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

B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O 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-GENEPROD O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O

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

O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O

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

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O

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

O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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: 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 : of snapshots of LimE - GFP distribution stress T for an amoeboid GFP - myo distribution for a separate amoeboid . Corresponding speeds are 7 . 4µm / min 4 . 5µm / min . sequences show both phases of protrusion retraction . arrows represent direction of motion .

O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O 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: 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 retraction speed for amoeboid , oscillatory fan - shaped , defined as average of pixels with 20 % lowest highest speed .

O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

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 high LimE - GFP GFP - myo . High was defined as 20 % brightest LimE - GFP GFP - myo pixels in kymographs while low consisted of remaining 80 % pixels .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O

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

O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

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

O O O O O O O O B-ORGANISM O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O

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

O O O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O

(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 of posterior midgut clones of indicated genotypes , marked by GFP ( dotted lines ) . Arrows mark ISCs / EBs , arrowheads mark ee , asterisks mark ECs . was visualized with DAPI ee were stained with Pros . Scale bars , 10 μm .

O O O O O B-EXP_ASSAY O O B-TISSUE O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE B-CELL_TYPE O O O B-GENEPROD O O O O O O O

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

O O O O O O O O O O O B-ORGANISM O O B-TISSUE O B-GENEPROD O B-ORGANISM O O O B-CELL_TYPE O O O O O B-GENEPROD O B-ORGANISM O O O B-CELL_TYPE O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O

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

B-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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) 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 carrying NICD OE , Hairless KD , Su ( H ) KD , dSir2 KD HP1c KD , statistical results ( F ) ( n = 4 biological replicates , mean ± s . d . ) . Data are evaluated with two - Student ' s t - ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

B-GENEPROD O B-GENEPROD O O B-CELL_LINE O B-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-CELL_LINE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

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

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O 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) 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 number of ISCs ee per unit ( 200 μm × 200 μm ) in ( D - F ) . More than twenty - five midguts were examined for each group . Error bars indicate . Data are evaluated with two - Student ' s t - ( * p < 0 . 05 , * * p < 0 . 01 ) .

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

O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-ORGANISM O O O O O O B-TISSUE O O B-DISEASE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O 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-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O B-DISEASE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

(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 showing 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 co - culture with MuSCs ) . Scale bar = 50 μm . ( D ) Graph showing fusion index of MuSCs in conditions described in ( C ) , ( n = 5 , biological replicates ) . Star ( * ) indicates statistical by Tukey relative to MuSCs cultured alone ( - ) , * * p < 0 . 01 ; Hash ( # ) indicates statistical by Tukey relative to MuSCs in co - cultured with FAPs not treated ( DMSO ) , # p < 0 . 05 ; § represents statistical by 2Way Anova ; § § § p < 0 , 001 . Data information : Nuclei were counterstained with DAPI ( blue ) . data correspond to average ± .

O O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-EXP_ASSAY O B-GENEPROD O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O 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-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O

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

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O

(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 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 fusion index of MuSCs in condition described in ( G ) , ( n = 3 , biological replicates ) . Star ( * ) indicates statistical by Tukey on % 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 by Tukey relative to EVs FAPs CTR , # # p < 0 . 01 , # # # p < 0 , 001 ; ns = not significant ; § represents statistical by 2way Anova , § § § § p < 0 , 0001 . Data information : Nuclei were counterstained with DAPI ( blue ) . data correspond to average ± .

O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-ORGANISM O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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-SUBCELLULAR O O B-GENEPROD O O B-CELL_TYPE O O O O B-SUBCELLULAR O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O

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

O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O

(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 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 ) , transfected with scramble ( Scr siRNA ) or Drosha siRNA ( Drosha siRNA ) prior to co - culture with MuSCs . Scale bar = 50 μm .

O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O

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

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O B-DISEASE O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

(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 of immunofluorescence for eMyHC ( red ) Laminin ( green ) in conditions described in ( E ) . ( H ) Graph showing of eMyHC positive of condition described in ( G ) .

O O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O 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) 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 of Masson ' s Trichrome in conditions described in ( E ) . ( J ) Graph showing fibrotic of conditions described in ( I ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O

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

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-SUBCELLULAR O O B-TISSUE O O B-TISSUE I-TISSUE O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 of CD90 ( green ) , Dapi ( blue ) miR - 206 - 3p ( violet ) immunohistochemistry in brachial biceps bioptic samples of DMD before ( CTR ) after Givinostat treatment ( GIV ) . Scale bar = 50 μm .

O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O B-DISEASE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O

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

O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O B-ORGANISM O O B-TISSUE O O O O O O O O O O O O B-DISEASE O B-DISEASE O O B-DISEASE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O

(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 of mdx injected with PKH67 - labeled EVs isolated from FAPs of 1 . 5 month old mdx mice exposed to TSA . left panel shows of PKH - 67 positive in whole muscle . right panel shows of PKH - 67 positive MuSCs .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-TISSUE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O

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

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O

(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 relative expression of miR - 206 - 3p in MuSCs isolated as described in ( A B ) in MuSCs from injected with PKH67 - labeled EVs isolated from FAPs of 1 . 5 month old mdx mice exposed to TSA , 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 , § § p < 0 , 01 .

O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

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

O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O 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) 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 expression of miR - 206 - 3p miR - 145 - 5p in FAPs - derived EVs treated or not with TSA after antagomiR treatment ( n = 3 , biological replicates ) . Star ( * ) indicates statistical by t - relative to FAPs - ; * p < 0 . 05 .

O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O O O 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) 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 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 antagomiR - 206 ( EVs - FAPs CTR A - 206 ) of EVs - FAPs TSA transfected with antagomiR - 206 antagomiR - 145 ( EVs - FAPs TSA A - 206 , EVs - FAPs TSA A - 145 ) . EVs were injected every seven days sacrificed after 21 days of treatment ( n = 5 for CTR , TSA EVs - FAPs CTR EVs - FAPs TSA while for AntagomiRs n = 3 , biological replicates ) . ( F ) Representative of immunofluorescence for embryonic myosin heavy chain ( eMyHC - red ) laminin ( Lam - cyan ) stainings . Scale bar = 50 μm . ( G ) Graph showing of cross - sectional ( CSA ) . ( H ) Graph showing of muscle regeneration ( eMyHC ) . Data information : Nuclei were counterstained with DAPI ( blue ) .

O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE I-TISSUE O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O B-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-SUBCELLULAR O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O 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-TISSUE O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE 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). (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 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 antagomiR - 206 ( EVs - FAPs CTR A - 206 ) of EVs - FAPs TSA transfected with antagomiR - 206 antagomiR - 145 ( EVs - FAPs TSA A - 206 , EVs - FAPs TSA A - 145 ) . EVs were injected every seven days sacrificed after 21 days of treatment ( n = 5 for CTR , TSA EVs - FAPs CTR EVs - FAPs TSA while for AntagomiRs n = 3 , biological replicates ) . ( I ) Representative of Masson ' s Trichrome . Scale bar = 50 μm . ( J ) Graph showing quantifications of muscle fiber fraction ( MFAF ) . ( K ) Graph showing quantifications of fibrotic . Data information : Nuclei were counterstained with DAPI ( blue ) .

B-EXP_ASSAY O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE I-TISSUE O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-SUBCELLULAR O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE 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). (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 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 antagomiR - 206 ( EVs - FAPs CTR A - 206 ) of EVs - FAPs TSA transfected with antagomiR - 206 antagomiR - 145 ( EVs - FAPs TSA A - 206 , EVs - FAPs TSA A - 145 ) . EVs were injected every seven days sacrificed after 21 days of treatment ( n = 5 for CTR , TSA EVs - FAPs CTR EVs - FAPs TSA while for AntagomiRs n = 3 , biological replicates ) . ( L ) Representative of myeloperoxidase ( MPO - red ) . Scale bar = 50 μm . ( M ) Graph showing quantifications of inflammation ( MPO ) . Data information : Nuclei were counterstained with DAPI ( blue ) .

B-EXP_ASSAY O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE I-TISSUE O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O B-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-SUBCELLULAR O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-DISEASE O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE 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 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 TSA ) transfected or not with Antagomir - 206 ( - A - 206 ) . ( n = 7 for samples , but antagomir ' s samples n = 3 , biological replicates ) . Scale bar = 50 μm .

O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) Representative images of 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 ) , 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 by Tukey 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 ; § § p > 0 , 01 .

O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O 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) 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 ) , 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 by Tukey 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 ; § § p > 0 , 01 .

O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O 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) 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 showing myofibers cultured alone ( Ctr ) or with extracellular

O O O O O B-CELL_TYPE O O O O O O O B-SUBCELLULAR

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


( F ) From left to right : representative of myofibers cultured for 24 hours alone ( - ) or with conditioned media of FAPs that were previously exposed or not to TSA GW4869 ( MEDIA - FAPs ) or extracellular vesicles ( EVs - FAPs ) collected from FAPs exposed or not to TSA in vivo ( CTR TSA ) 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 .

O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

(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 , cac alleles lack on transients at day 3 day 33 .

O O O O O O O O O B-GENEPROD O O O O O O O O O O

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


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

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


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

O O O O O O O O O O O B-CELL_TYPE O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O

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 photoreceptor terminal boxed in . Yellow arrow indicates double - membraned autophagosome , red indicates amphisome , blue indicates two fusion - primed AVs . Scale bars , 500 nm .

O O O O O O B-CELL_TYPE O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O

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 . of CPs , AZs , mitochondria per cartridge , respectively . Data are presented as means ± .

O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O

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


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

O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 ) poly - Ub antibody ( red ) . right panel is enlarged of boxed regions at left

O O O O O O B-ORGANISM B-TISSUE I-TISSUE O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O

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

O O O O B-GENEPROD O O O B-GENEPROD B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O

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 . sections of 1 - day - old 30 - days - old photoreceptor terminals of flies raised in 12 h / conditions . A , B , D , E . stj 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 fusion - primed AVs . J , K , M N . Vamp7 fab1 mutant photoreceptor terminals show highest increase in AV accumulation . C , F , I , L , O . of different AVs for each genotype ( * * : p < 0 . 01 ) . Eyes from three for each genotype at each were analyzed counted cartridge number were listed as " n = " in figure . Data are presented as means ± standard deviation ( SD ) . Scale bars , 1 μm .

O O O O B-GENEPROD O O O O O O 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-CELL_TYPE O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE 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-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O O O O 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 hematoxylin of mice cerebella indicates that Cacna1atg - la mice have progressive PC granule loss upon aging . Scale bar , 200 μm .

O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-TISSUE O O B-GENEPROD O O B-ORGANISM O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O

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

O O O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-ORGANISM O O O O O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

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

O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

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

O O O O B-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O B-GENEPROD O O B-ORGANISM O O 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. 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 cerebellar cultures of both CTL Cacna1atg - la mutants . Scale bars , 20 μm .

O O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O O

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 on Vacuolin - 1 enlarged LAMP1 positive lysosomes in cerebellar cultures of both CTL Cacna1atg - la mutants . Scale bars , 20 μm .

O O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-SUBCELLULAR O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O O

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

O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

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 lysosomal fractions isolated from mice cerebella with iodixanol gradient .

O O B-GENEPROD O O O O O O O B-ORGANISM B-TISSUE O B-SMALL_MOLECULE O O

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


A . LysoTracker Red of cerebella neurons from Cacna1atg - la mice WT controls . control show big bright , while mutant have much dimmer smaller . B , C D . of LysoTracker relative ( " n " represents counted number ) , LysoTracker positive number per ( " n " represents counted image number )

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

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 . Scale bar , 20 μm . F . of LC3 LAMP1 co - localization . Data are presented as means ± SD . ( ns : not significant ; * * * : p < 0 . 001 ) .

O O O O O B-GENEPROD O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O 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. 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 mutant . Scale bar , 20 μm . B . of DQ - BSA LAMP1 co - localization in CTL Cacna1atg - la . 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 . 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 . of DQ - BSA LAMP1 co - localization with ω - Aga TK or Bepridil treatment . Data are presented as means ± SD . ( ns : not significant ; * * : p < 0 . 001 ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O O O O O B-GENEPROD O O O O O 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-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O 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).


Representative current traces from oocytes expressing wild - type ( WT ) double cysteine mutant NMDARs before after DTE treatment . For each mutant , traces are normalized ( in height , not in current amplitude ) to maximal response obtained after DTE treatment on wild - type ( WT )

O B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O

Representative current traces from oocytes expressing wild-type (WT) and double cysteine mutant NMDARs before and after DTE treatment. For each mutant, traces are normalized (in height, not in current amplitude) to the maximal response obtained after DTE treatment on wild-type (WT) receptors


Summary of DTE - induced potentiation of WT double cysteine

O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE

Summary of the DTE-induced potentiation of WT and double cysteine mutants


Immunoblots from Xenopus oocytes expressing either wt or mutant subunits . M1 indicates GluN1 monomer ( ~ 110 kDa ) ; M2 GluN2B monomer ( ~ 180 kDa ) ; D1 / 1 GluN1 homodimer ( ~ 220 kDa ) ; D1 / 2 GluN1 / GluN2B heterodimer ( ~ 290 kDa ) . Lower panels : immunoblots in reducing conditions ( + β - mercaptoethanol ) . N . I . , non - injected

B-EXP_ASSAY O B-ORGANISM B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O

Immunoblots from Xenopus oocytes expressing either wt or mutant subunits. M1 indicates the GluN1 monomer (~110 kDa); M2 the GluN2B monomer (~180 kDa); D1/1 the GluN1 homodimer (~220 kDa); and D1/2 the GluN1/GluN2B heterodimer (~290 kDa). Lower panels: immunoblots in reducing conditions (+ β-mercaptoethanol). N.I., non-injected oocytes


Assessment of receptor channel activity using MK - 801 inhibition kinetics . Representative current traces from oocytes expressing either wild - type ( WT ) or GluN1 - E698C / GluN2B - L795C mutant receptors in response to 10 nM MK - 801 during agonist application . Responses were scaled to current amplitude obtained before MK - 801 application . Inset , mono - exponential fits of MK - 801 wash - in wash - out . Note strikingly faster kinetics in mutant receptors , both at onset offset of MK -

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE

Assessment of receptor channel activity using MK-801 inhibition kinetics. Representative current traces from oocytes expressing either wild-type (WT) or GluN1-E698C/GluN2B-L795C mutant receptors in response to 10 nM MK-801 during agonist application. Responses were scaled to the current amplitude obtained before MK-801 application. Inset, mono-exponential fits of MK-801 wash-in and wash-out. Note the strikingly faster kinetics in mutant receptors, both at the onset and offset of MK-801


Relative MK - 801 inhibition on - off - rate constants ( kon koff ) . values were normalized to value obtained for WT GluN1 / GluN2B

O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD

Relative MK-801 inhibition on- and off-rate constants (kon and koff). All values were normalized to the value obtained for WT GluN1/GluN2B receptors


Zinc , ifenprodil pH dose - response curves of wild - type ( WT ) GluN1 / GluN2B receptors mutant GluN1 - E698C / GluN2B - L795C receptors . For comparison , zinc ifenprodil dose - response curves of GluN1 / GluN2B receptors lacking whole GluN2B NTD ( GluN1 WT / GluN2B - delNTD ) are also shown ( dashed lines ; data from Rachline et al , 2005 )

B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O

Zinc, ifenprodil and pH dose-response curves of wild-type (WT) GluN1/GluN2B receptors and mutant GluN1-E698C/GluN2B-L795C receptors. For comparison, zinc and ifenprodil dose-response curves of GluN1/GluN2B receptors lacking the whole GluN2B NTD (GluN1 WT/GluN2B-delNTD) are also shown (dashed lines; data from Rachline et al, 2005)


Spermine ( 200 µM , pH 6 . 3 ) potentiation of WT GluN1 / GluN2B mutant GluN1 - E698C / GluN - L795C receptors . Spermine ( 200 µM , pH 6 . 5 ) sensitivity is also shown for WT GluN1 / GluN2B receptors receptors lacking either GluN1 ( GluN1 - delNTD / GluN2B WT ) or GluN2B ( GluN1 WT / GluN2B - delNTD ) NTD ( data from Mony et al , 2011 )

B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O

Spermine (200 µM, pH 6.3) potentiation of WT GluN1/GluN2B and mutant GluN1-E698C/GluN-L795C receptors. Spermine (200 µM, pH 6.5) sensitivity is also shown for WT GluN1/GluN2B receptors and receptors lacking either the GluN1 (GluN1-delNTD/GluN2B WT) or GluN2B (GluN1 WT/GluN2B-delNTD) NTD (data from Mony et al, 2011)


B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O

Glutamate and glycine dose-response curves of WT GluN1/GluN2B receptors and mutant GluN1-E698C/GluN2B-L795C receptors


MK - 801 inhibition kinetics . On - rate constants ( kon ) of inhibition by 10 nM MK - 801 on wild - type ( WT ) mutant receptors . values are normalized to that obtained for WT GluN1 / GluN2B

B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD

MK-801 inhibition kinetics. On-rate constants (kon) of inhibition by 10 nM MK-801 on wild-type (WT) and mutant receptors. All values are normalized to that obtained for WT GluN1/GluN2B receptors


Representative recordings of patches containing one single wild - type ( WT ) or GluN1 - E698C / GluN2B - L795C receptor . bottom trace is an expanded view . C , closed channel ; O , open

O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

Representative recordings of patches containing one single wild-type (WT) or GluN1-E698C/GluN2B-L795C receptor. The bottom trace is an expanded view. C, closed channel; O, open channel


Single channel properties ( equilibrium channel open probability , mean open time , mean closed time ) of WT super - active GluN1 - E698C / GluN2B - L795C

O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O

Single channel properties (equilibrium channel open probability, mean open time, mean closed time) of the WT and super-active GluN1-E698C/GluN2B-L795C receptors


Closed ( left ) open ( right ) time histograms for WT ( upper panels ) or super - active GluN1 - E698C / GluN2B - L795C ( lower panels ) receptors . Closed time histograms were best fit with 5 exponentials , whereas open time histograms were best fit with 2 exponentials ( see Online Methods Appendix Fig S8 ) . Smooth lines are associated exponential

O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Closed (left) and open (right) time histograms for WT (upper panels) or super-active GluN1-E698C/GluN2B-L795C (lower panels) receptors. Closed time histograms were best fit with 5 exponentials, whereas open time histograms were best fit with 2 exponentials (see Online Methods and Appendix Fig S8). Smooth lines are associated exponential fits


( A - C ) Measurement of autophagy during nutrient starvation in HT29 stably expressing YFP - LC3 . ( A ) Increased LC3 per following β ‐ catenin knockdown with siRNA compared with a non ‐ targeting ( NT ) control siRNA . Columns show autophagosome numbers ( mean ± s . e . m . , n > 200 in > 20 fields of view per experiment of three independent treatments ) assessed under normal starved ( 2 h ) conditions with β ‐ catenin or non ‐ targeting ( NT ) siRNA . Representative ( B ) of YFP - LC3 ( green ) DAPI nuclei ( blue ) are shown . ( C ) Upper panel : western blotting showed increased LC3 ‐ II expression in HT29 with β ‐ catenin knockdown compared to control after 2 h starvation . Lower panel : of LC3 ‐ II / β ‐ actin by densitometry ( mean ± s . e . m . of four independent treatments , * P = 0 . 011 in nutrient conditions , * P = 0 . 035 under starvation ) .

O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

(A-C) Measurement of autophagy during nutrient starvation in HT29 cells stably expressing YFP-LC3. (A) Increased LC3 puncta per cell following β‐catenin knockdown with siRNA compared with a non‐targeting (NT) control siRNA. Columns show autophagosome numbers (mean±s.e.m., n>200 cells in >20 fields of view per experiment of three independent treatments) assessed under normal and starved (2 h) conditions with β‐catenin or non‐targeting (NT) siRNA. Representative images (B) of YFP-LC3 puncta (green) and DAPI nuclei staining (blue) are shown. (C) Upper panel: western blotting showed increased LC3‐II expression in HT29 cells with β‐catenin knockdown compared to control cells after 2 h starvation. Lower panel: quantification of the LC3‐II/β‐actin ratio by densitometry (mean±s.e.m. of four independent treatments, *P=0.011 in nutrient conditions, *P=0.035 under starvation).


( D E ) HCT116 β ‐ cateninWT / − overexpressing a control or β ‐ cateninS33Y plasmid nutrient starved for 24 h . ( D ) Immunostaining of LC3 ( green ) with DAPI nuclei ( blue ) showed decreased LC3 with β ‐ cateninS33Y overexpression . ( E ) By western blotting , LC3 ‐ II decreased with β ‐ cateninS33Y overexpression ( upper panel ) . Lower panel : of LC3 ‐ II / β ‐ actin by densitometry ( mean ± s . e . m . of four independent treatments , * P = 0 . 015 ) .

O O O O B-CELL_LINE B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 and E) HCT116 β‐cateninWT/− cells overexpressing a control or β‐cateninS33Y plasmid nutrient starved for 24 h. (D) Immunostaining of LC3 (green) with DAPI nuclei staining (blue) showed decreased LC3 puncta with β‐cateninS33Y overexpression. (E) By western blotting, LC3‐II decreased with β‐cateninS33Y overexpression (upper panel). Lower panel: quantification of the LC3‐II/β‐actin ratio by densitometry (mean±s.e.m. of four independent treatments, *P=0.015).


( F ) Comparison of isogenic β ‐ cateninWT / − β ‐ cateninHCT116 . Upper panel : western blotting showed decreased LC3 ‐ II in HCT116β ‐ catenin . Lower panel : western blotting confirmed by using densitometry of LC3 ‐ II / β ‐ actin ( mean ± s . e . m . of three independent treatments , * P = 0 . 046 ) . Source data for this figure is available on online supplementary information page .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O 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) Comparison of isogenic β‐cateninWT/− and β‐cateninHCT116 cells. Upper panel: western blotting showed decreased LC3‐II in HCT116β‐catenin cells. Lower panel: western blotting confirmed by quantification using densitometry of the LC3‐II/β‐actin ratio (mean±s.e.m. of three independent treatments, *P=0.046).Source data for this figure is available on the online supplementary information page.


( A ) expression of p62 increased following β ‐ catenin knockdown under normal starved conditions in HT29 .

O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_LINE O

(A) Protein expression of p62 increased following β‐catenin knockdown under normal and starved conditions in HT29 cells.


( B ) Comparison of HCT116 expression in isogenic HCT116β ‐ cateninWT / − HCT116β ‐ catenin − / ΔS45 by western blotting showed decreased p62 expression in β ‐ catenin − / ΔS45 .

O O O O O O O O O B-CELL_LINE B-GENEPROD I-GENEPROD O O B-CELL_LINE B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

(B) Comparison of HCT116 expression in isogenic HCT116β‐cateninWT/− and HCT116β‐catenin−/ΔS45 cells by western blotting showed decreased p62 expression in β‐catenin−/ΔS45 cells.


( C ) LC3 number per during starvation nutrient addback post starvation . Left panel : LC3 number reduces in non ‐ targeting ( NT ) siRNA control with nutrient addback . Right panel : LC3 number reduces in β ‐ catenin knockdown with nutrient addback ( mean ± s . e . m . of three independent treatments ; n > 200 in > 20 fields of view per experiment ) . Western blotting for β ‐ catenin confirmed knockdown .

O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O O 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-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O

(C) LC3 puncta number per cell during starvation and nutrient addback post starvation. Left panel: LC3 puncta number reduces in non‐targeting (NT) siRNA control with nutrient addback. Right panel: LC3 puncta number reduces in β‐catenin knockdown cells with nutrient addback (mean±s.e.m. of three independent treatments; n>200 cells in >20 fields of view per experiment). Western blotting for β‐catenin confirmed knockdown.


( D ) Western blotting of LC3 p62 in HT29 following β ‐ catenin knockdown starvation with lysosomal inhibitors . LC3 ‐ II p62 expression showed an increase with β ‐ catenin siRNA in presence of autophagy flux inhibitors chloroquine ( 10 μM ) or bafilomycin A1 ( 100 nM ; applied for final 30 min of 8 h ) compared to β ‐ catenin knockdown alone . by densitometry of LC3 ‐ II / β ‐ actin ( mean ± s . e . m . of four independent experiments ) . See also Supplementary Figure S1A . Source data for this figure is available on online supplementary information page .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O 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) Western blotting of LC3 and p62 in HT29 cells following β‐catenin knockdown and starvation with lysosomal inhibitors. LC3‐II and p62 protein expression showed an increase with β‐catenin siRNA in the presence of autophagy flux inhibitors chloroquine (10 μM) or bafilomycin A1 (100 nM; applied for the final 30 min of the 8 h) compared to β‐catenin knockdown alone. Quantification by densitometry of the LC3‐II/β‐actin ratio (mean±s.e.m. of four independent experiments). See also Supplementary Figure S1A.Source data for this figure is available on the online supplementary information page.


( A - D ) LC3 ( red ) in mouse intestinal crypt villus epithelium increased ( arrowheads ) 2 days post β ‐ catenin deletion in β ‐ catenin − / lox ‐ villin ‐ creERT2 mice ( C D ) compared to control β ‐ catenin + / lox ‐ villin ‐ creERT2 mice ( A B ) . Further magnification of areas marked by squares is shown ( lower panels ) .

O O O O O B-GENEPROD O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

(A-D) LC3 staining (red) in mouse intestinal crypt and villus epithelium increased (white arrowheads) 2 days post β‐catenin deletion in β‐catenin−/lox‐villin‐creERT2 mice (C and D) compared to control β‐catenin+/lox‐villin‐creERT2 mice (A and B). Further magnification of the areas marked by white squares is shown (lower panels).


( E - H ) p62 ( green ) increased in crypt villus epithelium2 days after β ‐ catenin deletion in β ‐ catenin − / lox ‐ villin ‐ creERT2 mice ( G H ) compared to control in β ‐ catenin + / lox ‐ villin ‐ creERT2 mice ( E F ) . DAPI ( blue ) identifies nuclei . β ‐ Catenin deletion was confirmed by immunofluorescence ( Supplementary Figure S1B ) . Red green channel were adjusted post acquisition for clarity ( equal level changes applied across entire figure ) . Further magnification of areas marked by squares is shown ( lower panels ) .

O O O O O B-GENEPROD O O O O O B-TISSUE I-TISSUE I-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-TISSUE O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O 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-H)p62 staining (green) increased in the crypt and villus epithelium2 days after β‐catenin deletion in β‐catenin−/lox‐villin‐creERT2 mice (G and H) compared to control in β‐catenin+/lox‐villin‐creERT2 mice (E and F). DAPI staining (blue) identifies nuclei. β‐Catenin deletion was confirmed by immunofluorescence ( Supplementary Figure S1B). Red and green channel levels were adjusted post acquisition for clarity (equal level changes applied across the entire figure). Further magnification of the areas marked by white squares is shown (lower panels).


( I J ) Western blotting on extracts from mouse intestinal epithelial tissue demonstrated increased p62 LC3 ‐ II expression 2 ( I ) 4 ( J ) days post β ‐ catenin deletion in β ‐ catenin − / lox ‐ villin ‐ creERT2 mice compared to control β ‐ catenin + / lox ‐ villin ‐ creERT2 mice . Source data for this figure is available on online supplementary information page .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE I-TISSUE I-TISSUE O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O B-GENEPROD B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O

(I and J) Western blotting on extracts from mouse intestinal epithelial tissue demonstrated increased p62 and LC3‐II protein expression 2 (I) and 4 (J) days post β‐catenin deletion in β‐catenin−/lox‐villin‐creERT2 mice compared to control β‐catenin+/lox‐villin‐creERT2 mice.Source data for this figure is available on the online supplementary information page.


( A ) Relative p62 / SQSTM1 mRNA by quantitative real ‐ time ‐ PCR ( qRT - PCR ) increased following β ‐ catenin knockdown in HT29 ( mean ± s . e . m . , three independent experiments performed in triplicate , * * * P0 . 001 ) . β ‐ Catenin knockdown was confirmed by western blotting .

O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O

(A) Relative p62/SQSTM1 mRNA levels by quantitative real‐time‐PCR (qRT-PCR) increased following β‐catenin knockdown in HT29 cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). β‐Catenin knockdown was confirmed by western blotting.


( B ) Relative p62 mRNA by qRT - PCR decreased with β ‐ cateninS33Yoverexpression after 24 h of starvation in HCT116 β ‐ cateninWT / − ( mean ± s . e . m . , three independent experiments performed in triplicate , * * * P0 . 001 ) . β ‐ CateninS33Y overexpression was confirmed by western blotting .

O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_LINE B-GENEPROD I-GENEPROD I-GENEPROD O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O

(B) Relative p62 mRNA levels by qRT-PCR decreased with β‐cateninS33Yoverexpression after 24 h of starvation in HCT116 β‐cateninWT/− cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). β‐CateninS33Y overexpression was confirmed by western blotting.


( C ) increase in p62 expression induced by β ‐ catenin siRNA was attenuated in HT29 treated with 10 μg / μl cycloheximide starvation for 8 24 h compared to vehicle control . A complementary experiment using transcription inhibitor actinomycin D is shown in Supplementary Figure S1C .

O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O 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) The increase in p62 protein expression induced by β‐catenin siRNA was attenuated in HT29 cells treated with 10 μg/μl cycloheximide and starvation for 8 and 24 h compared to vehicle control. A complementary experiment using the transcription inhibitor actinomycin D is shown in Supplementary Figure S1C.


( D ) Overexpression of Fzd6 ( green , left panel ) increased p62 ( red , right panel ) expression in HEK293T . As an internal control , not overexpressing Fzd6 are shown in field of view .

O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O

(D) Overexpression of Fzd6 (green, left panel) increased p62 (red, right panel) protein expression in HEK293T cells. As an internal control, cells not overexpressing Fzd6 are shown in the field of view.


( E ) Doxycycline induction ( 1 μg / ml ) of DNTCF4 increased p62 LC3 ‐ II expression in doxycycline ‐ inducible DNTCF4 LS174T ‐ L8 . LGR5 downregulation by DNTCF4 confirmed inhibition of β ‐ catenin / TCF4 signalling .

O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O

(E) Doxycycline induction (1 μg/ml) of DNTCF4 increased p62 and LC3‐II protein expression in doxycycline‐inducible DNTCF4 LS174T‐L8 cells. LGR5 downregulation by DNTCF4 confirmed inhibition of β‐catenin/TCF4 signalling.


( F ) Relative p62 mRNA ( 48 h ) by qRT - PCR increased following doxycycline induction of DNTCF4 in LS174T ‐ L8 ( mean ± s . e . m . , three independent experiments performed in triplicate , * * * P0 . 001 ) .

O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O 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) Relative p62 mRNA levels (48 h) by qRT-PCR increased following doxycycline induction of DNTCF4 in LS174T‐L8 cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001).


( G ) Upper panel : Wnt3a ( 200 ng / ml ) treatment of HCT116 β ‐ cateninWT / − ( 24 h ) decreased LC3 ‐ II p62 expression by western blotting . Lower panel : by densitometry of LC3 ‐ II / β ‐ actin ( mean ± s . e . m . of three independent experiments ) . Source data for this figure is available on online supplementary information page .

O O O O O O B-GENEPROD O O O O O O O O O B-CELL_LINE B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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) Upper panel: Wnt3a (200 ng/ml) treatment of HCT116 β‐cateninWT/− cells (24 h) decreased LC3‐II and p62 protein expression by western blotting. Lower panel: quantification by densitometry of the LC3‐II/β‐actin ratio (mean±s.e.m. of three independent experiments).Source data for this figure is available on the online supplementary information page.


( A ) immunoprecipitation ( ChIP ) demonstrates β ‐ catenin TCF4 binding to p62 promoter . HT29 were grown in normal or starvation conditions for 2 h subjected to ChIP with indicated antibodies ( IgG was used as a negative control ) . Binding of Pol II , β ‐ catenin TCF4 to p62 promoter region was measured by quantitative PCR expressed as enrichment relative to input . During nutrient deprivation , binding of Pol II to p62 promoter increased ; binding of β ‐ catenin to p62 promoter decreased ; TCF4 binding did not change ( data are from one representative experiment of at least three independent experiments performed in triplicate ) . PCR products subjected to agarose gel electrophoresis are shown in Supplementary Figure S2A .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O 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) Chromatin immunoprecipitation (ChIP) demonstrates β‐catenin and TCF4 binding to the p62 promoter. HT29 cells were grown in normal or starvation conditions for 2 h and subjected to ChIP analysis with the indicated antibodies (IgG was used as a negative control). Binding of RNA Pol II, β‐catenin and TCF4 to the p62 promoter region was measured by quantitative PCR and expressed as percent enrichment relative to the input chromatin. During nutrient deprivation, binding of RNA Pol II to the p62 promoter increased; binding of β‐catenin to the p62 promoter decreased; TCF4 binding did not change (data are from one representative experiment of at least three independent experiments performed in triplicate). PCR products subjected to agarose gel electrophoresis are shown in Supplementary Figure S2A.


( B ) Relative p62 mRNA expression by qRT - PCR increases ∼ 14 ‐ fold after 24 h starvation in HT29 ( mean ± s . e . m . , three independent experiments performed in triplicate , * * * P0 . 001 ) .

O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-CELL_LINE O 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) Relative p62 mRNA expression by qRT-PCR increases ∼14‐fold after 24 h starvation in HT29 cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001).


( C ) Binding of acetyl ‐ Histone H3 to p62 promoter increased under starvation conditions , suggesting p62 gene derepression ( data are from one representative experiment of at least three independent experiments performed in triplicate ) .

O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

(C) Binding of acetyl‐Histone H3 to the p62 promoter increased under starvation conditions, suggesting p62 gene derepression (data are from one representative experiment of at least three independent experiments performed in triplicate).


( D ) Binding of β ‐ catenin to p62 promoter ( relative to β ‐ catenin binding to a non ‐ target gene promoter , GAPDH ) significantly decreased under starvation conditions ( mean ± s . e . m . , three independent experiments performed in triplicate , * * P0 . 01 ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O 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) Binding of β‐catenin to the p62 promoter (relative to β‐catenin binding to a non‐target gene promoter, GAPDH) significantly decreased under starvation conditions (mean±s.e.m., three independent experiments performed in triplicate, **P0.01).


( A ) Western blotting showing that autophagy was induced after 2 h starvation , as evidenced by decreased p62 increased LC3 ‐ II expression . β ‐ Catenin expression decreased over a 24 ‐ h period of starvation in HT29 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-CELL_LINE O

(A) Western blotting showing that autophagy was induced after 2 h starvation, as evidenced by decreased p62 protein and increased LC3‐II protein expression. β‐Catenin protein expression decreased over a 24‐h period of starvation in HT29 cells.


( B ) Autophagy decreased following Atg7 knockdown as shown by increased p62 decreased LC3 ‐ II expression . β ‐ Catenin expression increased following Atg7 siRNA . Reduction of LC3 with Atg7 siRNA was confirmed by immunofluorescence is shown in Supplementary Figure S2B .

O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O

(B) Autophagy decreased following Atg7 knockdown as shown by increased p62 and decreased LC3‐II protein expression. β‐Catenin protein expression increased following Atg7 siRNA. Reduction of LC3 puncta with Atg7 siRNA was confirmed by immunofluorescence and is shown in Supplementary Figure S2B.


( C , D ) Reduction of TopFlash activity in HT29 after 24 h of autophagy induction using ( C ) starvation or ( D ) 100 nM mTOR inhibitor PP242 ( C D , mean ± s . e . m . , three independent experiments performed in triplicate , * * * P0 . 001 ) . Autophagy induction was confirmed by western blotting is shown in Supplementary Figures S2C S2D ) .

O O O O O O O B-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Reduction of TopFlash activity in HT29 cells after 24 h of autophagy induction using (C) starvation or (D) 100 nM mTOR inhibitor PP242 (C and D, mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). Autophagy induction was confirmed by western blotting and is shown in Supplementary Figures S2C and S2D).


( E , F ) qRT - PCR shows reduction of Wnt ‐ target gene expression ( E ) Axin2 ( * P = 0 . 017 ) ( F ) Cyclin D1 ( * * P = 0 . 0036 ) after 8 h starvation ( mean ± s . e . m . , three ( Axin2 ) or six ( Cyclin D1 ) independent experiments performed in triplicate ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD O O 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-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O

(E, F) qRT-PCR shows reduction of Wnt‐target gene expression (E) Axin2 (*P=0.017) and (F) Cyclin D1 (**P=0.0036) after 8 h starvation (mean±s.e.m., three (Axin2) or six (Cyclin D1) independent experiments performed in triplicate).


( G - I ) Inhibition of Wnt ‐ induced TopFlash activity by autophagy induction in ( G ) HCT116 β ‐ cateninWT / − ( mean ± s . e . m . , three independent experiments performed in triplicate , * * P0 . 01 ) ( H ) RKO ( mean ± s . e . m . , three independent experiments performed in triplicate , * P0 . 05 ) . ( I ) Reduction of Wnt3a ‐ induced Cyclin D1 gene expression RKO after 12 h treatment with autophagy induction using starvation or PP242 ( mean ± s . e . m . , two independent experiments ) .

O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-CELL_LINE B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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-CELL_LINE O O O O 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-GENEPROD O O B-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

(G-I) Inhibition of Wnt‐induced TopFlash activity by autophagy induction in (G) HCT116 β‐cateninWT/− cells (mean±s.e.m., three independent experiments performed in triplicate, **P0.01) and (H) RKO cells (mean±s.e.m., three independent experiments performed in triplicate, *P0.05). (I) Reduction of Wnt3a‐induced Cyclin D1 gene expression RKO cells after 12 h treatment with autophagy induction using starvation or PP242 (mean±s.e.m., two independent experiments).