PBMC transplanted mice (n=5 independent mice).


M FACS analysis of total human CD4 + cell engraftment in mouse lungs following transplantation of mitocepted PBMC or control PBMC cells ( n = 3 , with a total of 25 and 27 mice per group , 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 O

M FACS analysis of total human CD4+ cell engraftment in mouse lungs following transplantation of mitocepted PBMC or control PBMC cells (n=3, with a total of 25 and 27 mice per group, respectively).


a , b , Immunoblot for LC3 conversion in isolated primary intestinal epithelial cells ( a ) ( n = 5 / 4 ) and for autophagy proteins in primary intestinal epithelial cell scrapings ( b ) ( n = 3 ) .

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a, b, Immunoblot for LC3 conversion in isolated primary intestinal epithelial cells (a) (n = 5/4) and for autophagy proteins in primary intestinal epithelial cell scrapings (b) (n = 3).


c , Transmission electron microscopy ( TEM ) of crypts . Note autophagic vacuoles in various stages of evolution in Xbp1 Dgr ; IEC hypomorphic Paneth cells .

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c, Transmission electron microscopy (TEM) of crypts. Note autophagic vacuoles in various stages of evolution in Xbp1 Dgr;IEC hypomorphic Paneth cells.


d , e , Crypt showing GFP - LC3 punctae ( d ) , quantified in e ( n = 10 ; unpaired Student ' s t - test ; mean ± s . e . m . ) . Scale bar , 5 µm .

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d, e, Crypt showing GFP-LC3 punctae (d), quantified in e (n = 10; unpaired Student's t-test; mean ± s.e.m.). Scale bar, 5 µm.


f , g , Immunoblot of silenced MODE - K cells ( f ) and primary intestinal epithelial cell scrapings ( g ) for the PERK - eIF2α branch ( n = 3 ) .

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f, g, Immunoblot of silenced MODE-K cells (f) and primary intestinal epithelial cell scrapings (g) for the PERK-eIF2α branch (n = 3).


j , GFP - LC3 punctae per crypt after treatment with tamoxifen , and vehicle or salubrinal ( n = 10 ; one - way ANOVA with post - hoc Bonferroni ; mean ± s . e . m . ) .

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

j, GFP-LC3 punctae per crypt after treatment with tamoxifen, and vehicle or salubrinal (n = 10; one-way ANOVA with post-hoc Bonferroni; mean ± s.e.m.).


k , Enteritis histology score after salubrinal and tamoxifen co - treatment ( n = 12 / 14 / 13 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . Results represent three ( a , f , g ) or two ( c , e , h , i ) independent experiments . * P 0 . 05 , * * * P 0 . 001 .

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

k, Enteritis histology score after salubrinal and tamoxifen co-treatment (n = 12/14/13; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). Results represent three (a, f, g) or two (c, e, h, i) independent experiments. *P 0.05, ***P 0.001.


a , TEM of crypts of Lieberkühn ( n = 2 ) . Scale bars , 2 µm .

a, TEM of crypts of Lieberkühn (n = 2). Scale bars, 2 µm.


b , Representative haematoxylin and eosin stainings . Note transmural inflammation extending through muscularis propria ( white arrow ) into serosa ( black arrow ) in Atg7 / Xbp1Dgr ; IEC and Atg16l1 / Xbp1Dgr ; IEC mice scored in c and e . Scale bars , 50 µm , except for lower two panels ( 10 µm ) . c , Enteritis histology score ( n = 26 / 12 / 18 / 27 ; 10 - 18 weeks ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . e , Enteritis histology score ( n = 11 ; 18 weeks ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . NS , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 haematoxylin and eosin stainings. Note transmural inflammation extending through muscularis propria (white arrow) into serosa (black arrow) in Atg7/Xbp1Dgr;IEC and Atg16l1/Xbp1Dgr;IEC mice scored in c and e. Scale bars, 50 µm, except for lower two panels (10 µm). c, Enteritis histology score (n = 26/12/18/27; 10-18 weeks; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test).e, Enteritis histology score (n = 11; 18 weeks; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). NS, not significant.


d , CryptTEM in indicated genotypes ( n = 2 ) . Scale bars , 2 µm .

d, CryptTEM in indicated genotypes (n = 2). Scale bars, 2 µm.


f , Xbp1 mRNA splicing ( Xbp1u , unspliced ; Xbp1s , spliced ) of crypts ; densitometry in g ( n = 7 ; unpaired Student ' s t - test ; mean ± s . e . m . ) .

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f, Xbp1 mRNA splicing (Xbp1u, unspliced; Xbp1s, spliced) of crypts; densitometry in g (n = 7; unpaired Student's t-test; mean ± s.e.m.).


h , GRP78 ( green ) immunofluorescence , white arrows indicate GRP78 + crypts ( DAPI , blue ; n = 5 ) . Scale bars , 10 µm . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

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h, GRP78 (green) immunofluorescence, white arrows indicate GRP78+ crypts (DAPI, blue; n = 5). Scale bars, 10 µm. *P 0.05, **P 0.01, ***P 0.001.


a , shCtrl or shXbp1 MODE - K cells were co - silenced for Atg16l1 ( siAtg16l1 ) or with scrambled siRNA ( siCtrl ) , and analysed by flow cytometry for annexin V and propidium iodide ( PI ; n = 3 ; one - way ANOVA with post - hoc Bonferroni ; mean ± s . e . m . ) .

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a, shCtrl or shXbp1 MODE-K cells were co-silenced for Atg16l1 (siAtg16l1) or with scrambled siRNA (siCtrl), and analysed by flow cytometry for annexin V and propidium iodide (PI; n = 3; one-way ANOVA with post-hoc Bonferroni; mean ± s.e.m.).


b , Immunoblot of primary intestinal epithelial cell scrapings ( n = 3 ) .

b, Immunoblot of primary intestinal epithelial cell scrapings (n = 3).


c , Immunoblot of cytoplasmic extracts from shCtrl or shXbp1 MODE - K cells after TNF stimulation .

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c, Immunoblot of cytoplasmic extracts from shCtrl or shXbp1 MODE-K cells after TNF stimulation.


d , TUNEL + intestinal epithelial cells ( IECs ) per 100 crypts after BAY11 - 7082 or vehicle treatment ( n = 3 / 4 / 4 ; one - way ANOVA with post - hoc Holm ' s - corrected unpaired Student ' s t - test ; mean ± s . e . m . ) . e , Enteritis histology score of mice treated with BAY11 - 7082 or vehicle ( n = 10 / 10 / 9 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O 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, TUNEL+ intestinal epithelial cells (IECs) per 100 crypts after BAY11-7082 or vehicle treatment (n = 3/4/4; one-way ANOVA with post-hoc Holm's-corrected unpaired Student's t-test; mean ± s.e.m.). e, Enteritis histology score of mice treated with BAY11-7082 or vehicle (n = 10/10/9; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test).


f , Immunoblot of intestinal epithelial cell scrapings for ( p - ) IRE1α ; after IRE1α ; immunoprecipitation ( IP ) . beta - actin , loading control of whole lysates .

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f, Immunoblot of intestinal epithelial cell scrapings for (p-)IRE1α; after IRE1α; immunoprecipitation (IP). beta-actin, loading control of whole lysates.


g , Immunoblot of primary intestinal epithelial cell scrapings ( n = 4 ) .

g, Immunoblot of primary intestinal epithelial cell scrapings (n = 4).


h , i , Enteritis histology score of indicated genotypes ( h , n = 15 / 16 / 14 / 15 ; i , n = 5 / 10 / 12 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) .

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h, i, Enteritis histology score of indicated genotypes (h, n = 15/16/14/15; i, n = 5/10/12; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test).


j , Enteritis histology score of specific pathogen - free ( SPF ) and germ free ( GF ) housed mice ( n = 10 / 9 / 7 / 7 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . k , Representative images of p - IκBα immunoreactivity under conditions as in j ( n = 4 ) . Scale bars , 20 µm . Results represent four ( f ) , three ( c ) or two ( a , b ) independent experiments . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

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j, Enteritis histology score of specific pathogen-free (SPF) and germ free (GF) housed mice (n = 10/9/7/7; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). k, Representative images of p-IκBα immunoreactivity under conditions as in j (n = 4). Scale bars, 20 µm. Results represent four (f), three (c) or two (a, b) independent experiments. *P  0.05, **P  0.01, ***P  0.001.


a , Immunoblot of primary intestinal epithelial cell scrapings from mice treated with or without rapamycin for 14 consecutive days ( n = 3 ) . b , Enteritis histology score for experiment as in a ( n = 4 ; median shown ; Mann - Whitney U - test ) .

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a, Immunoblot of primary intestinal epithelial cell scrapings from mice treated with or without rapamycin for 14 consecutive days (n = 3). b, Enteritis histology score for experiment as in a (n = 4; median shown; Mann-Whitney U-test).


c , Representative images of EYFP - Rosa26 / D6 - cre + / - reporter mice and EYFP - Rosa26 ( controls ) . Co - localization of Defa6 Cre - driven EYFP expression ( yellow ) with lysozyme - expressing Paneth cells ( red ; n = 3 ) . DAPI , blue ; scale bars , 50 µm .

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c, Representative images of EYFP-Rosa26/D6-cre+/- reporter mice and EYFP-Rosa26 (controls). Co-localization of Defa6 Cre-driven EYFP expression (yellow) with lysozyme-expressing Paneth cells (red; n = 3). DAPI, blue; scale bars, 50 µm.


d , Immunoblots of crypt intestinal epithelial cells from Xbp1Dgr ; PC and wild - type controls ( n = 2 ) .

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d, Immunoblots of crypt intestinal epithelial cells from Xbp1Dgr;PC and wild-type controls (n = 2).


e , f , Representative confocal images of lysozyme ( green ) expressing Paneth cells ( e ) with quantification of crypts with indicated number of lysozyme + granulated dots in f ( n = 5 ; unpaired Student ' s t - test ; mean ± s . e . m . ) . DAPI , blue ; scale bars , 10 µm .

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e, f, Representative confocal images of lysozyme (green) expressing Paneth cells (e) with quantification of crypts with indicated number of lysozyme+ granulated dots in f (n = 5; unpaired Student's t-test; mean ± s.e.m.). DAPI, blue; scale bars, 10 µm.


g , Immunohistochemistry for p - eIF2α ( n = 3 ) . Scale bars , 20 µm .

g, Immunohistochemistry for p-eIF2α (n = 3). Scale bars, 20 µm.


h , Representative haematoxylin and eosin images of Xbp1Dgr ; PC and wild - type mice scored in i . Scale bars , 50 µm . i , Enteritis scoring in Xbp1fl / fl ( WTfl ) , Defa6 - cre + ( WTcre ) and Defa6 - cre + ; Xbp1fl / fl ( Xbp1Dgr ; PC ) mice ( n = 21 / 26 / 29 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . Results represent two ( b , d ) independent experiments . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

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

h, Representative haematoxylin and eosin images of Xbp1Dgr;PC and wild-type mice scored in i. Scale bars, 50 µm. i, Enteritis scoring in Xbp1fl/fl (WTfl), Defa6-cre+ (WTcre) and Defa6-cre+;Xbp1fl/fl (Xbp1Dgr;PC) mice (n = 21/26/29; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). Results represent two (b, d) independent experiments. *P 0.05, **P 0.01, ***P 0.001.


The syntheses of the ligands PR - 924 , 9 , 14 , 16 , 17 and 18 have been described previously ( de Bruin et al , 2014 ) . All ligands share the peptide scaffold shown in the upper left corner with P1 , P2 and P3 side chains and an N - cap ; they are equipped with a l - Ala ( stereochemistry ( S ) ) or d - Ala ( stereochemistry ( R ) ) residue at P3 ( red ) and either an N - acylmorpholine or a 3 - methyl - 1H - indene group ( green ) . Furthermore , compounds differ in their P2 residue ( either methoxytyrosine or tryptophane ; blue ) . Inhibitor 9 , also termed LU - 015i , is endowed with an exceptional cyclohexyl residue ( gray ) , which is associated with increased selectivity for human β5i .

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

The syntheses of the ligands PR-924, 9, 14, 16, 17 and 18 have been described previously (de Bruin et al, 2014). All ligands share the peptide scaffold shown in the upper left corner with P1, P2 and P3 side chains and an N-cap; they are equipped with a l-Ala (stereochemistry (S)) or d-Ala (stereochemistry (R)) residue at P3 (red) and either an N-acylmorpholine or a 3-methyl-1H-indene group (green). Furthermore, compounds differ in their P2 residue (either methoxytyrosine or tryptophane; blue). Inhibitor 9, also termed LU-015i, is endowed with an exceptional cyclohexyl residue (gray), which is associated with increased selectivity for human β5i.


A . Inhibition of human β5c and β5i by ONX 0914 and PR - 924 . IC50 values are deduced from the fitted data .

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

A. Inhibition of human β5c and β5i by ONX 0914 and PR-924. IC50 values are deduced from the fitted data.


B . IC50 values [ µM ] for all compounds towards the yeast , human and mouse β5 subunits .

B. IC50 values [µM] for all compounds towards the yeast, human and mouse β5 subunits.


E . Serial dilutions of yeast cells were spotted on YPD plates and grown for 2 days either at 30 ° C or 37 ° C . Mutant yeasts are significantly retarded in growth and show increased temperature sensitivity .

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E. Serial dilutions of yeast cells were spotted on YPD plates and grown for 2 days either at 30 °C or 37 °C. Mutant yeasts are significantly retarded in growth and show increased temperature sensitivity.


F . Surface illustration of the yCP . The β5 and β6 subunits are depicted in dark gray , with the residue ranges that correspond to mammalian β5 and β6 subunits in the chimeric proteasomes highlighted in gold and blue , respectively . The β5 ' and β6 ' subunits of the second β - ring are hidden on the back of 20S proteasome .

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

F. Surface illustration of the yCP. The β5 and β6 subunits are depicted in dark gray, with the residue ranges that correspond to mammalian β5 and β6 subunits in the chimeric proteasomes highlighted in gold and blue, respectively. The β5' and β6' subunits of the second β-ring are hidden on the back of 20S proteasome.


A . Superposition of the β5i / β6 active sites of genuine mouse iCP and the hβ5i / hβ6 human / yeast chimera illustrates that Met45 adopts the same ' open ' conformation in both structures . Amino acid side chains important for substrate binding are depicted as sticks . The catalytic Thr1 is colored in black . Met31 , the single amino acid difference between mouse and human β5i active sites , is highlighted in green .

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A. Superposition of the β5i/β6 active sites of genuine mouse iCP and the hβ5i/hβ6 human/yeast chimera illustrates that Met45 adopts the same 'open' conformation in both structures. Amino acid side chains important for substrate binding are depicted as sticks. The catalytic Thr1 is colored in black. Met31, the single amino acid difference between mouse and human β5i active sites, is highlighted in green.


B . Superposition of the genuine mouse and the human chimeric β5c / β6 active sites according to panel A proves the proper orientation of Met45 in the ' closed ' conformation .

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B. Superposition of the genuine mouse and the human chimeric β5c/β6 active sites according to panel A proves the proper orientation of Met45 in the 'closed' conformation.


C . Superposition of the β5c / β6 and β5i / β6 active sites of the natural mouse cCP and iCP according to panel A . Note the distinct conformations of Met45 ( Huber et al , 2012 ) .

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C. Superposition of the β5c/β6 and β5i/β6 active sites of the natural mouse cCP and iCP according to panel A. Note the distinct conformations of Met45 (Huber et al, 2012).


D . Superposition of the hβ5c / hβ6 and the hβ5i / hβ6 chimeric proteasomes confirm the distinct positions of Met45 analogously to the natural mouse subunits shown in panel C .

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D. Superposition of the hβ5c/hβ6 and the hβ5i/hβ6 chimeric proteasomes confirm the distinct positions of Met45 analogously to the natural mouse subunits shown in panel C.


A . Complex structures of the human β5c / β6 ( left ) and β5i / β6 ( right ) chimeric proteasomes with PR - 924 . The ligand binds in a linear ( l ) manner to the β5c / β6 substrate binding channel , but in a kinked ( k ) conformation to the β5i / β6 active site . The 2FO - FC electron density maps for the ligands bound to Thr1 of the chimeric β5 active sites are shown as blue meshes contoured at 1σ . The inhibitor and Thr1 have been omitted for phasing .

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A. Complex structures of the human β5c/β6 (left) and β5i/β6 (right) chimeric proteasomes with PR-924. The ligand binds in a linear (l) manner to the β5c/β6 substrate binding channel, but in a kinked (k) conformation to the β5i/β6 active site. The 2FO-FC electron density maps for the ligands bound to Thr1 of the chimeric β5 active sites are shown as blue meshes contoured at 1σ. The inhibitor and Thr1 have been omitted for phasing.


B . Comparison of the conformations of ONX 0914 , PR - 924 , 14 , 16 , 17 and 18 bound to the chimeric hβ5 / hβ6 substrate binding channels . The l amino acid compounds ONX 0914 and 14 display a linear ( l ) binding mode to the hβ5c / hβ6 ( left ) and the hβ5i / hβ6 ( right ) chimeras , whereas the ligands 16 and 17 ( P3 - d - Ala and morpholine cap ) adopt a kinked ( k ) conformation in both β5 substrate binding channels . Remarkably , the orientation of PR - 924 and its analogue 18 ( P3 - d - Ala and 3 - methyl - 1H - indene cap ) in the hβ5c / hβ6 chimera ( and in WT yβ5 ; see Appendix Figure S5 ) ) differs from that observed with the hβ5i / hβ6 chimera . Note that the inhibitors are rotated by about 45 ° compared to panel A .

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B. Comparison of the conformations of ONX 0914, PR-924, 14, 16, 17 and 18 bound to the chimeric hβ5/hβ6 substrate binding channels. The l amino acid compounds ONX 0914 and 14 display a linear (l) binding mode to the hβ5c/hβ6 (left) and the hβ5i/hβ6 (right) chimeras, whereas the ligands 16 and 17 (P3-d-Ala and morpholine cap) adopt a kinked (k) conformation in both β5 substrate binding channels. Remarkably, the orientation of PR-924 and its analogue 18 (P3-d-Ala and 3-methyl-1H-indene cap) in the hβ5c/hβ6 chimera (and in WT yβ5; see Appendix Figure S5)) differs from that observed with the hβ5i/hβ6 chimera. Note that the inhibitors are rotated by about 45 ° compared to panel A.


A . Complex structures of the mouse chimeric proteasome ( left ) and the hβ5i - V31M / hβ6 mutant ( right ) with PR - 924 visualize the kinked ( k ) orientation . The 2FO - FC electron density maps for the ligands bound to Thr1 of the chimeric β5 active sites are depicted according to Fig 5A . Steric hindrance of Met31 ( green ) with the bulky 3 - methyl - 1H - indene cap is indicated by black double arrows .

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A. Complex structures of the mouse chimeric proteasome (left) and the hβ5i-V31M/hβ6 mutant (right) with PR-924 visualize the kinked (k) orientation. The 2FO-FC electron density maps for the ligands bound to Thr1 of the chimeric β5 active sites are depicted according to Fig 5A. Steric hindrance of Met31 (green) with the bulky 3-methyl-1H-indene cap is indicated by black double arrows.


B . Superposition of inhibitors bound to the mouse chimeric proteasome ( left ) and the hβ5i - V31M / hβ6 mutant ( right ) . Compounds display the same binding modes ( kinked ( k ) or linear ( l ) ) to the mβ5i / mβ6 active site as to the corresponding human β5i / β6 subunit ( Fig . 5 ) and to the hβ5i - V31M / hβ6 mutant . Note that the inhibitors are rotated by about 45 ° compared to panel A .

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B. Superposition of inhibitors bound to the mouse chimeric proteasome (left) and the hβ5i-V31M/hβ6 mutant (right). Compounds display the same binding modes (kinked (k) or linear (l)) to the mβ5i/mβ6 active site as to the corresponding human β5i/β6 subunit (Fig. 5) and to the hβ5i-V31M/hβ6 mutant. Note that the inhibitors are rotated by about 45° compared to panel A.


C . Superposition of chimeric mammalian and WT yeast β5 active sites bound to the P3 - d - Ala compounds PR - 924 and 18 . The nucleophilic Thr1 is depicted as well as the S1 pocket forming residues 31 and 45 . Residue 31 impacts on the position of the 3 - methyl - 1H - indene cap of PR - 924 and 18 . Due to steric hindrance , the N - cap is shifted in the mouse and in the hβ5i - V31M / hβ6 chimera compared to the hβ5i / hβ6 mutant proteasome crystal structures ( black double arrow ) . Note that the structures are rotated by 90 ° compared to panel B .

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C. Superposition of chimeric mammalian and WT yeast β5 active sites bound to the P3-d-Ala compounds PR-924 and 18. The nucleophilic Thr1 is depicted as well as the S1 pocket forming residues 31 and 45. Residue 31 impacts on the position of the 3-methyl-1H-indene cap of PR-924 and 18. Due to steric hindrance, the N-cap is shifted in the mouse and in the hβ5i-V31M/hβ6 chimera compared to the hβ5i/hβ6 mutant proteasome crystal structures (black double arrow). Note that the structures are rotated by 90° compared to panel B.


ONX 0914 binds in a linear mode to all active sites . Changing the stereochemistry at the P3 site from l - Ala ( S ) to d - Ala ( R ) leads to a kinked conformation in the β5 substrate binding channels ( compound 16 ) . Substitution of the morpholine cap of 16 by a larger 3 - metyhl - 1H - indene moiety yields PR - 924 and results in different inhibitor conformations at the yβ5 / hβ5c and the mammalian β5i active sites . Hereby , the selectivity for hβ5i over hβ5c is drastically increased ( IC50 ratio hβ5c / hβ5i : 255 ) compared to ONX 0914 ( IC50 ratio hβ5c / hβ5i : 20 ) and compound 16 ( IC50 ratio hβ5c / hβ5i : 5 . 2 , Fig 2 ) . Residue Met31 of mβ5i sterically hinders the kinked binding mode of PR - 924 , thereby creating 7 - fold selectivity for human β5i .

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ONX 0914 binds in a linear mode to all active sites. Changing the stereochemistry at the P3 site from l-Ala (S) to d-Ala (R) leads to a kinked conformation in the β5 substrate binding channels (compound 16). Substitution of the morpholine cap of 16 by a larger 3-metyhl-1H-indene moiety yields PR-924 and results in different inhibitor conformations at the yβ5/hβ5c and the mammalian β5i active sites. Hereby, the selectivity for hβ5i over hβ5c is drastically increased (IC50 ratio hβ5c/hβ5i: 255) compared to ONX 0914 (IC50 ratio hβ5c/hβ5i: 20) and compound 16 (IC50 ratio hβ5c/hβ5i: 5.2, Fig 2). Residue Met31 of mβ5i sterically hinders the kinked binding mode of PR-924, thereby creating 7-fold selectivity for human β5i.


A : Snapshots of LimE - GFP , GFP - myo , and corresponding kymographs for type 1 cells . Corresponding speeds are 10 . 8µm / min and 9 . 4µm / min . White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint , as extracted from the corresponding stress kymographs . C : Snapshots of LimE - GFP , GFP - myo , and corresponding kymographs for type 2 cells . Corresponding speeds are 5 . 6µm / min and 8 . 7µm / min . White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint .

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A: Snapshots of LimE-GFP, GFP-myo, and corresponding kymographs for type 1 cells. Corresponding speeds are 10.8µm/min and 9.4µm/min. White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint, as extracted from the corresponding stress kymographs. C: Snapshots of LimE-GFP, GFP-myo, and corresponding kymographs for type 2 cells. Corresponding speeds are 5.6µm/min and 8.7µm/min. White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint.


B : Stress maps quantifying the magnitude of the force per area using a color scale with blue / red corresponding to small / large stresses and the direction of forces using vectors . Fan - shaped cells were rotated so that the vertical ( x ) axis is the direction of motion ( see Methods ) . Shown are the overall stress T , the stress in the direction of motion Tx and the stress perpendicular to motion Ty for the type 1 cell with the LimE marker , and the corresponding Tx and Ty kymographs along the cells outlines . D : Stress maps of T , Tx and Ty for the type 2 cell with the LimE marker , and corresponding Tx and Ty kymographs along the cells outlines .

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B: Stress maps quantifying the magnitude of the force per area using a color scale with blue/red corresponding to small/large stresses and the direction of forces using vectors. Fan-shaped cells were rotated so that the vertical (x) axis is the direction of motion (see Methods). Shown are the overall stress T, the stress in the direction of motion Tx and the stress perpendicular to motion Ty for the type 1 cell with the LimE marker, and the corresponding Tx and Ty kymographs along the cells outlines. D: Stress maps of T, Tx and Ty for the type 2 cell with the LimE marker, and corresponding Tx and Ty kymographs along the cells outlines.


A : Sequence of snapshots of the LimE - GFP distribution and stress T for an oscillatory cell together with snapshots of the GFP - myo distribution for a separate oscillatory cell . Corresponding speeds are 7 . 0µm / min and 4 . 6µm / min . The sequences show an entire cycle of oscillation ( spreading and contraction ) . White arrows represent the direction of motion before the cell spreads and contracts during which no net motion exists .

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A: Sequence of snapshots of the LimE-GFP distribution and stress T for an oscillatory cell together with snapshots of the GFP-myo distribution for a separate oscillatory cell. Corresponding speeds are 7.0µm/min and 4.6µm/min. The sequences show an entire cycle of oscillation (spreading and contraction). White arrows represent the direction of motion before the cell spreads and contracts during which no net motion exists.


A : Sequence of snapshots of the LimE - GFP distribution and stress T for an amoeboid cell and the GFP - myo distribution for a separate amoeboid cell . Corresponding speeds are 7 . 4µm / min and 4 . 5µm / min . The sequences show both the phases of protrusion and retraction . White arrows represent the direction of motion .

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A: Sequence of snapshots of the LimE-GFP distribution and stress T for an amoeboid cell and the GFP-myo distribution for a separate amoeboid cell. Corresponding speeds are 7.4µm/min and 4.5µm/min. The sequences show both the phases of protrusion and retraction. White arrows represent the direction of motion.


A : Protrusion and retraction speed for amoeboid , oscillatory and fan - shaped cells , defined as the average of the pixels with the 20 % lowest and highest membrane speed .

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A: Protrusion and retraction speed for amoeboid, oscillatory and fan-shaped cells, defined as the average of the pixels with the 20% lowest and highest membrane speed.


C : Average edge velocity in regions of low and high LimE - GFP and GFP - myo fluorescence . High fluorescence was defined as the 20 % brightest LimE - GFP and GFP - myo pixels in the kymographs while low fluorescence consisted of the remaining 80 % pixels .

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C: Average edge velocity in regions of low and high LimE-GFP and GFP-myo fluorescence. High fluorescence was defined as the 20% brightest LimE-GFP and GFP-myo pixels in the kymographs while low fluorescence consisted of the remaining 80% pixels.


( C - G ) Resembling the phenotype caused by Notch overexpression ( D ) or dSir2 mutation ( E ) , HP1c knockdown ( F ) and mutant ( G ) flies also exhibit supernumerary anterior scutellar ( aSC ) bristles on the notum , as indicated by the enlarged views in the left bottom panes . Scale bars , 100 μm .

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(C-G) Resembling the phenotype caused by Notch overexpression (D) or dSir2 mutation (E), HP1c knockdown (F) and mutant (G) flies also exhibit supernumerary anterior scutellar (aSC) bristles on the notum, as indicated by the enlarged views in the left bottom panes. Scale bars, 100 μm.


( H - M ) Compared with control flies ( H and K ) , HP1c knockdown ( J ) and mutant ( M ) flies exhibit an ectopic vein at the distal of L4 and L5 ( arrows ) and a broken posterior cross vein ( arrowhead ) , which is similar to the phenotype caused by Notch overexpression ( I ) and dSir2 mutant ( L ) . Scale bars , 500 μm .

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(H-M) Compared with control flies (H and K), HP1c knockdown (J) and mutant (M) flies exhibit an ectopic vein at the distal of L4 and L5 (arrows) and a broken posterior cross vein (arrowhead), which is similar to the phenotype caused by Notch overexpression (I) and dSir2 mutant (L). Scale bars, 500 μm.


( A - D ) The posterior midguts of 10 - day - old control ( A ) , HP1c knockdown ( B ) , HP1c null mutant ( C ) and HP1c overexpression flies ( D ) . Arrows mark ISCs , arrowheads mark ee cells , circles mark ISC - EB , EB - EB or ISC - ISC nests . ISCs were visualized with esg - GFP alone , EBs were stained with both esg - GFP and Su ( H ) - lacZ , while ee cells were labeled by Pros and DNA was marked by DAPI . Scale bars , 30 μm .

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(A-D) The posterior midguts of 10-day-old control (A), HP1c knockdown (B), HP1c null mutant (C) and HP1c overexpression flies (D). Arrows mark ISCs, arrowheads mark ee cells, circles mark ISC-EB, EB-EB or ISC-ISC nests. ISCs were visualized with esg-GFP alone, EBs were stained with both esg-GFP and Su(H)-lacZ, while ee cells were labeled by Pros and DNA was marked by DAPI. Scale bars, 30 μm.


( E - I ) Confocal images of the posterior midgut clones of the indicated genotypes , marked by GFP ( dotted lines ) . Arrows mark ISCs / EBs , arrowheads mark ee cells , and asterisks mark ECs . DNA was visualized with DAPI and ee cells were stained with Pros . Scale bars , 10 μm .

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(E-I) Confocal images of the posterior midgut clones of the indicated genotypes, marked by GFP (dotted lines). Arrows mark ISCs/EBs, arrowheads mark ee cells, and asterisks mark ECs. DNA was visualized with DAPI and ee cells were stained with Pros. Scale bars, 10 μm.


( A - D ) Compared with the control ( A ) flies , posterior midguts of Notch OE flies exhibit decreased ISCs and ee cells ( B ) , while HP1c OE flies show increased ISCs and ee cells ( C ) , and the phenotype of Notch OE can be rescued by HP1c OE ( D ) . Arrows mark ISCs , arrowheads mark ee cells , circles mark ISC - ISC , ISC - EB or EB - EB nests . ISCs were visualized with esg - GFP alone , EBs were stained with both esg - GFP and Su ( H ) - lacZ , while ee cells were labeled by Pros and DNA was marked by DAPI . Scale bars , 30 μm .

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(A-D) Compared with the control (A) flies, posterior midguts of Notch OE flies exhibit decreased ISCs and ee cells (B), while HP1c OE flies show increased ISCs and ee cells (C), and the phenotype of Notch OE can be rescued by HP1c OE (D). Arrows mark ISCs, arrowheads mark ee cells, circles mark ISC-ISC, ISC-EB or EB-EB nests. ISCs were visualized with esg-GFP alone, EBs were stained with both esg-GFP and Su(H)-lacZ, while ee cells were labeled by Pros and DNA was marked by DAPI. Scale bars, 30 μm.


ChIP - qPCR results show the association of HP1c with Notch target genes compared to RFP control . The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin ( equivalent to 1 ) . Error bars indicate SEM . n = 3 technical replicates . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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ChIP-qPCR results show the association of HP1c with Notch target genes compared to RFP control. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 1). Error bars indicate SEM. n=3 technical replicates. Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).


( B ) RT - qPCR analysis determined the expression of Notch target genes when HP1c was depleted ( n = 3 technical replicates , , mean ± s . d . ) . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * * p < 0 . 001 ) .

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(B) RT-qPCR analysis determined the expression of Notch target genes when HP1c was depleted (n=3 technical replicates,, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (*p < 0.05, ***p < 0.001).


NRE - GFP signals of S2 cells carrying NICD OE , Hairless KD , Su ( H ) KD , dSir2 KD and HP1c KD , and the statistical results ( F ) ( n = 4 biological replicates , mean ± s . d . ) . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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NRE-GFP signals of S2 cells carrying NICD OE, Hairless KD, Su(H) KD, dSir2 KD and HP1c KD , and the statistical results (F) (n=4 biological replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).


( D , E ) The interaction between HP1c and Su ( H ) is further confirmed through co - immunoprecipitation ( Co - IP ) . Plasmids expressing HP1c - eGFP and Su ( H ) - flag were transfected into S2 cells . Immunoprecipitation and western blotting were performed with the indicated antibodies .

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(D, E) The interaction between HP1c and Su(H) is further confirmed through co-immunoprecipitation (Co-IP). Plasmids expressing HP1c-eGFP and Su(H)-flag were transfected into S2 cells. Immunoprecipitation and western blotting were performed with the indicated antibodies.


( B ) The relative binding of HP1c with Notch target genes when Su ( H ) KD using ChIP - qPCR analysis ( n = 3 technical replicates , mean ± s . d . ) . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * p < 0 . 01 ) . ( C ) The relative binding of HP1c on Notch target genes when NICD OE using ChIP - qPCR analysis . ( n = 3 technical replicates , mean ± s . d . ) . Data are evaluated with two - tailed Student ' s t - test ( * * * p < 0 . 001 ) .

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(B) The relative binding of HP1c with Notch target genes when Su(H) KD using ChIP-qPCR analysis (n=3 technical replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01). (C) The relative binding of HP1c on Notch target genes when NICD OE using ChIP-qPCR analysis. (n=3 technical replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (***p < 0.001).


( M ) Statistical results of the number of ISCs and ee cells per unit area ( 200 μm × 200 μm ) in ( D - F ) . More than twenty - five midguts were examined for each group . Error bars indicate SEM . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * p < 0 . 01 ) .

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(M) Statistical results of the number of ISCs and ee cells per unit area (200 μm × 200 μm) in (D-F). More than twenty-five midguts were examined for each group. Error bars indicate SEM. Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01).


( A ) Representative images showing the myogenic differentiation , as assessed by immunostaining for MyHC ( green ) , of human MuSCs ( hMuSCs ) isolated from muscle biopsies of DMD patients , either before the beginning of the clinical trial ( hMuSCs ( - ) ) or after 1 year of treatment with Givinostat ( hMuSCs Giv ( - ) ) . Cells were cultured alone or ( only for hMuSCs - ) in transwell co - culture with FAPs isolated from the same patients and exposed in vitro to Giv ( + ehFAPs Giv Vitro ) or control vehicle ( + ehFAPs ) . The upper panels represent myogenic differentiation of DMD - 1 patient ; bottom panels represent myogenic differentiation of DMD - 2 patient . Dose and timing of Giv treatment as described in Bettica et al . , 2016 ( 23 ) . Scale bar = 50 μm .

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(A) Representative images showing the myogenic differentiation, as assessed by immunostaining for MyHC (green), of human MuSCs (hMuSCs) isolated from muscle biopsies of DMD patients, either before the beginning of the clinical trial (hMuSCs (-)) or after 1 year of treatment with Givinostat (hMuSCs Giv (-)). Cells were cultured alone or (only for hMuSCs -) in transwell co-culture with FAPs isolated from the same patients and exposed in vitro to Giv (+ehFAPs Giv Vitro) or control vehicle (+ehFAPs). The upper panels represent myogenic differentiation of DMD-1 patient; bottom panels represent myogenic differentiation of DMD-2 patient. Dose and timing of Giv treatment as described in Bettica et al., 2016 (23). Scale bar = 50 μm.


( C ) Representative images showing the myogenic differentiation of MuSCs isolated from 1 . 5 month old mdx mice , as assessed by immunostaining for MyHC ( green ) . MuSCs were cultured alone ( - ) or in transwell co - culture with mdx FAPs , which were pretreated with DMSO or GW4869 ( GW 10mM added to FAPs 30 minutes before starting the co - culture with MuSCs ) . Scale bar = 50 μm . ( D ) Graph showing the fusion index of MuSCs in the conditions described in ( C ) , ( n = 5 , biological replicates ) . Star ( * ) indicates statistical analysis by Tukey test relative to MuSCs cultured alone ( - ) , * * p < 0 . 01 ; Hash ( # ) indicates statistical analysis by Tukey test relative to MuSCs in co - cultured with FAPs not treated ( DMSO ) , # p < 0 . 05 ; § represents statistical analysis by 2Way Anova test ; § § § p < 0 , 001 . Data information : Nuclei were counterstained with DAPI ( blue ) . All data correspond to the average ± SEM .

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(C) Representative images showing the myogenic differentiation of MuSCs isolated from 1.5 month old mdx mice, as assessed by immunostaining for MyHC (green). MuSCs were cultured alone (-) or in transwell co-culture with mdx FAPs, which were pretreated with DMSO or GW4869 (GW 10mM added to FAPs 30 minutes before starting the co-culture with MuSCs). Scale bar = 50 μm. (D) Graph showing the fusion index of MuSCs in the conditions described in (C), (n=5, biological replicates). Star (*) indicates statistical analysis by Tukey test relative to MuSCs cultured alone (-), ** p < 0.01; Hash (#) indicates statistical analysis by Tukey test relative to MuSCs in co-cultured with FAPs not treated (DMSO), # p<0.05; § represents statistical analysis by 2Way Anova test; §§§ p < 0,001. Data information: Nuclei were counterstained with DAPI (blue). All data correspond to the average ± SEM.


( E ) Representative images of PKH - 67 ( green ) and DAPI ( blue ) staining in a transwell co - culture between MuSCs and FAPs isolated from 1 . 5 month old mdx mice . FAPs exposed to PKH - 67 prior to the co - culture with MuSCs . Scale bar = 50 μm . ( F ) Graph showing the percentage of PKH - 67 positive MuSCs after the co - culture with FAPs , ( n = 3 , biological replicates ) . Star ( * ) indicates statistical analysis by t - test ; * * * * p < 0 . 0001 . Data information : Nuclei were counterstained with DAPI ( blue ) . All data correspond to the average ± SEM .

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(E) Representative images of PKH-67 (green) and DAPI (blue) staining in a transwell co-culture between MuSCs and FAPs isolated from 1.5 month old mdx mice. FAPs exposed to PKH-67 prior to the co-culture with MuSCs. Scale bar = 50 μm. (F) Graph showing the percentage of PKH-67 positive MuSCs after the co-culture with FAPs, (n=3, biological replicates). Star (*) indicates statistical analysis by t-test;**** p < 0.0001. Data information: Nuclei were counterstained with DAPI (blue). All data correspond to the average ± SEM.


( G ) Representative images of myogenic differentiation of mdx MuSCs , as assessed by immunostaining for MyHC ( green ) . MuSCs were cultured either alone ( - ) or incubated with EVs isolated from 1 . 5 month old mdx mice ( EVs FAPs ) treated with vehicle ( CTR ) or exposed to TSA in vivo ( TSA ) or in vitro ( TSA vitro ) . Scale bar = 50 μm . ( H ) Graph showing the fusion index of MuSCs in the condition described in ( G ) , ( n = 3 , biological replicates ) . Star ( * ) indicates statistical analysis by Tukey test on the % of n > 5 ( % of nuclei that were MyHC + inside myotubes containing more than 5 nuclei ) relative to MuSCs cultured alone ( - ) , * * p < 0 . 01 , * * * * p < 0 , 0001 ; Hash ( # ) indicates statistical analysis by Tukey test relative to EVs FAPs CTR , # # p < 0 . 01 , # # # p < 0 , 001 ; ns = not significant ; § represents statistical analysis by 2way Anova test , § § § § p < 0 , 0001 . Data information : Nuclei were counterstained with DAPI ( blue ) . All data correspond to the average ± SEM .

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(G) Representative images of myogenic differentiation of mdx MuSCs, as assessed by immunostaining for MyHC (green). MuSCs were cultured either alone (-) or incubated with EVs isolated from 1.5 month old mdx mice (EVs FAPs) treated with vehicle (CTR) or exposed to TSA in vivo (TSA) or in vitro (TSA vitro). Scale bar = 50 μm. (H) Graph showing the fusion index of MuSCs in the condition described in (G), (n=3, biological replicates). Star (*) indicates statistical analysis by Tukey test on the % of n>5 (% of nuclei that were MyHC+ inside myotubes containing more than 5 nuclei) relative to MuSCs cultured alone (-), **p <0.01, ****p<0,0001; Hash (#) indicates statistical analysis by Tukey test relative to EVs FAPs CTR, ##p <0.01, ###p<0,001; ns= not significant; § represents statistical analysis by 2way Anova test, §§§§p < 0,0001. Data information: Nuclei were counterstained with DAPI (blue). All data correspond to the average ± SEM.


( A ) Graph showing the relative expression of Drosha in FAPs after Drosha down - regulation by siRNA ( n = 4 , biological replicates ) . Values correspond to the average ± SEM . Star ( * ) indicates statistical analysis by t - test relative to FAPs TSA treated and transfected with scramble ( Scr ) ; * * p < 0 . 01 .

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(A) Graph showing the relative expression of Drosha in FAPs after Drosha down-regulation by siRNA (n=4, biological replicates). Values correspond to the average ± SEM. Star (*) indicates statistical analysis by t-test relative to FAPs TSA treated and transfected with scramble (Scr); **p<0.01.


( B ) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC ( green ) . MuSCs were cultured alone ( - ) or in transwell co - culture with FAPs isolated from mdx mice treated either with vehicle ( + FAPs CTR ) or TSA ( 0 . 6 mg / kg / day for 15 days by i . p . ) ( + FAPs TSA ) , and transfected with scramble ( Scr siRNA ) or Drosha siRNA ( Drosha siRNA ) prior to co - culture with MuSCs . Scale bar = 50 μm .

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(B) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC (green). MuSCs were cultured alone (-) or in transwell co-culture with FAPs isolated from mdx mice treated either with vehicle (+FAPs CTR) or TSA (0.6 mg/kg/day for 15 days by i.p.) (+FAPs TSA), and transfected with scramble (Scr siRNA) or Drosha siRNA (Drosha siRNA) prior to co-culture with MuSCs. Scale bar = 50 μm.


( E ) Representative images of miR - 206 - 3p ( violet ) immunohistochemistry in vastus medialis bioptic samples of Control and DMD patients at different ages : 1 year ( n = 2 ) , 4 years ( n = 3 ) and 9 years old ( n = 2 ) . ( F ) Graph representing miR - 206 - 3p violet area quantification relative to ( E ) .

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(E) Representative images of miR-206-3p (violet) immunohistochemistry in vastus medialis bioptic samples of Control and DMD patients at different ages: 1 year (n=2), 4 years (n=3) and 9 years old (n=2). (F) Graph representing miR-206-3p violet area quantification relative to (E).


( G ) Representative images of immunofluorescence for eMyHC ( red ) and Laminin ( green ) in conditions described in ( E ) . ( H ) Graph showing the percentage of eMyHC positive fibers of condition described in ( G ) .

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(G) Representative images of immunofluorescence for eMyHC (red) and Laminin (green) in conditions described in (E). (H) Graph showing the percentage of eMyHC positive fibers of condition described in (G).


( I ) Representative images of Masson ' s Trichrome staining in conditions described in ( E ) . ( J ) Graph showing the fibrotic area quantification of conditions described in ( I ) .

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(I) Representative images of Masson's Trichrome staining in conditions described in (E). (J) Graph showing the fibrotic area quantification of conditions described in (I).


( A ) Representative images of Sca - 1 ( green ) , Laminin ( white ) , DAPI ( blue ) immunofluorescence , and miR - 206 - 3p ( violet ) immunohistochemistry on 10 uM sequencial cryosections of Tibialis Anterior ( TA ) of young mdx mice ( CTR ) and after TSA treatment ( TSA ) ( 0 . 6 mg / kg / day for 15 days by i . p . ) ( n = 6 ) . Scale bar = 25 μm .

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(A) Representative images of Sca-1 (green), Laminin (white), DAPI (blue) immunofluorescence, and miR-206-3p (violet) immunohistochemistry on 10 uM sequencial cryosections of Tibialis Anterior (TA) of young mdx mice (CTR) and after TSA treatment (TSA) (0.6 mg/kg/day for 15 days by i.p.) (n=6). Scale bar = 25 μm.


( C ) Graph representing the miR - 206 - 3p relative expression in EVs isolated from muscle interstitium of tibialis anterior from wild type mice ( CTR ) , mdx young and old mice ( control - CTR - and TSA treated ) ( n = 4 ) . Star ( * ) indicates Tukey analysis compared to wild type mice ( CTR ) , * * * * p < 0 . 0001 ; hash ( # ) indicates Tukey analysis compared to mdx young CTR , # # # # p < 0 . 0001 . § represents statistical analysis by Anova test . § § § § p < 0 , 0001 .

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(C) Graph representing the miR-206-3p relative expression in EVs isolated from muscle interstitium of tibialis anterior from wild type mice (CTR), mdx young and old mice (control -CTR- and TSA treated) (n=4). Star (*) indicates Tukey analysis compared to wild type mice (CTR), ****p < 0.0001; hash (#) indicates Tukey analysis compared to mdx young CTR, #### p<0.0001. § represents statistical analysis by Anova test. §§§§ p<0,0001.


( D ) Representative images of CD90 ( green ) , Dapi ( blue ) and miR - 206 - 3p ( violet ) immunohistochemistry in brachial biceps bioptic samples of DMD patient before ( CTR ) and after Givinostat treatment ( GIV ) . Scale bar = 50 μm .

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(D) Representative images of CD90 (green), Dapi (blue) and miR-206-3p (violet) immunohistochemistry in brachial biceps bioptic samples of DMD patient before (CTR) and after Givinostat treatment (GIV). Scale bar = 50 μm.


( F ) Graph showing the relative expression of miR - 206 - 3p in EVs isolated from a human population of muscle resident cells enriched in FAPs ( ehFAPs ) isolated from biopsies of two DMD patients ( DMD - 1 and DMD - 2 ) before ( CTR ) and after ( GIV ) Givinostat treatment , Two technical replicates are shown .

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(F) Graph showing the relative expression of miR-206-3p in EVs isolated from a human population of muscle resident cells enriched in FAPs (ehFAPs) isolated from biopsies of two DMD patients (DMD-1 and DMD-2) before (CTR) and after (GIV) Givinostat treatment, Two technical replicates are shown.


( A ) Flow cytometry analysis of mdx muscles injected with PKH67 - labeled EVs isolated from FAPs of 1 . 5 month old mdx mice exposed to TSA . The left panel shows the percentage of PKH - 67 positive cells in whole muscle . The right panel shows the percentage of the PKH - 67 positive MuSCs .

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(A) Flow cytometry analysis of mdx muscles injected with PKH67-labeled EVs isolated from FAPs of 1.5 month old mdx mice exposed to TSA. The left panel shows the percentage of PKH-67 positive cells in whole muscle. The right panel shows the percentage of the PKH-67 positive MuSCs.


( B ) Representative images of PKH - 67 ( green ) and DAPI ( blue ) staining in MuSCs freshly isolated from muscles previously injected with PKH67 - labeled EVs . Scale bar = 25 μm . Data information : Nuclei were counterstained with DAPI ( blue ) .

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(B) Representative images of PKH-67 (green) and DAPI (blue) staining in MuSCs freshly isolated from muscles previously injected with PKH67-labeled EVs. Scale bar = 25 μm. Data information: Nuclei were counterstained with DAPI (blue).


( C ) Graph showing the relative expression of miR - 206 - 3p in MuSCs isolated as described in ( A and B ) and in MuSCs from muscles injected with PKH67 - labeled EVs isolated from FAPs of 1 . 5 month old mdx mice exposed to TSA , and then transfected or not with Drosha siRNA ( siRNA Drosha ) ( n = 4 ) . Star ( * ) means significance relative to MuSCs that not uptake PKH67 - labled EVs , * * p < 0 . 01 . Hash ( # ) means significance compared to MuSCs that uptake EVs - FAPs TSA , # # p < 0 . 01 . § indicates Anova analysis , § § p < 0 , 01 .

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(C) Graph showing the relative expression of miR-206-3p in MuSCs isolated as described in (A and B) and in MuSCs from muscles injected with PKH67-labeled EVs isolated from FAPs of 1.5 month old mdx mice exposed to TSA, and then transfected or not with Drosha siRNA (siRNA Drosha) (n=4). Star (*) means significance relative to MuSCs that not uptake PKH67-labled EVs, **p < 0.01. Hash (#) means significance compared to MuSCs that uptake EVs-FAPs TSA, ##p < 0.01. § indicates Anova analysis, §§p<0,01.


( D ) Graph showing the expression levels of miR - 206 - 3p and miR - 145 - 5p in FAPs treated or not with TSA after antagomiR treatment ( n = 3 , biological replicates ) . Star ( * ) indicates statistical analysis by t - test relative to FAPs - ; * p < 0 . 05 , * * p < 0 .

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(D) Graph showing the expression levels of miR-206-3p and miR-145-5p in FAPs treated or not with TSA after antagomiR treatment (n=3, biological replicates). Star (*) indicates statistical analysis by t-test relative to FAPs -; *p<0.05, **p<0.01


( E ) Graph showing the expression levels of miR - 206 - 3p and miR - 145 - 5p in FAPs - derived EVs treated or not with TSA after antagomiR treatment ( n = 3 , biological replicates ) . Star ( * ) indicates statistical analysis by t - test relative to FAPs - ; * p < 0 . 05 .

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(E) Graph showing the expression levels of miR-206-3p and miR-145-5p in FAPs-derived EVs treated or not with TSA after antagomiR treatment (n=3, biological replicates). Star (*) indicates statistical analysis by t-test relative to FAPs -; *p<0.05.


Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1 . 5 months old mdx mice treated daily for 21 days with intra - peritoneal injection of vehicle ( CTR ) or TSA , or once a week with intramuscular injections ( Tibialis Anterior ) of EVs derived from FAPs ( EVs - FAPs ) exposed or not to TSA in vivo ( EVs - FAPs CTR - , EVs - FAPs TSA - ) EVs derived from FAPs control transfected with the antagomiR - 206 ( EVs - FAPs CTR A - 206 ) and of EVs - FAPs TSA transfected with antagomiR - 206 and antagomiR - 145 ( EVs - FAPs TSA A - 206 , EVs - FAPs TSA A - 145 ) . EVs were injected every seven days and sacrificed after 21 days of treatment ( n = 5 for CTR , TSA EVs - FAPs CTR and EVs - FAPs TSA while for AntagomiRs n = 3 , biological replicates ) . ( F ) Representative images of immunofluorescence for embryonic myosin heavy chain ( eMyHC - red ) and laminin ( Lam - cyan ) stainings . Scale bar = 50 μm . ( G ) Graph showing the quantification of cross - sectional area ( CSA ) . ( H ) Graph showing the quantification of muscle regeneration ( eMyHC ) . Data information : Nuclei were counterstained with DAPI ( blue ) .

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Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1.5 months old mdx mice treated daily for 21 days with intra-peritoneal injection of vehicle (CTR) or TSA, or once a week with intramuscular injections (Tibialis Anterior) of EVs derived from FAPs (EVs-FAPs) exposed or not to TSA in vivo (EVs-FAPs CTR -, EVs-FAPs TSA -) EVs derived from FAPs control transfected with the antagomiR-206 (EVs-FAPs CTR A-206) and of EVs-FAPs TSA transfected with antagomiR-206 and antagomiR-145 (EVs-FAPs TSA A-206, EVs-FAPs TSA A-145). EVs were injected every seven days and sacrificed after 21 days of treatment (n=5 for CTR, TSA EVs-FAPs CTR and EVs-FAPs TSA while for AntagomiRs n=3, biological replicates). (F) Representative images of immunofluorescence for embryonic myosin heavy chain (eMyHC-red) and laminin (Lam-cyan) stainings. Scale bar = 50 μm. (G) Graph showing the quantification of cross-sectional area (CSA). (H) Graph showing the quantification of muscle regeneration (eMyHC). Data information: Nuclei were counterstained with DAPI (blue).


Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1 . 5 months old mdx mice treated daily for 21 days with intra - peritoneal injection of vehicle ( CTR ) or TSA , or once a week with intramuscular injections ( Tibialis Anterior ) of EVs derived from FAPs ( EVs - FAPs ) exposed or not to TSA in vivo ( EVs - FAPs CTR - , EVs - FAPs TSA - ) EVs derived from FAPs control transfected with the antagomiR - 206 ( EVs - FAPs CTR A - 206 ) and of EVs - FAPs TSA transfected with antagomiR - 206 and antagomiR - 145 ( EVs - FAPs TSA A - 206 , EVs - FAPs TSA A - 145 ) . EVs were injected every seven days and sacrificed after 21 days of treatment ( n = 5 for CTR , TSA EVs - FAPs CTR and EVs - FAPs TSA while for AntagomiRs n = 3 , biological replicates ) . ( I ) Representative images of Masson ' s Trichrome staining . Scale bar = 50 μm . ( J ) Graph showing the quantifications of muscle fiber area fraction ( MFAF ) . ( K ) Graph showing the quantifications of fibrotic area . Data information : Nuclei were counterstained with DAPI ( blue ) .

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Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1.5 months old mdx mice treated daily for 21 days with intra-peritoneal injection of vehicle (CTR) or TSA, or once a week with intramuscular injections (Tibialis Anterior) of EVs derived from FAPs (EVs-FAPs) exposed or not to TSA in vivo (EVs-FAPs CTR -, EVs-FAPs TSA -) EVs derived from FAPs control transfected with the antagomiR-206 (EVs-FAPs CTR A-206) and of EVs-FAPs TSA transfected with antagomiR-206 and antagomiR-145 (EVs-FAPs TSA A-206, EVs-FAPs TSA A-145). EVs were injected every seven days and sacrificed after 21 days of treatment (n=5 for CTR, TSA EVs-FAPs CTR and EVs-FAPs TSA while for AntagomiRs n=3, biological replicates). (I) Representative images of Masson's Trichrome staining. Scale bar = 50 μm. (J) Graph showing the quantifications of muscle fiber area fraction (MFAF). (K) Graph showing the quantifications of fibrotic area. Data information: Nuclei were counterstained with DAPI (blue).


Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1 . 5 months old mdx mice treated daily for 21 days with intra - peritoneal injection of vehicle ( CTR ) or TSA , or once a week with intramuscular injections ( Tibialis Anterior ) of EVs derived from FAPs ( EVs - FAPs ) exposed or not to TSA in vivo ( EVs - FAPs CTR - , EVs - FAPs TSA - ) EVs derived from FAPs control transfected with the antagomiR - 206 ( EVs - FAPs CTR A - 206 ) and of EVs - FAPs TSA transfected with antagomiR - 206 and antagomiR - 145 ( EVs - FAPs TSA A - 206 , EVs - FAPs TSA A - 145 ) . EVs were injected every seven days and sacrificed after 21 days of treatment ( n = 5 for CTR , TSA EVs - FAPs CTR and EVs - FAPs TSA while for AntagomiRs n = 3 , biological replicates ) . ( L ) Representative images of myeloperoxidase staining ( MPO - red ) . Scale bar = 50 μm . ( M ) Graph showing the quantifications of inflammation ( MPO ) . Data information : Nuclei were counterstained with DAPI ( blue ) .

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

Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1.5 months old mdx mice treated daily for 21 days with intra-peritoneal injection of vehicle (CTR) or TSA, or once a week with intramuscular injections (Tibialis Anterior) of EVs derived from FAPs (EVs-FAPs) exposed or not to TSA in vivo (EVs-FAPs CTR -, EVs-FAPs TSA -) EVs derived from FAPs control transfected with the antagomiR-206 (EVs-FAPs CTR A-206) and of EVs-FAPs TSA transfected with antagomiR-206 and antagomiR-145 (EVs-FAPs TSA A-206, EVs-FAPs TSA A-145). EVs were injected every seven days and sacrificed after 21 days of treatment (n=5 for CTR, TSA EVs-FAPs CTR and EVs-FAPs TSA while for AntagomiRs n=3, biological replicates). (L) Representative images of myeloperoxidase staining (MPO-red). Scale bar = 50 μm. (M) Graph showing the quantifications of inflammation (MPO). Data information: Nuclei were counterstained with DAPI (blue).


A ) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC ( green ) . Nuclei were counterstained with DAPI ( blue ) . MuSCs were cultured alone ( - ) or with EVs ( EVs FAPs ) isolated from FAPs in vivo exposed or not to TSA ( CTR and TSA ) and transfected or not with Antagomir - 206 ( - and A - 206 ) . ( n = 7 for all samples , but antagomir ' s samples n = 3 , biological replicates ) . Scale bar = 50 μm .

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A) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC (green). Nuclei were counterstained with DAPI (blue). MuSCs were cultured alone (-) or with EVs (EVs FAPs) isolated from FAPs in vivo exposed or not to TSA (CTR and TSA) and transfected or not with Antagomir-206 (- and A-206). (n=7 for all samples, but antagomir's samples n=3, biological replicates). Scale bar =50 μm.


( C ) Graph relative to Notch3 expression in MuSCs isolated from 1 . 5 month old mdx mice treated either with vehicle ( CTR ) or TSA ( TSA ) , and in EVs derived from FAPs ( EVs - FAPs ) isolated from 1 . 5 month old mdx mice treated either with vehicle ( CTR ) or TSA ( TSA ) ( n = 3 ) . Star ( * ) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice ( CTR ) ; * p < 0 , 05 ; . hash ( # ) means significance compared to EVs - FAPs CTR , # # p < 0 , 01 . § indicates significance by Anova test ; § § p > 0 , 01 .

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(C) Graph relative to Notch3 expression in MuSCs isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA), and in EVs derived from FAPs (EVs-FAPs) isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA) (n=3). Star (*) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice (CTR); *p<0,05;. hash (#) means significance compared to EVs-FAPs CTR, ##p<0,01. § indicates significance by Anova test; §§ p>0,01.


( D ) Graph relative to Notch1 expression in MuSCs isolated from 1 . 5 month old mdx mice treated either with vehicle ( CTR ) or TSA ( TSA ) , and in EVs derived from FAPs ( EVs - FAPs ) isolated from 1 . 5 month old mdx mice treated either with vehicle ( CTR ) or TSA ( TSA ) n = 3 . Star ( * ) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice ( CTR ) ; * p < 0 , 05 ; . hash ( # ) means significance compared to EVs - FAPs CTR ; * p < 0 . 05 . § indicates significance by Anova test ; § § p > 0 , 01 .

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(D) Graph relative to Notch1 expression in MuSCs isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA), and in EVs derived from FAPs (EVs-FAPs) isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA) n=3. Star (*) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice (CTR); *p<0,05;. hash (#) means significance compared to EVs-FAPs CTR; *p < 0.05. § indicates significance by Anova test; §§ p>0,01.


( E ) Representative images showing myofibers cultured alone ( Ctr ) or with extracellular

(E) Representative images showing myofibers cultured alone (Ctr) or with extracellular vesicles


( F ) From left to right : representative images of myofibers cultured for 24 hours alone ( - ) or with conditioned media of FAPs that were previously exposed or not to TSA and GW4869 ( MEDIA - FAPs ) or extracellular vesicles ( EVs - FAPs ) collected from FAPs exposed or not to TSA in vivo ( CTR and TSA ) and transfected with antagomiR - 206 ( TSA A - 206 ) . Myofibers have been stained for EdU ( Cyan ) , immunofluorescence with Pax7 ( green ) , MyoD ( red ) . Nuclei were counterstained with DAPI ( blue ) . ( n = 3 ) . Scale bar = 25 μm .

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(F) From left to right: representative images of myofibers cultured for 24 hours alone (-) or with conditioned media of FAPs that were previously exposed or not to TSA and GW4869 (MEDIA-FAPs) or extracellular vesicles (EVs-FAPs) collected from FAPs exposed or not to TSA in vivo (CTR and TSA) and transfected with antagomiR-206 (TSA A-206). Myofibers have been stained for EdU(Cyan), immunofluorescence with Pax7 (green), MyoD (red). Nuclei were counterstained with DAPI (blue). (n=3). Scale bar = 25 μm.


A . In response to a 1 s light pulse , cac alleles lack on transients at day 3 and day 33 .

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A. In response to a 1 s light pulse, cac alleles lack on transients at day 3 and day 33.


B . Quantification of depolarization amplitudes ( top ) and on amplitudes ( bottom ) . Data are presented as means ± standard error of the mean ( SEM ) .

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B. Quantification of depolarization amplitudes (top) and on amplitudes (bottom). Data are presented as means ± standard error of the mean (SEM).


A . TEM sections of 3 - and 27 - days - old photoreceptor terminals of flies . Terminals of 27 - days - old flies show terminal expansion , loss of capitate projections ( CPs ) and active zones ( AZs ) and increased number of mitochondria per terminal . Scale bars , 500 nm .

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A. TEM sections of 3- and 27-days-old photoreceptor terminals of flies. Terminals of 27-days-old flies show terminal expansion, loss of capitate projections (CPs) and active zones (AZs) and increased number of mitochondria per terminal. Scale bars, 500 nm.


B . Inset from A representing the photoreceptor terminal boxed in white . Yellow arrow indicates double - membraned autophagosome , red indicates amphisome , and blue indicates two fusion - primed AVs . Scale bars , 500 nm .

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B. Inset from A representing the photoreceptor terminal boxed in white. Yellow arrow indicates double-membraned autophagosome, red indicates amphisome, and blue indicates two fusion-primed AVs. Scale bars, 500 nm.


C - E . Quantification of CPs , AZs , and mitochondria per cartridge , respectively . Data are presented as means ± SEM .

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C-E. Quantification of CPs, AZs, and mitochondria per cartridge, respectively. Data are presented as means ± SEM.


F . Quantification of the different AVs per cartridge . The mutant terminals accumulate late stage AVs and fusion - primed AVs ( ns : not significant ; * : p < 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 ; * * * * : p < 0 . 0001 ) . Data are presented as means ±

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F. Quantification of the different AVs per cartridge. The mutant terminals accumulate late stage AVs and fusion-primed AVs (ns: not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001). Data are presented as means ± SEM


G . 30 - days old fly eye brain complexes stained with neuronal marker Elav ( blue ) and poly - Ub antibody ( red ) . The right panel is the enlarged images of the boxed regions at the left

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G. 30-days old fly eye brain complexes stained with neuronal marker Elav (blue) and poly-Ub antibody (red). The right panel is the enlarged images of the boxed regions at the left side


H . The level of p62 is increased in cacJ brain . I . Quantification of H . Data are presented as means ± SEM . ( * : p < 0 . 05 ) .

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H. The level of p62 is increased in cacJ brain. I. Quantification of H. Data are presented as means ± SEM. (*: p < 0.05).


Autophagy defects observed in cac mutants are not due to defects in neurotransmitter release , but due to defects in lysosomal fusion . TEM sections of 1 - day - old and 30 - days - old photoreceptor terminals of flies raised in 12 h light / dark conditions . A , B , D , and E . stj and Vha100 - 1 ( V0 ) mutant terminals accumulate AVs upon aging . G , H . n - Syb mutant photoreceptor terminals show increased SVs , but not a significant accumulation of AVs and fusion - primed AVs . J , K , M and N . Vamp7 and fab1 mutant photoreceptor terminals show the highest increase in AV accumulation . C , F , I , L , O . Quantification of the different AVs for each genotype ( * * : p < 0 . 01 ) . Eyes from three animals for each genotype at each age were analyzed and the counted cartridge number were listed as " n = " in the figure . Data are presented as means ± standard deviation ( SD ) . Scale bars , 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Autophagy defects observed in cac mutants are not due to defects in neurotransmitter release, but due to defects in lysosomal fusion.TEM sections of 1-day-old and 30-days-old photoreceptor terminals of flies raised in 12 h light/dark conditions. A, B, D, and E. stj and Vha100-1 (V0) mutant terminals accumulate AVs upon aging. G, H. n-Syb mutant photoreceptor terminals show increased SVs, but not a significant accumulation of AVs and fusion-primed AVs. J, K, M and N. Vamp7 and fab1 mutant photoreceptor terminals show the highest increase in AV accumulation. C, F, I, L, O. Quantification of the different AVs for each genotype (**: p < 0.01). Eyes from three animals for each genotype at each age were analyzed and the counted cartridge number were listed as "n = " in the figure. Data are presented as means ± standard deviation (SD). Scale bars, 1 μm.


A . Calbindin and hematoxylin staining of mice cerebella indicates that Cacna1atg - la mice have progressive PC and granule cell loss upon aging . Scale bar , 200 μm .

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A. Calbindin and hematoxylin staining of mice cerebella indicates that Cacna1atg-la mice have progressive PC and granule cell loss upon aging. Scale bar, 200 μm.


. B , D . Calbindin ( green ) and 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ) ( blue ) staining of micecerebellum indicates that there are swollen PC axons ( arrows ) in the granular layers of the Cacna1atg - lamicecerebellum at day 25 ( B ) and the Cacna2d2du - 2Jmice at day 50 ( D ) . Scale bar , 20

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. B, D. Calbindin (green) and 4',6-diamidino-2-phenylindole (DAPI)(blue) staining of micecerebellum indicates that there are swollen PC axons (arrows) in the granular layers of the Cacna1atg-lamicecerebellum at day 25 (B) and the Cacna2d2du-2Jmice at day 50 (D). Scale bar, 20 μm


C , E . The TEM of the axons at the granular layers of the cerebellum showing that many PC axons in Cacna1atg - la and Cacna2d2du - 2J mice are swollen and accumulated with numerous mitochondria , autophagosome / MVBs - like structures ( red arrows ) , and various cytoplasmic vesicles or aberrant membrane structures , including stacks of cisternal membranes ( blue arrows ) , and folded ER ( yellow arrows ) . Scale bars for right two panels are 2 μm . Other scale bars are 500

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C, E. The TEM of the axons at the granular layers of the cerebellum showing that many PC axons in Cacna1atg-la and Cacna2d2du-2J mice are swollen and accumulated with numerous mitochondria, autophagosome/MVBs-like structures (red arrows), and various cytoplasmic vesicles or aberrant membrane structures, including stacks of cisternal membranes (blue arrows), and folded ER (yellow arrows). Scale bars for right two panels are 2 μm. Other scale bars are 500 nm


F , G . LC3II and p62 are slightly increased in 15 - days - old Cacna1atg - la mice and 50 - days - old Cacna2d2du - 2J mice . b , d are quantification of a and c . Data are presented as means ± SEM . ( * : p < 0 . 05 ) .

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F, G. LC3II and p62 are slightly increased in 15-days-old Cacna1atg-la mice and 50-days-old Cacna2d2du-2J mice. b, d are quantification of a and c. Data are presented as means ± SEM. (*: p < 0.05).


A . CACNA1A co - localizes with LAMP1 in primary cerebellar cultures of both CTL and Cacna1atg - la mutants . Scale bars , 20 μm .

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A. CACNA1A co-localizes with LAMP1 in primary cerebellar cultures of both CTL and Cacna1atg-la mutants. Scale bars, 20 μm.


B . CACNA1A is present as punctae on the Vacuolin - 1 enlarged LAMP1 positive lysosomes in primary cerebellar cultures of both CTL and Cacna1atg - la mutants . Scale bars , 20 μm .

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B. CACNA1A is present as punctae on the Vacuolin-1 enlarged LAMP1 positive lysosomes in primary cerebellar cultures of both CTL and Cacna1atg-la mutants. Scale bars, 20 μm.


C . Quantification of LAMP1 positive organelles that are also CACNA1A positive . Data are presented as means ± SD . ( ns : not significant ) .

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C. Quantification of LAMP1 positive organelles that are also CACNA1A positive. Data are presented as means ± SD. (ns: not significant).


D . CACNA1A is concentrated in the lysosomal fractions isolated from mice cerebella with iodixanol gradient .

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D. CACNA1A is concentrated in the lysosomal fractions isolated from mice cerebella with iodixanol gradient.


A . LysoTracker Red staining of the primary cerebella neurons from Cacna1atg - la mice and WT controls . The control cells show big bright punctae , while the mutant cells have much dimmer and smaller punctae . B , C and D . Quantification of LysoTracker relative intensity ( " n " represents the counted cell number ) , LysoTracker positive punctae number and size per cell ( " n " represents the counted image number )

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A. LysoTracker Red staining of the primary cerebella neurons from Cacna1atg-la mice and WT controls. The control cells show big bright punctae, while the mutant cells have much dimmer and smaller punctae. B, C and D. Quantification of LysoTracker relative intensity ("n" represents the counted cell number), LysoTracker positive punctae number and size per cell ("n" represents the counted image number)


. E . Lysosomal marker LAMP1 co - localizes with autophagosome marker LC3 in CTL but not Cacna1atg - la mutant cells . Scale bar , 20 μm . F . Quantification of LC3 and LAMP1 co - localization . Data are presented as means ± SD . ( ns : not significant ; * * * : p < 0 . 001 ) .

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. E. Lysosomal marker LAMP1 co-localizes with autophagosome marker LC3 in CTL but not Cacna1atg-la mutant cells. Scale bar, 20 μm. F. Quantification of LC3 and LAMP1 co-localization. Data are presented as means ± SD. (ns: not significant; ***: p < 0.001).


A . DQ - BSA labeled late endosomes fuse with LAMP1 labeled lysosomes in CTL but not the mutant cells . Scale bar , 20 μm . B . Quantification of DQ - BSA and LAMP1 co - localization in CTL and Cacna1atg - la cells . Data are presented as means ± SD . ( ns : not significant ; * * * : p < 0 . 001 ) C . Bepridil but not ω - agatoxin TK ( ω - Aga TK ) treatment reduces lysosomal fusion in cultured cerebellar cells . DQ - BSA ( Green ) labeled late endosomes co - localize with LAMP1 ( Red ) labeled lysosomes with / without ω - Aga TK ( 1 µM ) or Bepridil ( 10 µM ) treatment . Scale bar , 20 μm . D . Quantification of DQ - BSA and LAMP1 co - localization with ω - Aga TK or Bepridil treatment . Data are presented as means ± SD . ( ns : not significant ; * * : p < 0 . 001 ) .

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

A. DQ-BSA labeled late endosomes fuse with LAMP1 labeled lysosomes in CTL but not the mutant cells. Scale bar, 20 μm. B. Quantification of DQ-BSA and LAMP1 co-localization in CTL and Cacna1atg-la cells. Data are presented as means ± SD. (ns: not significant; ***: p < 0.001) C. Bepridil but not ω-agatoxin TK (ω-Aga TK) treatment reduces lysosomal fusion in cultured cerebellar cells. DQ-BSA (Green) labeled late endosomes co-localize with LAMP1 (Red) labeled lysosomes with/without ω-Aga TK (1 µM) or Bepridil (10 µM) treatment. Scale bar, 20 μm. D. Quantification of DQ-BSA and LAMP1 co-localization with ω-Aga TK or Bepridil treatment. Data are presented as means ± SD. (ns: not significant; **: p < 0.001).