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

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


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

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


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

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


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

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


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

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


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

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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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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 ) 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 )

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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 the DTE - induced potentiation of WT and double cysteine

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


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

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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 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 -

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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 - and off - rate constants ( kon and koff ) . All values were normalized to the value obtained for WT GluN1 / GluN2B

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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 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 )

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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 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 )

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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)


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

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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 ) and mutant receptors . All values are normalized to that obtained for WT GluN1 / GluN2B

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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 . The bottom trace is an expanded view . C , closed channel ; O , open

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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 the WT and super - active GluN1 - E698C / GluN2B - L795C

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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 ) 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

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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 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 ) .

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(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 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 ) .

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(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 / − 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 .

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

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(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 / − and HCT116β ‐ catenin − / ΔS45 cells by western blotting showed decreased p62 expression in β ‐ catenin − / ΔS45 cells .

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(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 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 .

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(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 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 .

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(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 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 ) .

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(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 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 ) .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 ) 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 .

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(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 ) protein expression in HEK293T cells . As an internal control , cells not overexpressing Fzd6 are shown in the field of view .

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(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 and LC3 ‐ II protein expression in doxycycline ‐ inducible DNTCF4 LS174T ‐ L8 cells . LGR5 downregulation by DNTCF4 confirmed inhibition of β ‐ catenin / TCF4 signalling .

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(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 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 ) .

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(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 / − 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 .

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(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 ) 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 .

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(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 cells ( mean ± s . e . m . , three independent experiments performed in triplicate , * * * P0 . 001 ) .

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(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 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 ) .

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(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 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 ) .

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(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 protein and increased LC3 ‐ II protein expression . β ‐ Catenin protein expression decreased over a 24 ‐ h period of starvation in HT29 cells .

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(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 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 .

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(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 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 ) .

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(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 ) 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 ) .

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(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 / − 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 ) .

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(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).


( J , K ) TopFlash activity following Atg7 knockdown in ( J ) HT29 cells ( mean ± s . e . m . , four independent experiments performed in triplicate , * P = 0 . 0488 ) and ( K ) RKO cells with 24 hWnt3a treatment ( mean ± s . e . m . , three independent experiments performed in triplicate , * P = 0 . 0282 ) .

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(J, K) TopFlash activity following Atg7 knockdown in (J) HT29 cells (mean±s.e.m., four independent experiments performed in triplicate, *P=0.0488) and (K) RKO cells with 24 hWnt3a treatment (mean±s.e.m., three independent experiments performed in triplicate, *P=0.0282).


( L ) A representative western blot of RKO cells following Wnt3a treatment and Atg7 siRNA .

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(L) A representative western blot of RKO cells following Wnt3a treatment and Atg7 siRNA.


( M ) Relative β ‐ catenin mRNA levels did not change after 8 h starvation by qRT - PCR ( mean ± s . e . m . , three independent experiments performed in triplicate ) .

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(M) Relative β‐catenin mRNA levels did not change after 8 h starvation by qRT-PCR (mean±s.e.m., three independent experiments performed in triplicate).


( N ) Western blot showing prevented β ‐ catenin protein degradation during starvation in the presence of lysosomal autophagy inhibitors chloroquine ( 10 μM ) and bafilomycin A1 ( 100 nM ) compared to starvation alone .

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(N) Western blot showing prevented β‐catenin protein degradation during starvation in the presence of lysosomal autophagy inhibitors chloroquine (10 μM) and bafilomycin A1 (100 nM) compared to starvation alone.


( O ) Inhibition of autophagy using wortmannin ( 50 nM ) prevented β ‐ catenin protein degradation during starvation .

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(O) Inhibition of autophagy using wortmannin (50 nM) prevented β‐catenin protein degradation during starvation.


( P ) Western blot analysis of RKO cells expressing a myc - tagged β ‐ catenin mutant ( S33A , S37A , T41A , S45A ) resistant to proteasomal degradation ( myc - β ‐ cateninAAAA ) . RKO cells were subject to nutrient starvation for 2 , 8 and 24 h , and autophagy induction was confirmed by increased LC3 ‐ II and decreased p62 protein expression . Both endogenous β ‐ catenin and myc - β ‐ cateninAAAA protein expression decreased during starvation .

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(P) Western blot analysis of RKO cells expressing a myc-tagged β‐catenin mutant (S33A, S37A, T41A, S45A) resistant to proteasomal degradation (myc-β‐cateninAAAA). RKO cells were subject to nutrient starvation for 2, 8 and 24 h, and autophagy induction was confirmed by increased LC3‐II and decreased p62 protein expression. Both endogenous β‐catenin and myc-β‐cateninAAAA protein expression decreased during starvation.


( Q ) Proteasome inhibition with MG132 ( 10 μM ) did not prevent starvation ‐ induced decrease in β ‐ catenin protein expression . Source data for this figure is available on the online supplementary information page .

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(Q) Proteasome inhibition with MG132 (10 μM) did not prevent starvation‐induced decrease in β‐catenin protein expression.Source data for this figure is available on the online supplementary information page.


LC3 and β ‐ catenin co ‐ localise in the mouseintestinal epithelium . ( A and B ) Immunofluorescence of LC3 ( red ) and β ‐ catenin ( green ) expression in the intestinal epithelium following 2 daystamoxifen treatment in control ( A : β ‐ catenin + / lox ‐ villin ‐ creERT2 ) and β ‐ catenin deleted ( B : β ‐ catenin − / lox ‐ villin ‐ creERT2 ) mice . ( C and D ) Magnified areas from B revealing co ‐ localisation of β ‐ catenin ( red ) and β ‐ catenin ( green ) . Arrowheads indicate co ‐ localisation of LC3 and β ‐ catenin . ( E ) Linescan analyses from C and D showing staining intensity of indicated co ‐ localised puncta . Red and green channel levels were adjusted post acquisition ( equal changes applied across the entire figure ) and the blue channel was removed for clarity .

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LC3 and β‐catenin co‐localise in the mouseintestinal epithelium. (A and B) Immunofluorescence of LC3 (red) and β‐catenin (green) expression in the intestinal epithelium following 2 daystamoxifen treatment in control (A: β‐catenin+/lox‐villin‐creERT2) and β‐catenin deleted (B: β‐catenin−/lox‐villin‐creERT2) mice. (C and D) Magnified areas from B revealing co‐localisation of β‐catenin (red) and β‐catenin (green). Arrowheads indicate co‐localisation of LC3 and β‐catenin. (E) Linescan analyses from C and D showing staining intensity of indicated co‐localised puncta. Red and green channel levels were adjusted post acquisition (equal changes applied across the entire figure) and the blue channel was removed for clarity.


( A ) Co ‐ immunoprecipitation of YFP - LC3 or negative control YFP in HT29 cells . Binding of endogenous β ‐ catenin to YFP - LC3 was detected after 8 h of autophagy induction by starvation and starvation in the presence of lysosomal autophagy flux inhibitor chloroquine ( 10 μM ) . Input and immunodepleted lysates are shown .

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(A) Co‐immunoprecipitation of YFP-LC3 or negative control YFP in HT29 cells. Binding of endogenous β‐catenin to YFP-LC3 was detected after 8 h of autophagy induction by starvation and starvation in the presence of lysosomal autophagy flux inhibitor chloroquine (10 μM). Input and immunodepleted lysates are shown.


( B ) Immunoprecipitation of endogenous LC3 in HT29 cells . IgG was used as a negative control .

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(B) Immunoprecipitation of endogenous LC3 in HT29 cells. IgG was used as a negative control.


( D ) Pulldown assays using recombinant GST or GST - LC3B and lysates from HEK293 cells expressing HA - tagged wild ‐ type β ‐ catenin ( HA - β ‐ cateninWT ) or W504A / I507A β ‐ catenin ( HA - β ‐ cateninW504A / I507A ) . HA - β ‐ cateninW504A / I507A exhibited reduced GST - LC3B binding compared to HA - β ‐ cateninWT .

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(D) Pulldown assays using recombinant GST or GST-LC3B and lysates from HEK293 cells expressing HA-tagged wild‐type β‐catenin (HA-β‐cateninWT) or W504A/I507A β‐catenin (HA-β‐cateninW504A/I507A). HA-β‐cateninW504A/I507A exhibited reduced GST-LC3B binding compared to HA-β‐cateninWT.


( E ) Recombinant myc - tagged His - LC3 ( His - LC3 - myc ) interacted with recombinant GST - β ‐ cateninWTin vitro , but binding to GST - β ‐ cateninW504A / I507A was reduced ( upper panel ) . Input recombinant proteins visualised with Coomassie blue are shown ( lower panels ) .

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(E) Recombinant myc-tagged His-LC3 (His-LC3-myc) interacted with recombinant GST-β‐cateninWTin vitro, but binding to GST-β‐cateninW504A/I507A was reduced (upper panel). Input recombinant proteins visualised with Coomassie blue are shown (lower panels).


( F ) Co ‐ immunoprecipitation experiments using lysates from HEK293 cells transiently expressing YFP - LC3 with β ‐ cateninWT or β ‐ cateninW504A / I507A starved for 8 h with chloroquine ( 10 μM ) . YFP - LC3 immunoprecipitated β ‐ cateninWT but not β ‐ cateninW504A / I507A . Input lysates are shown and immunoprecipitated p62 and YFP - LC3 served as positive controls .

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(F) Co‐immunoprecipitation experiments using lysates from HEK293 cells transiently expressing YFP-LC3 with β‐cateninWT or β‐cateninW504A/I507A starved for 8 h with chloroquine (10 μM). YFP-LC3 immunoprecipitated β‐cateninWT but not β‐cateninW504A/I507A. Input lysates are shown and immunoprecipitated p62 and YFP-LC3 served as positive controls.


( G ) HA - β ‐ cateninS33A / S37A / T41A / S45A / W504A / I507A ( HA - β ‐ catenin6A ) was more resistant to the starvation ‐ induced reduction in β ‐ catenin protein expression than HA - β ‐ cateninS33Y .

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(G) HA-β‐cateninS33A/S37A/T41A/S45A/W504A/I507A (HA-β‐catenin6A) was more resistant to the starvation‐induced reduction in β‐catenin protein expression than HA-β‐cateninS33Y.


( H ) Relative cell death in HT29 cells subject to nutrient starvation for 24 h . Atg7 knockdown increased cell survival . The increase in cell survival following Atg7 knockdown was significantly reversed by simultaneously depleting β ‐ catenin ( double knockdown of Atg7 and β ‐ catenin ) . Data are the mean ± s . e . m . of three independent experiments performed in triplicate , * * * P0 . 001 ; * * P0 . 01 . Source data for this figure is available on the online supplementary information page .

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(H) Relative cell death in HT29 cells subject to nutrient starvation for 24 h. Atg7 knockdown increased cell survival. The increase in cell survival following Atg7 knockdown was significantly reversed by simultaneously depleting β‐catenin (double knockdown of Atg7 and β‐catenin). Data are the mean±s.e.m. of three independent experiments performed in triplicate, ***P0.001; **P0.01.Source data for this figure is available on the online supplementary information page.


Identification of Rtt105 - TAP associated proteins . Purified proteins were resolved on a 4 - 12 % gel , revealed by silver staining , and identified by mass spectrometry ( Table EV1 ) . * Non - specific band also in control .

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Identification of Rtt105-TAP associated proteins. Purified proteins were resolved on a 4-12% gel, revealed by silver staining, and identified by mass spectrometry (Table EV1). *Non-specific band also in control.


Cells containing Rfa1 - TAP were synchronized at G1 , S , and G2 / M phase and used for the TAP purification . Rfa1 - TAP associated protein complexes were analyzed by Western blot using CBP , Rfa2 , and Rtt105 antibodies ( left panel ) . DNA content was monitored by flow cytometry ( right panel ) .

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Cells containing Rfa1-TAP were synchronized at G1, S, and G2/M phase and used for the TAP purification. Rfa1-TAP associated protein complexes were analyzed by Western blot using CBP, Rfa2, and Rtt105 antibodies (left panel). DNA content was monitored by flow cytometry (right panel).


MBP - Rtt105 pulled down recombinant RPA , resolved on SDS - PAGE gels and visualized by Coomassie Brilliant Blue ( CBB ) staining . MBP was used as a negative control .

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MBP-Rtt105 pulled down recombinant RPA, resolved on SDS-PAGE gels and visualized by Coomassie Brilliant Blue (CBB) staining. MBP was used as a negative control.


GST - tagged full length ( FL ) and Rtt105 deletion mutants ( Schematic of the Rtt105 truncations is shown in the upper panel ) were purified and used to pull down Rfa1 protein . GST proteins were used as a negative control . Isolated protein complexes were resolved on 15 % SDS - PAGE gels and visualized by CBB staining .

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GST-tagged full length (FL) and Rtt105 deletion mutants (Schematic of the Rtt105 truncations is shown in the upper panel) were purified and used to pull down Rfa1 protein. GST proteins were used as a negative control. Isolated protein complexes were resolved on 15% SDS-PAGE gels and visualized by CBB staining.


Rfa1 - TAP purification was performed using Rfa1 - TAP rtt105 ∆ strains expressing WT and indicated Rtt105 mutant forms . pRS313 serves as vector control ( Vc ) .

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Rfa1-TAP purification was performed using Rfa1-TAP rtt105∆ strains expressing WT and indicated Rtt105 mutant forms. pRS313 serves as vector control (Vc).


Snapshots of Rfa1 ChIP - Seq peak at ARS305 from WT and rtt105 ∆ cells released into HU - medium . The average Rfa1 ChIP - Seq read density from cells released into HU - medium around ACS sites . ACS : ARS consensus sequence .

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Snapshots of Rfa1 ChIP-Seq peak at ARS305 from WT and rtt105∆ cells released into HU-medium. The average Rfa1 ChIP-Seq read density from cells released into HU-medium around ACS sites. ACS: ARS consensus sequence.


Snapshots of Rfa2 ChIP - Seq peaks at ARS305 from cells released at 16 ° C for 72 min . The average Rfa2 ChIP - Seq read density surrounding ACS sites from cells released at 16 ° C for 72 min .

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Snapshots of Rfa2 ChIP-Seq peaks at ARS305 from cells released at 16°C for 72 min. The average Rfa2 ChIP-Seq read density surrounding ACS sites from cells released at 16°C for 72 min.


Rfa1 occupancy analysis of cells progression through S phase . G1 - arrested cells were released into fresh YPD medium to allow cell progression through S phase at standard growth temperature ( 25 ° C ) . Equal amounts of cells were collected at each time point . Rfa1 ChIP were performed using IgG beads for TAP - tag . ARS305 and ARS607 , early fired replication origins ; ARS607 + 14kb , a corresponding 14kb away region of ARS607 . The mean and standard deviation ( SD ) of three biological replicates were shown .

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Rfa1 occupancy analysis of cells progression through S phase. G1-arrested cells were released into fresh YPD medium to allow cell progression through S phase at standard growth temperature (25°C). Equal amounts of cells were collected at each time point. Rfa1 ChIP were performed using IgG beads for TAP-tag. ARS305 and ARS607, early fired replication origins; ARS607+14kb, a corresponding 14kb away region of ARS607. The mean and standard deviation (SD) of three biological replicates were shown.


The nuclear localization of Rfa1 proteins is altered in rtt105 ∆ mutant cells . Rfa1 tagged with GFP at its C terminus was used to analyze the localization of Rfa1 - GFP fusion protein ( Green ) and the nuclear envelop is visualized with the Nup49 - mCherry fusion protein ( Red ) using fluorescence microscopy . DIC : differential interference contrast . Scale bar : 5μm .

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The nuclear localization of Rfa1 proteins is altered in rtt105∆ mutant cells. Rfa1 tagged with GFP at its C terminus was used to analyze the localization of Rfa1-GFP fusion protein (Green) and the nuclear envelop is visualized with the Nup49-mCherry fusion protein (Red) using fluorescence microscopy. DIC: differential interference contrast. Scale bar: 5μm.


NLS - RFA1 - GFP rescues the nuclear localization of Rfa1 in rtt105 ∆ mutant cells . The wild type RFA1 gene was fused with an SV40 large T Antigen nuclear localization sequence ( NLS ) at its 5 ' - end and a GFP gene at its 3 ' end to obtain a construct , driven by the RFA1 promoter to express NLS - RFA1 - GFP fusion protein . The engineered construct was then transformed into WT or rtt105 ∆ mutant yeast cells to replace its endogenous RFA1 expression . The resulting yeast cells were then visualized under microscope , and GFP signals enriched in the nuclei were scored . RFA1 - GFP lacking the NLS sequence was transformed into rtt105 ∆ rfa1 ∆ mutant cells as a control . DAPI staining indicates nuclear DNA .

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NLS-RFA1-GFP rescues the nuclear localization of Rfa1 in rtt105∆ mutant cells. The wild type RFA1 gene was fused with an SV40 large T Antigen nuclear localization sequence (NLS) at its 5'-end and a GFP gene at its 3' end to obtain a construct, driven by the RFA1 promoter to express NLS-RFA1-GFP fusion protein. The engineered construct was then transformed into WT or rtt105∆ mutant yeast cells to replace its endogenous RFA1 expression. The resulting yeast cells were then visualized under microscope, and GFP signals enriched in the nuclei were scored. RFA1-GFP lacking the NLS sequence was transformed into rtt105∆ rfa1∆ mutant cells as a control. DAPI staining indicates nuclear DNA.


An increase in nuclear localization of Rfa1 could not rescue the RPA binding defects at the replication regions in rtt105 ∆ cells . The percentage of Rfa2 ChIP - DNA over the total input DNA was calculated . The mean and standard error ( SE ) of three biological replicates were shown . Statistical significance was evaluated based on Student ' s t - tests ( * : 0 . 01 ≤ P value < 0 . 05 ) .

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An increase in nuclear localization of Rfa1 could not rescue the RPA binding defects at the replication regions in rtt105∆ cells. The percentage of Rfa2 ChIP-DNA over the total input DNA was calculated. The mean and standard error (SE) of three biological replicates were shown. Statistical significance was evaluated based on Student's t-tests (*: 0.01≤P value<0.05).


Purified RPA and Rtt105 - RPA complex were resolved on 15 % SDS - PAGE gels and analyzed by silver staining .

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Purified RPA and Rtt105-RPA complex were resolved on 15% SDS-PAGE gels and analyzed by silver staining.


EMSA assay was performed to analyze ssDNA binding ability of RPA or Rtt105 - RPA complex . Cy3 - labeled 30nt ssDNA was used as the substrate , and reaction mixtures were resolved on native PAGE gels . Quantification of the RPA - bound ssDNA is shown in ( C ) .

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EMSA assay was performed to analyze ssDNA binding ability of RPA or Rtt105-RPA complex. Cy3-labeled 30nt ssDNA was used as the substrate, and reaction mixtures were resolved on native PAGE gels. Quantification of the RPA-bound ssDNA is shown in (C).


Recombinant Rtt105 - RPA complex was mixed with a 30nt - ssDNA in a 2 : 1 ratio , and the reaction product were subjected to gel filtration chromatography . Equal volume of fraction 5 - 10 were resolved on SDS - PAGE for detecting proteins by Ruby staining ( upper panel ) or were resolved by native agarose gel for detection of Cy3 - ssDNA signal ( lower panel ) .

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

Recombinant Rtt105-RPA complex was mixed with a 30nt-ssDNA in a 2:1 ratio, and the reaction product were subjected to gel filtration chromatography. Equal volume of fraction 5-10 were resolved on SDS-PAGE for detecting proteins by Ruby staining (upper panel) or were resolved by native agarose gel for detection of Cy3-ssDNA signal (lower panel).