( a ) RT - PCR expression and quantification of IGF1 , IGF1Rβ and IGF2 levels in untreated C2C12 and shCD20 C2C12 myoblasts and 10 nM IGF1 - treated C2C12 and over - expressing CD20 C2C12 myoblasts . Each experiment was replicated independently four times .

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(a) RT-PCR expression and quantification of IGF1, IGF1Rβ and IGF2 levels in untreated C2C12 and shCD20 C2C12 myoblasts and 10 nM IGF1-treated C2C12 and over-expressing CD20 C2C12 myoblasts. Each experiment was replicated independently four times.


( b ) Immunofluorescence for IGF2 ( in green ) in untreated and 10 nM IGF1 - treated C2C12 myoblasts . Scale bars = 75 μm .

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

(b) Immunofluorescence for IGF2 (in green) in untreated and 10 nM IGF1-treated C2C12 myoblasts. Scale bars = 75 μm.


Representative CD20 immunoprecipitation using anti - pSer + pThr in ( c ) C2C12 cells treated with IGF1 for two hours ( 2 h ) or overnight ( ON ) as indicated . Densitometric analysis of data are expressed as the ratio of CD20 / vinculin or pSer + pThr / CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . Each experiment was performed in triplicate wells . All values are expressed as the mean ± SEM .

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

Representative CD20 immunoprecipitation using anti-pSer+pThr in (c) C2C12 cells treated with IGF1 for two hours (2 h) or overnight (ON) as indicated. Densitometric analysis of data are expressed as the ratio of CD20/vinculin or pSer+pThr/CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two-way ANOVA. *p<0.05; **p<0.01; ****p<0.0001. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM.


Representative CD20 immunoprecipitation using anti - pSer + pThr in ( d ) sh - empty ( shCTR ) - and shCD20 - treated C2C12 cells treated with anti - Flag and 10 nM anti - IGF2R , as indicated . Densitometric analysis of data are expressed as the ratio of CD20 / vinculin or pSer + pThr / CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . Each experiment was performed in triplicate wells . All values are expressed as the mean ± SEM .

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

Representative CD20 immunoprecipitation using anti-pSer+pThr in (d) sh-empty (shCTR)- and shCD20-treated C2C12 cells treated with anti-Flag and 10 nM anti-IGF2R, as indicated. Densitometric analysis of data are expressed as the ratio of CD20/vinculin or pSer+pThr/CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two-way ANOVA. *p<0.05; **p<0.01; ****p<0.0001. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM.


Representative CD20 immunoprecipitation using anti - pSer + pThr in ( e ) C2C12 cells treated with IGF1 and IGF2 , as indicated . Densitometric analysis of data are expressed as the ratio of CD20 / vinculin or pSer + pThr / CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . Each experiment was performed in triplicate wells . All values are expressed as the mean ± SEM .

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

Representative CD20 immunoprecipitation using anti-pSer+pThr in (e) C2C12 cells treated with IGF1 and IGF2, as indicated. Densitometric analysis of data are expressed as the ratio of CD20/vinculin or pSer+pThr/CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two-way ANOVA. *p<0.05; **p<0.01; ****p<0.0001. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM.


( f ) Representative WB of IGF2R and phosphorylated IGF2R ( pIGF2R ) in untreated , shCD20 - treated and anti - IGF2R - treated C2C12 cells . Densitometric analysis of data are expressed as the ratio of IGF2R / actin or pIGF2R / IGF2R in arbitrary units in the lower panels . One - way ANOVA . * p < 0 . 05 ; Each experiment was performed in triplicate wells . All values are expressed as the mean ± SEM .

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(f) Representative WB of IGF2R and phosphorylated IGF2R (pIGF2R) in untreated, shCD20-treated and anti-IGF2R-treated C2C12 cells. Densitometric analysis of data are expressed as the ratio of IGF2R/actin or pIGF2R/IGF2R in arbitrary units in the lower panels. One-way ANOVA. *p<0.05; Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM.


( g ) IGF2R immunoprecipitation products were immunoblotted for Gαi2 and WB expression of IGF2R in untreated , IGF2 - treated , anti - IGF2R - treated and IGF2 + anti - IGF2R treated C2C12 cells . Densitometric analysis of data are expressed as the ratio of Gαi2 / IGF2R in arbitrary units in the lower panel .

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

(g) IGF2R immunoprecipitation products were immunoblotted for Gαi2 and WB expression of IGF2R in untreated, IGF2-treated, anti-IGF2R-treated and IGF2+anti-IGF2R treated C2C12 cells. Densitometric analysis of data are expressed as the ratio of Gαi2/IGF2R in arbitrary units in the lower panel.


( h ) Representative WB of IGF1Rβ and IGF1Rβ immunoprecipitation products immunoblotted for p - Tyr in untreated , IGF2 - treated and IGF2 + anti - IGF2R - treated ( 1 : 500 , 1 : 1000 and 1 : 2000 dilutions of anti - IGF2R ) C2C12 cells ( cells were treated for 2 and 24 h ) . Densitometric analysis of data are expressed as the ratio of pTYR / IGF1Rβ in arbitrary units in the lower panel . Each experiment was performed in triplicate wells . All values are expressed as the mean ± SEM .

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

(h) Representative WB of IGF1Rβ and IGF1Rβ immunoprecipitation products immunoblotted for p-Tyr in untreated, IGF2-treated and IGF2+anti-IGF2R-treated (1:500, 1:1000 and 1:2000 dilutions of anti-IGF2R) C2C12 cells (cells were treated for 2 and 24 h). Densitometric analysis of data are expressed as the ratio of pTYR/IGF1Rβ in arbitrary units in the lower panel. Each experiment was performed in triplicate wells. All values are expressed as the mean±SEM.


( d ) Over - expressing CD20 in C2C12 myoblasts that co - expressed CD20 ( in green ) and IGF2R ( in red ) . DAPI - labelled nuclei are shown in blue . Scale bars = 75 μm .

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

(d) Over-expressing CD20 in C2C12 myoblasts that co-expressed CD20 (in green) and IGF2R (in red). DAPI-labelled nuclei are shown in blue. Scale bars=75 μm.


( e ) Representative CD20 and IGF2R immunoprecipitation products immunoblotted for IGF2R and CD20 , respectively , in UNTREATED and shCD20 - treated C2C12 cell membranes and whole lysates of proteins . The immunoprecipitation output is shown as IP neg .

(e) Representative CD20 and IGF2R immunoprecipitation products immunoblotted for IGF2R and CD20, respectively, in UNTREATED and shCD20-treated C2C12 cell membranes and whole lysates of proteins. The immunoprecipitation output is shown as IP neg.


( f ) Myotubes immunofluorescence of cells in proliferation medium ( P . M . ) and after 2 , 4 and 6 days of myogenic differentiation in serum free medium . Control ( untreated ) , shCD20 - treated C2C12 cells , C2C12 and HSkM myoblasts pre - treated with anti - IGF2R for 24 hours were stained . Desmin - positive myotubes are shown in green . Scale bars = 75 μm .

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

(f) Myotubes immunofluorescence of cells in proliferation medium (P.M.) and after 2, 4 and 6 days of myogenic differentiation in serum free medium. Control (untreated), shCD20-treated C2C12 cells, C2C12 and HSkM myoblasts pre-treated with anti-IGF2R for 24 hours were stained. Desmin-positive myotubes are shown in green. Scale bars=75 μm.


( g ) Fusion index quantification after 6 days of differentiation . One - way ANOVA . * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . Each experiment was performed in triplicate wells . All values are expressed as mean ± SEM .

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

(g) Fusion index quantification after 6 days of differentiation. One-way ANOVA. **p<0.01; ***p<0.001; ****p<0.0001. Each experiment was performed in triplicate wells. All values are expressed as mean±SEM.


( h ) Representative WB of anti - myogenin , anti - Myf5 , anti - MyHC and anti - β - actin in total protein lysates obtained from untreated , shCD20 - treated C2C12 cells , and C2C12 and HSkM myoblasts pre - treated with anti - IGF2R for 24 hours ; cells were collected under P . M . and after 2 , 4 and 6 days of myogenic differentiation . Densitometric analysis of WB data expressed as the ratio of the indicated antibody / β - actin in arbitrary units . Two - way ANOVA test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 in comparison to the results obtained in untreated cells at the corresponding time point . # # p < 0 . 01 ; # # # p < 0 . 001 ; # # # # p < 0 . 0001 in comparison to the results obtained in shCD20 - treated C2C12 cells at the corresponding time point . Each experiment was performed in triplicate wells . All values are expressed as the mean ± SEM .

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

(h) Representative WB of anti-myogenin, anti-Myf5, anti-MyHC and anti-β-actin in total protein lysates obtained from untreated, shCD20-treated C2C12 cells, and C2C12 and HSkM myoblasts pre-treated with anti-IGF2R for 24 hours; cells were collected under P.M. and after 2, 4 and 6 days of myogenic differentiation. Densitometric analysis of WB data expressed as the ratio of the indicated antibody/β-actin in arbitrary units. Two-way ANOVA test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 in comparison to the results obtained in untreated cells at the corresponding time point. ##p<0.01; ###p<0.001; ####p<0.0001 in comparison to the results obtained in shCD20-treated C2C12 cells at the corresponding time point. Each experiment was performed in triplicate wells. All values are expressed as the mean±SEM.


Immunofluorescence analysis of CD20 and IGF2R expression ( in red ) in the TA muscles of three - month - old C57Bl6 / J and mdx mice . Scale bars = 75μm . ( a ) .

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Immunofluorescence analysis of CD20 and IGF2R expression (in red) in the TA muscles of three-month-old C57Bl6/J and mdx mice. Scale bars=75μm. (a).


IGF2R ( green ) and sarcomeric actin ( magenta ) expression in the VM muscles of two DMD patients . Scale bars = 25μm . ( b ) .

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IGF2R (green) and sarcomeric actin (magenta) expression in the VM muscles of two DMD patients. Scale bars=25μm. (b).


Representative WB analysis of CD20 and β - actin ( c ) expression in the TA and VM muscles of C57Bl6 / J and mdx mice ( n = 10 mice per group ) . Densitometry analysis of WB data are expressed as the CD20 / β - actin ratio , in arbitrary units in the lower panels One - way ANOVA . * * * * p < 0 . 0001 .

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

Representative WB analysis of CD20 and β-actin (c) expression in the TA and VM muscles of C57Bl6/J and mdx mice (n=10 mice per group). Densitometry analysis of WB data are expressed as the CD20/β-actin ratio, in arbitrary units in the lower panels One-way ANOVA. ****p<0.0001.


Representative WB analysis of pSer + pThr and GADPH ( d ) expression in the TA and VM muscles of C57Bl6 / J and mdx mice ( n = 10 mice per group ) . Densitometry analysis of WB data are expressed as the pSer + pThr / CD20 ratio in arbitrary units in the lower panels One - way ANOVA . * * * * p < 0 . 0001 .

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

Representative WB analysis of pSer+pThr and GADPH (d) expression in the TA and VM muscles of C57Bl6/J and mdx mice (n=10 mice per group). Densitometry analysis of WB data are expressed as the pSer+pThr/CD20 ratio in arbitrary units in the lower panels One-way ANOVA. ****p<0.0001.


( e ) Representative WB analysis of IGF2R and GAPDH expression in total protein lysates of the human ( healthy and DMD ) and murine ( C57Bl6 / J and mdx ) TA and VM muscles ( n = 2 per healthy and DMD ; n = 10 mice per group ) ; quantifications are expressed as the IGF2R / GAPDH ratio in arbitrary units .

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

(e) Representative WB analysis of IGF2R and GAPDH expression in total protein lysates of the human (healthy and DMD) and murine (C57Bl6/J and mdx) TA and VM muscles (n=2 per healthy and DMD; n=10 mice per group); quantifications are expressed as the IGF2R/GAPDH ratio in arbitrary units.


( f ) Representative WB analysis of IGF2R expression in sarcolemma protein lysates of the TA and VM muscles of healthy , DMD , C57Bl6 / J and mdx ( n = 2 per healthy and DMD ; n = 10 mice per group ) . Densitometry analysis of IGF2R expression , results are shown as a fold increase , as indicated in the lower panel in arbitrary units . One - way ANOVA . * p < 0 . 05 ; * * * * p < 0 . 0001 . All values are expressed as the mean ± SEM .

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

(f) Representative WB analysis of IGF2R expression in sarcolemma protein lysates of the TA and VM muscles of healthy, DMD, C57Bl6/J and mdx (n=2 per healthy and DMD; n=10 mice per group). Densitometry analysis of IGF2R expression, results are shown as a fold increase, as indicated in the lower panel in arbitrary units. One-way ANOVA. *p<0.05; ****p<0.0001. All values are expressed as the mean±SEM.


Quantification of the relative frequency of myofibre cross - sectional areas ( CSAs ) expressed as a frequency distribution in the TA and VM muscles of untreated mdx mice or mdx mice treated with a low or high dose of anti - IGF2R for 4 and 9 weeks ( a ) .

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

Quantification of the relative frequency of myofibre cross-sectional areas (CSAs) expressed as a frequency distribution in the TA and VM muscles of untreated mdx mice or mdx mice treated with a low or high dose of anti-IGF2R for 4 and 9 weeks (a).


( b ) Quantification of regenerating myofibres as a proportion of centrally nucleated fibres in the total number of fibres after anti - IGF2R administration . The quantification of regenerating and M - cadherin - positive satellite cells per section is shown . One - way ANOVA test , * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 regenerating myofibres as a proportion of centrally nucleated fibres in the total number of fibres after anti-IGF2R administration. The quantification of regenerating and M-cadherin-positive satellite cells per section is shown. One-way ANOVA test, **p<0.01; ***p<0.001; ****p<0.0001.


( c ) Representative WB analysis of neonatal myosin heavy chain ( neonatal MHC ) expression and quantification expressed as the ratio of neonatal MHC / β - actin in untreated mdx mice and mdx mice treated with low and high doses of anti - IGF2R for 4 and 9 weeks . One - way ANOVA . * p < 0 . 05 ; * * * p < 0 . 0001 ( n = 10 mice per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative WB analysis of neonatal myosin heavy chain (neonatal MHC) expression and quantification expressed as the ratio of neonatal MHC/β-actin in untreated mdx mice and mdx mice treated with low and high doses of anti-IGF2R for 4 and 9 weeks. One-way ANOVA. *p<0.05; ***p<0.0001 (n=10 mice per group).


( d ) Specific force ( maximal tetanic force normalized to cross - sectional area , Po / CSA ) from TA and VM muscles in C57Bl6 / J mice , untreated mdx mice , and mdx mice treated with low and high doses of anti - IGF2R for 4 and 9 weeks . One - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ( n = 10 mice per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Specific force (maximal tetanic force normalized to cross-sectional area, Po/CSA) from TA and VM muscles in C57Bl6/J mice, untreated mdx mice, and mdx mice treated with low and high doses of anti-IGF2R for 4 and 9 weeks. One-way ANOVA. *p<0.05; **p<0.01; ***p<0.001 (n=10 mice per group).


SERCA activity quantified as ATPase hydrolysis activity vs . min ( e ) in C57Bl6 / J mice , untreated mdx mice , and mdx mice treated with low and high doses of anti - IGF2R for 4 and 9 weeks . One - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ( n = 10 mice per group ) .

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SERCA activity quantified as ATPase hydrolysis activity vs. min (e) in C57Bl6/J mice, untreated mdx mice, and mdx mice treated with low and high doses of anti-IGF2R for 4 and 9 weeks. One-way ANOVA. *p<0.05; **p<0.01; ***p<0.001 (n=10 mice per group).


( a ) Representative WB analysis of active 48 kDa and inactive 60 kDa calcineurin A and β - actin expression in the TA and VM muscles in untreated C57BL mice , untreated mdx mice and mdx treated with low and high doses of anti - IGF2R for 4 and 9 weeks . Densitometry analysis of data expressed as ratios of 48 kDa and 60 kDa calcineurin A / β - actin in arbitrary units . Two - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 ( n = 10 mice per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 WB analysis of active 48 kDa and inactive 60 kDa calcineurin A and β-actin expression in the TA and VM muscles in untreated C57BL mice, untreated mdx mice and mdx treated with low and high doses of anti-IGF2R for 4 and 9 weeks. Densitometry analysis of data expressed as ratios of 48 kDa and 60 kDa calcineurin A/β-actin in arbitrary units. Two-way ANOVA. *p<0.05; **p<0.01; ****p<0.0001 (n=10 mice per group).


( b ) Representative WB analysis of CAMKII , p - CAMKII and GAPDH expression quantified and shown as CAMKII / GAPDH and p - CAMKII / CAMKII ratios in arbitrary units . Two - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ( n = 10 mice per group ) .

(b) Representative WB analysis of CAMKII, p-CAMKII and GAPDH expression quantified and shown as CAMKII/GAPDH and p-CAMKII/CAMKII ratios in arbitrary units. Two-way ANOVA. *p<0.05; **p<0.01 (n=10 mice per group).


( c ) Representative WB analysis of PKCα , p - PKCα and β - actin expression levels quantified and expressed as the PKCα / β - actin and p - PKCα / PKCα ratios in arbitrary units ( n = 10 mice per group ) . All values are expressed as the mean ± SEM .

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(c) Representative WB analysis of PKCα, p-PKCα and β-actin expression levels quantified and expressed as the PKCα/β-actin and p-PKCα/PKCα ratios in arbitrary units (n=10 mice per group). All values are expressed as the mean±SEM.


WB analysis performed in TA and VM muscles of mdx mice treated with anti - IGF2R using antibodies against CD20 , pSer + pThr and vinculin . Densitometry analysis of data expressed as the pSer + pThr / CD20 and CD20 / vinculin ratio in arbitrary units . One - way ANOVA * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 for the comparison with untreated mice ( n = 10 mice per group ) ( a ) .

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

WB analysis performed in TA and VM muscles of mdx mice treated with anti-IGF2R using antibodies against CD20, pSer+pThr and vinculin. Densitometry analysis of data expressed as the pSer+pThr/CD20 and CD20/vinculin ratio in arbitrary units. One-way ANOVA *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 for the comparison with untreated mice (n=10 mice per group)(a).


Representative WB analysis performed on TA and VM muscles of mdx mice treated with anti - IGF2R using anti - IGF1R , anti - pospho - IGF1R and GAPDH . Densitometry analysis of data expressed as the pIGF1R / IGF1R ratio normalized to GAPDH content in arbitrary units . One - way ANOVA . * * * * p < 0 . 0001 for the comparison with untreated mice ( n = 10 mice per group ) ( b ) . All values are expressed as the mean ± SEM .

Representative WB analysis performed on TA and VM muscles of mdx mice treated with anti-IGF2R using anti-IGF1R, anti-pospho-IGF1R and GAPDH. Densitometry analysis of data expressed as the pIGF1R/IGF1R ratio normalized to GAPDH content in arbitrary units. One-way ANOVA. ****p<0.0001 for the comparison with untreated mice (n=10 mice per group) (b). All values are expressed as the mean±SEM.


Corrosion casting results showing the linearization of capillaries in IGF2R - treated VM mdx muscles . Pericytes around capillaries are shown in the insert box ( a ) . Scale bars = 100μm .

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Corrosion casting results showing the linearization of capillaries in IGF2R-treated VM mdx muscles. Pericytes around capillaries are shown in the insert box (a). Scale bars=100μm.


Representative immunofluorescence results ( b ) of longitudinal sections of VM mdx muscles for the CD146 - and NG2 - positive capillaries are shown .

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Representative immunofluorescence results (b) of longitudinal sections of VM mdx muscles for the CD146- and NG2-positive capillaries are shown.


Representative immunofluorescence quantification ( c ) of longitudinal sections of VM mdx muscles for the CD146 - and NG2 - positive capillaries are shown . Scale bars = 75μm . One - way ANOVA . * * * * p < 0 . 0001 for the comparison with untreated mice ( n = 10 mice per group ) . All values are expressed as the mean ± SEM .

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

Representative immunofluorescence quantification (c) of longitudinal sections of VM mdx muscles for the CD146- and NG2-positive capillaries are shown. Scale bars=75μm. One-way ANOVA. ****p<0.0001 for the comparison with untreated mice (n=10 mice per group). All values are expressed as the mean± SEM.


( d ) Representative WB showing CD146 and NG2 expression in total protein lysates of TA and VM muscles of mdx mice treated with anti - IGF2R . Densitometry analysis of data expressed as the ratio of the indicated antibody / β - actin in arbitrary units . One - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 for the comparison with untreated mice ( n = 10 mice per group ) . All values are expressed as the mean ± SEM .

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(d) Representative WB showing CD146 and NG2 expression in total protein lysates of TA and VM muscles of mdx mice treated with anti-IGF2R. Densitometry analysis of data expressed as the ratio of the indicated antibody/β-actin in arbitrary units. One-way ANOVA. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 for the comparison with untreated mice (n=10 mice per group). All values are expressed as the mean ± SEM.


( e ) Representative Ve - cadherin ( red ) and CD31 ( green ) expression in small vessels of VM muscles from untreated mdx and mdx treated with low dose of anti - IGF2R for 4 weeks . Scale bars = 25 μm .

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(e) Representative Ve-cadherin (red) and CD31 (green) expression in small vessels of VM muscles from untreated mdx and mdx treated with low dose of anti-IGF2R for 4 weeks. Scale bars=25 μm.


C , Phosphorylation of TOC1 is eliminated or largely reduced in 5X and S175A . Total protein extracts ( ZT13 ) from 10 - day - old seedlings grown under 12h / 12h light / dark cycles were subject to heat ( 30ºC 20min ) , pyrophosphatase ( PP ) and NaF / Na3VO4 ( phosphatase inhibitors ) treatments . TOC1 - GFP at different phosphorylation states were separated by 8 % SDS - PAGE ( acrylamide : bisacrylamide 150 : 1 ) and subsequently detected by α - GFP .

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

C, Phosphorylation of TOC1 is eliminated or largely reduced in 5X and S175A. Total protein extracts (ZT13) from 10-day-old seedlings grown under 12h/12h light/dark cycles were subject to heat (30ºC 20min), pyrophosphatase (PP) and NaF/Na3VO4 (phosphatase inhibitors) treatments. TOC1-GFP at different phosphorylation states were separated by 8% SDS-PAGE (acrylamide:bisacrylamide 150:1) and subsequently detected by α-GFP.


A - F Shown are hypocotyl lengths of cRL - grown 4 - day - old seedlings of toc1 complemented with wild - type TOC1 ( A ) , 5X ( B ) , T135A ( C ) , S175A ( D ) driven by TOC1 native promoter and wild - type TOC1 ( E ) , 5X ( F ) driven by 35S promoter at sequential fluent rates . Different letters indicate statistically significant differences between genotypes ( p < 0 . 01 , one - way ANOVA followed by Tukey - Kramer HSD test ) , error bars indicate SEM . Data are representative of three biological trials with similar results .

A-F Shown are hypocotyl lengths of cRL-grown 4-day-old seedlings of toc1 complemented with wild-type TOC1 (A), 5X (B), T135A (C), S175A (D) driven by TOC1 native promoter and wild-type TOC1 (E), 5X (F) driven by 35S promoter at sequential fluent rates. Different letters indicate statistically significant differences between genotypes (p<0.01, one-way ANOVA followed by Tukey-Kramer HSD test), error bars indicate SEM. Data are representative of three biological trials with similar results.


Hypocotyl lengths in SD condition of Col - 0 , toc1 - 101 and two independent native or 35S promoter lines of wild - type TOC1 and each phosphosite mutants . The middle line of the box represents the median , the x in the box represents the mean . The bottom line and the top line of the box represent 1st and 3rd quartile , respectively . The whiskers extend from the ends of the box to the minimum value and maximum value . Data information : 18 to 27 seedlings per line were entrained in 12h / 12h light / dark cycles for 7 d and next transferred to constant red light at ZT2 for image acquisition with 2 - h intervals for 1 week .

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Hypocotyl lengths in SD condition of Col-0, toc1-101 and two independent native or 35S promoter lines of wild-type TOC1 and each phosphosite mutants. The middle line of the box represents the median, the x in the box represents the mean. The bottom line and the top line of the box represent 1st and 3rd quartile, respectively. The whiskers extend from the ends of the box to the minimum value and maximum value. Data information: 18 to 27 seedlings per line were entrained in 12h/12h light/dark cycles for 7 d and next transferred to constant red light at ZT2 for image acquisition with 2-h intervals for 1 week.


C - E , Average CCA1 : LUC bioluminescence traces of indicated plant lines . Data information : 18 to 27 seedlings per line were entrained in 12h / 12h light / dark cycles for 7 d and next transferred to constant red light at ZT2 for image acquisition with 2 - h intervals for 1 week . White and gray regions indicate subjective light and dark period . Data are representative of at least two independent trials with similar results . Different letters indicate statistically significant differences in period between genotypes ( p < 0 . 01 , one - way ANOVA followed by Tukey - Kramer HSD test ) .

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C-E, Average CCA1:LUC bioluminescence traces of indicated plant lines. Data information: 18 to 27 seedlings per line were entrained in 12h/12h light/dark cycles for 7 d and next transferred to constant red light at ZT2 for image acquisition with 2-h intervals for 1 week. White and gray regions indicate subjective light and dark period. Data are representative of at least two independent trials with similar results. Different letters indicate statistically significant differences in period between genotypes (p<0.01, one-way ANOVA followed by Tukey-Kramer HSD test).


A - C , PIL1 , AT5G02580 and CDF5 expression pattern in 3 - d - old SD - grown seedlings from indicated plant lines at night . Data is representative of 3 biological trials .

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A-C, PIL1, AT5G02580 and CDF5 expression pattern in 3-d-old SD-grown seedlings from indicated plant lines at night. Data is representative of 3 biological trials.


A , Co - IP assay of PIF3 - TAP with TOC1 - GFP vs . 5X - GFP in N . benthamiana . Protein extracts were immunoprecipitated by IgG resins followed by HRV - 3C protease digestion . PIF3 and TOC1 were detected by anti - MYC and anti - GFP , respectively . B , Quantification of protein interaction in A . Results from 3 independent trials were averaged . Error bars indicate SEM , asterisks indicate significant difference ( * p < 0 . 05 , student ' s t test ) .

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A, Co-IP assay of PIF3-TAP with TOC1-GFP vs. 5X-GFP in N. benthamiana. Protein extracts were immunoprecipitated by IgG resins followed by HRV-3C protease digestion. PIF3 and TOC1 were detected by anti-MYC and anti-GFP, respectively. B, Quantification of protein interaction in A. Results from 3 independent trials were averaged. Error bars indicate SEM, asterisks indicate significant difference (*p<0.05, student's t test).


C , ChIP - qPCR assay of TOC1 binding to promoter of pre - dawn - phased PIF3 target genes at ZT14 in Col - 0 , native promoter TOC1 and 5X lines . D , ChIP - qPCR assay of the chromatin residence of native promoter TOC1 - GFP at target promoters in toc1 vs . pif3 / 4 / 5 toc1 ( pif345t1 ) mutant backgrounds . Data information : 3 - d - old SD - grown seedlings were harvested at ZT14 , TOC1 - GFP was immunoprecipitated by α - GFP and magnetic protein G beads . Data from 3 independent trials were averaged , error bars indicate SEM . Asterisks indicates significant differences ( * * p < 0 . 001 , * * * p < 0 . 0001 , Student ' s t test ) , n . s = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, ChIP-qPCR assay of TOC1 binding to promoter of pre-dawn-phased PIF3 target genes at ZT14 in Col-0, native promoter TOC1 and 5X lines. D, ChIP-qPCR assay of the chromatin residence of native promoter TOC1-GFP at target promoters in toc1 vs. pif3/4/5 toc1 (pif345t1) mutant backgrounds. Data information: 3-d-old SD-grown seedlings were harvested at ZT14, TOC1-GFP was immunoprecipitated by α-GFP and magnetic protein G beads. Data from 3 independent trials were averaged, error bars indicate SEM. Asterisks indicates significant differences (** p<0.001, *** p<0.0001, Student's t test), n.s=not significant.


B , Measurement of TOC1 - GFP protein turn - over in TOC1 , 5X , T135A , S175A native promoter lines by cycloheximide ( CHX ) treatment . 10 - d - old seedlings grown in 12h / 12h light / dark cycles were subject to cycloheximide ( CHX ) or ethanol ( Mock ) treatment for indicated time length . Data from 3 independent trials were fitted to non - linear Weibull regression curves . Data information : TOC1 protein was detected by α - GFP , ADK was used as loading control . TOC1 / ADK ratio was calculated by the intensities of TOC1 - GFP bands normalized by the intensities of ADK bands using ImageJ .

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

B, Measurement of TOC1-GFP protein turn-over in TOC1, 5X, T135A, S175A native promoter lines by cycloheximide (CHX) treatment. 10-d-old seedlings grown in 12h/12h light/dark cycles were subject to cycloheximide (CHX) or ethanol (Mock) treatment for indicated time length. Data from 3 independent trials were fitted to non-linear Weibull regression curves. Data information: TOC1 protein was detected by α-GFP, ADK was used as loading control. TOC1/ADK ratio was calculated by the intensities of TOC1-GFP bands normalized by the intensities of ADK bands using ImageJ.


C , Estimates of TOC1 - GFP protein half - life in B . Error bars indicate SEM , statistical analyses were performed with student ' s t test , different letters indicate significant differences ( p < 0 . 05 ) . Data from 3 independent trials were averaged .

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C, Estimates of TOC1-GFP protein half-life in B. Error bars indicate SEM, statistical analyses were performed with student's t test, different letters indicate significant differences (p<0.05). Data from 3 independent trials were averaged.


B , Quantification of interactions between TOC1 / 5X - TAP and NF - YC proteins in presence or absence of NF - YB subunits . Proteins were transiently expressed in N . benthamiana . TOC1 / 5X were immunoprecipitated with IgG resins followed by HRV - 3C protease digestion . TOC1 / 5X and NF - YB / C were detected by α - MYC and α - HA , respectively . Protein band intensity was measured by ImageJ and interactions were exhibited as ratio of co - immunoprecipitated NF - YC to immunoprecipitated TOC1 / 5X to NF - YC input . Results from 3 independent trials were averaged . Error bars indicate SEM , different letters denote significant differences ( p < 0 . 05 , Student ' s t test ) .

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B, Quantification of interactions between TOC1/5X-TAP and NF-YC proteins in presence or absence of NF-YB subunits. Proteins were transiently expressed in N. benthamiana. TOC1/5X were immunoprecipitated with IgG resins followed by HRV-3C protease digestion. TOC1/5X and NF-YB/C were detected by α-MYC and α-HA, respectively. Protein band intensity was measured by ImageJ and interactions were exhibited as ratio of co-immunoprecipitated NF-YC to immunoprecipitated TOC1/5X to NF-YC input. Results from 3 independent trials were averaged. Error bars indicate SEM, different letters denote significant differences (p<0.05, Student's t test).


C , ChIP - qPCR assay of native promoter driven TOC1 - GFP and 5X - GFP ' s residence at target promoters in toc1 vs . nf - yc3 / 4 / 9 toc1 ( c349t1 ) mutants . 3 - d - old SD - grown seedlings were harvested at ZT14 , TOC1 and 5X were immunoprecipitated by α - GFP and magnetic protein G beads . Data from 3 independent trials were averaged . Error bars indicate SEM , asterisks indicate significant differences ( * p < 0 . 05 , * * * p < 0 . 0001 , Student ' s t test ) , n . s = not significant .

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C, ChIP-qPCR assay of native promoter driven TOC1-GFP and 5X-GFP's residence at target promoters in toc1 vs. nf-yc3/4/9 toc1 (c349t1) mutants. 3-d-old SD-grown seedlings were harvested at ZT14, TOC1 and 5X were immunoprecipitated by α-GFP and magnetic protein G beads. Data from 3 independent trials were averaged. Error bars indicate SEM, asterisks indicate significant differences (* p<0.05, *** p<0.0001, Student's t test), n.s= not significant.


A , Co - IP assay of HDA15 - TAP with TOC1 - GFP and 5X - GFP in N . benthamiana . HDA15 - TAP was immunoprecipitated with IgG resins and digested by HRV - 3C protease . HDA15 and TOC1 were detected by α - MYC and α - GFP , respectively . B , Quantification of the interaction strength in A . Results from 5 independent trials were averaged . Error bar indicates SEM , asterisk indicates significant difference ( * p < 0 . 05 , student ' s t test ) .

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A, Co-IP assay of HDA15-TAP with TOC1-GFP and 5X-GFP in N. benthamiana. HDA15-TAP was immunoprecipitated with IgG resins and digested by HRV-3C protease. HDA15 and TOC1 were detected by α-MYC and α-GFP, respectively. B, Quantification of the interaction strength in A. Results from 5 independent trials were averaged. Error bar indicates SEM, asterisk indicates significant difference (*p<0.05, student's t test).


C and D , Hypocotyl lengths and free - running periods of indicated plants lines . Data of ~ 75 plants from 3 independent experiments were averaged , different letters indicate statistically significant differences ( p < 0 . 01 , one - way ANOVA followed by Tukey - Kramer HSD test ) . The middle line of the box represents the median , the x in the box represents the mean . The bottom line and the top line of the box represent 1st and 3rd quartile , respectively . The whiskers extend from the ends of the box to the minimum value and maximum value .

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C and D, Hypocotyl lengths and free-running periods of indicated plants lines. Data of ~75 plants from 3 independent experiments were averaged, different letters indicate statistically significant differences (p<0.01, one-way ANOVA followed by Tukey-Kramer HSD test). The middle line of the box represents the median, the x in the box represents the mean. The bottom line and the top line of the box represent 1st and 3rd quartile, respectively. The whiskers extend from the ends of the box to the minimum value and maximum value.


E , Expression of PIL1 , AT5G02580 and CDF5 in 3 - d - old SD - grown seedlings at ZT18 . Data from 3 independent experiments were averaged . Error bars indicate SEM . Different letters denote statistically significant differences based on Wilcoxon test ( p < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Expression of PIL1, AT5G02580 and CDF5 in 3-d-old SD-grown seedlings at ZT18. Data from 3 independent experiments were averaged. Error bars indicate SEM. Different letters denote statistically significant differences based on Wilcoxon test (p<0.05).


A and B , ChIP - qPCR assay of HDA15 ' s ( A ) and NF - YC9 ' s ( B ) residence at target promoters in presence or absence of TOC1 and PRR5 . GFP - HDA15 and GFP - NF - YC9 driven by 35S promoter were transiently expressed in protoplasts isolated from Col - 0 , toc1 single and prr5 toc1 double mutant grown in SD condition . Results were normalized by % input of PIL1 coding region ( PIL1 CDS ) . Data from at least 3 independent experiments were averaged , error bars indicate SEM . Asterisks indicate significant differences ( * p < 0 . 05 , * * p < 0 . 001 , * * * p < 0 . 0001 , Student ' s t test ) .

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A and B, ChIP-qPCR assay of HDA15's (A) and NF-YC9's (B) residence at target promoters in presence or absence of TOC1 and PRR5. GFP-HDA15 and GFP-NF-YC9 driven by 35S promoter were transiently expressed in protoplasts isolated from Col-0, toc1 single and prr5 toc1 double mutant grown in SD condition. Results were normalized by % input of PIL1 coding region (PIL1 CDS). Data from at least 3 independent experiments were averaged, error bars indicate SEM. Asterisks indicate significant differences (* p<0.05, ** p<0.001, *** p<0.0001, Student's t test).


C and D , Dual luciferase assay of the repression activity of GFP - TOC1 and GFP - 5X on promoter of AT5G02580 ( C ) and CDF5 ( D ) in protoplasts of toc1 and hda15toc1 ( h15t1 ) mutants . AT5G02580 : LUC , CDF5 : LUC reporters and the effectors ( empty GFP , GFP - TOC1 , and GFP - 5X ) were co - transfected to toc1 and hda15 toc1 protoplasts with 35S : Renilla - LUC as internal control . Data are means ± SEM ( n = 5 biological trials ) relative to empty GFP which is set as 1 . Different letters indicate statistically significant differences ( student ' s t test , p < 0 . 05 ) .

C and D, Dual luciferase assay of the repression activity of GFP-TOC1 and GFP-5X on promoter of AT5G02580 (C) and CDF5 (D) in protoplasts of toc1 and hda15toc1 (h15t1) mutants. AT5G02580:LUC, CDF5:LUC reporters and the effectors (empty GFP, GFP-TOC1, and GFP-5X) were co-transfected to toc1 and hda15 toc1 protoplasts with 35S:Renilla-LUC as internal control. Data are means ± SEM (n = 5 biological trials) relative to empty GFP which is set as 1. Different letters indicate statistically significant differences (student's t test, p<0.05).


A , Quantification of interactions between TAP - PRR5 and NF - YC proteins in presence or absence of NF - YB subunits . Proteins were transiently expressed in N . benthamiana . TAP - PRR5 was immunoprecipitated with IgG resins followed by HRV - 3C protease digestion . PRR5 and NF - YB / C were detected by α - MYC and α - HA , respectively . Protein band intensity was measured by ImageJ and interactions were exhibited as ratio of co - immunoprecipitated NF - YC to immunoprecipitated PRR5 to NF - YC input . Results from 3 independent trials were averaged . Error bars indicate SEM , different letters denote significant differences ( p < 0 . 05 , student ' s t test ) .

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A, Quantification of interactions between TAP-PRR5 and NF-YC proteins in presence or absence of NF-YB subunits. Proteins were transiently expressed in N. benthamiana. TAP-PRR5 was immunoprecipitated with IgG resins followed by HRV-3C protease digestion. PRR5 and NF-YB/C were detected by α-MYC and α-HA, respectively. Protein band intensity was measured by ImageJ and interactions were exhibited as ratio of co-immunoprecipitated NF-YC to immunoprecipitated PRR5 to NF-YC input. Results from 3 independent trials were averaged. Error bars indicate SEM, different letters denote significant differences (p<0.05, student's t test).


C , ChIP - qPCR assay of native promoter TOC1 - GFP and 5X - GFP binding to the target promoters at ZT14 in toc1 vs . prr5 toc1 ( p5t1 ) mutant . 3 - d - old SD - grown seedlings were harvested at ZT14 , TOC1 and 5X were immunoprecipitated by α - GFP . Data from 3 independent trials were averaged , error bars indicate SEM . Asterisks indicate significant differences ( Student ' s t test ; * p < 0 . 05 ) , n . s = not significant .

C, ChIP-qPCR assay of native promoter TOC1-GFP and 5X-GFP binding to the target promoters at ZT14 in toc1 vs. prr5 toc1 (p5t1) mutant. 3-d-old SD-grown seedlings were harvested at ZT14, TOC1 and 5X were immunoprecipitated by α-GFP. Data from 3 independent trials were averaged, error bars indicate SEM. Asterisks indicate significant differences (Student's t test; * p<0.05), n.s= not significant.


D , Expression of PIL1 , AT5G02580 and CDF5 in 3 - d - old SD - grown seedlings at ZT18 . Data from 3 biological trials were averaged . Error bars indicate SEM . Different letters denote statistically significant differences based on Wilcoxon test ( p < 0 . 05 ) .

D, Expression of PIL1, AT5G02580 and CDF5 in 3-d-old SD-grown seedlings at ZT18. Data from 3 biological trials were averaged. Error bars indicate SEM. Different letters denote statistically significant differences based on Wilcoxon test (p<0.05).


A . Rabies virus infection kinetics in the central nervous system of infected mice . Tha - RABV load ( copies / µg of RNA ) detected in the thoracolumbar spinal cord , in the brainstem plus cerebellum and in the cerebral cortex of infected mice according to different time - points post - infection ( n = 4 per time - point ) . nd : not detected . Horizontal lines indicate the median .

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A. Rabies virus infection kinetics in the central nervous system of infected mice. Tha-RABV load (copies/µg of RNA) detected in the thoracolumbar spinal cord, in the brainstem plus cerebellum and in the cerebral cortex of infected mice according to different time-points post-infection (n=4 per time-point). nd: not detected. Horizontal lines indicate the median.


B . Follow - up of the clinical signs of mice under different treatments . Heatmaps were established based on a progressive 0 - 7 clinical score scale ( 0 : no apparent changes | 1 : ruffled fur | 2 : slow movement , hind limb ataxia | 3 : apathy | 4 : monoplegia | 5 : hind limb paralysis , tremors | 6 : paralysis , conjunctivitis / keratitis , urine staining of the haircoat of the perineum | 7 : death ) . Each line represents one animal throughout time .

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B. Follow-up of the clinical signs of mice under different treatments. Heatmaps were established based on a progressive 0-7 clinical score scale (0: no apparent changes | 1: ruffled fur | 2: slow movement, hind limb ataxia | 3: apathy | 4: monoplegia | 5: hind limb paralysis, tremors | 6: paralysis, conjunctivitis/ keratitis, urine staining of the haircoat of the perineum | 7: death). Each line represents one animal throughout time.


C . Body weight progression of mice under different treatments . Mice were weighted on a daily basis during the treatment administration , and then twice a week up to 100 days post - infection . All the mice were equipped with iPrecio pumps weighting ca . 3 . 3 g . Each line represents one animal throughout time .

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C. Body weight progression of mice under different treatments. Mice were weighted on a daily basis during the treatment administration, and then twice a week up to 100 days post-infection. All the mice were equipped with iPrecio pumps weighting ca. 3.3 g. Each line represents one animal throughout time.


D - E . Mouse behavioral testing . The tested animals were non - infected ( n = 3 mice with iPrecio pump + n = 4 age - related mice without iPrecio pump ) , infected non - treated ( n = 5 ) , infected and treated at 7 dpi ( n = 5 , mice # 9 to # 13 ) , and infected and treated at 8 dpi ( n = 5 , mice # 24 to # 28 ) . ( D ) Open field test performed at 7 dpi ( prodromal phase ) and after the remission of clinical signs for the survivors ( 80 dpi ) . ( E ) Rotarod motor performance at 7 dpi ( prodromal phase ) , 8 dpi ( symptomatic phase ) and after the remission of clinical signs for the survivors ( 20 , 30 and 60 dpi ) . Horizontal lines indicate the median . Kruskal - Wallis followed by the Dunn ' multiple comparisons test ( 7 and 8 days post - infection ) and Mann - Whitney test ( 20 , 30 and 60 days post - infection ) . The rotarod performance presented a negative correlation ( Spearman r = - 0 . 768 ) with the clinical score at 8 dpi . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Treatments description = NI : non - infected ( n = 5 ) ; NT : infected , non - treated ( n = 5 ) ; infected , treated at 6 dpi ( 2 + 2 mg / kg ) ( n = 4 ) ; infected , treated at 7 dpi ( 2 + 2 mg / kg ) ( n = 9 ) ; infected , treated at 8 dpi ( 2 + 2 mg / kg ) ( n = 15 ) ; infected , treated at 8 dpi ( 10 + 10 mg / kg ) ( n = 4 ) .

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D-E. Mouse behavioral testing. The tested animals were non-infected (n=3 mice with iPrecio pump + n=4 age-related mice without iPrecio pump), infected non-treated (n=5), infected and treated at 7 dpi (n=5, mice #9 to #13), and infected and treated at 8 dpi (n=5, mice #24 to #28). (D) Open field test performed at 7 dpi (prodromal phase) and after the remission of clinical signs for the survivors (80 dpi). (E) Rotarod motor performance at 7 dpi (prodromal phase), 8 dpi (symptomatic phase) and after the remission of clinical signs for the survivors (20, 30 and 60 dpi). Horizontal lines indicate the median. Kruskal-Wallis followed by the Dunn' multiple comparisons test (7 and 8 days post-infection) and Mann-Whitney test (20, 30 and 60 days post-infection). The rotarod performance presented a negative correlation (Spearman r = -0.768) with the clinical score at 8 dpi. *P<0.05, **P<0.01, ***P<0.001. Treatments description= NI: non-infected (n=5); NT: infected, non-treated (n=5); infected, treated at 6 dpi (2+2 mg/kg) (n=4); infected, treated at 7 dpi (2+2 mg/kg) (n=9); infected, treated at 8 dpi (2+2 mg/kg) (n=15); infected, treated at 8 dpi (10+10 mg/kg) (n=4).


A . Cumulative Kaplan - Meier survival curves of mice treated at different time - points post - infection . Log - rank ( Mantel - Cox ) test to compare treated groups with the infected , non - treated group . * * P < 0 . 01 , * * * P < 0 . 001 . Treatments description = infected , non - treated ( n = 5 ) ; infected , treated at 6 dpi ( 2 + 2 mg / kg ) ( n = 4 ) ; infected , treated at 7 dpi ( 2 + 2 mg / kg ) ( n = 9 ) ; infected , treated at 8 dpi ( 2 + 2 mg / kg ) ( n = 15 ) ; infected , treated at 8 dpi ( 10 + 10 mg / kg ) ( n = 4 ) .

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A. Cumulative Kaplan-Meier survival curves of mice treated at different time-points post-infection. Log-rank (Mantel-Cox) test to compare treated groups with the infected, non-treated group. **P<0.01, ***P<0.001. Treatments description= infected, non-treated (n=5); infected, treated at 6 dpi (2+2 mg/kg) (n=4); infected, treated at 7 dpi (2+2 mg/kg) (n=9); infected, treated at 8 dpi (2+2 mg/kg) (n=15); infected, treated at 8 dpi (10+10 mg/kg) (n=4).


B . Cumulative Kaplan - Meier survival curves of all the treated animals that presented with clinical signs ( n = 31 , all treatments combined ) . Log - rank ( Mantel - Cox ) test to compare treated groups with the infected , non - treated group . * * * * P < 0 . 0001 .

B. Cumulative Kaplan-Meier survival curves of all the treated animals that presented with clinical signs (n=31, all treatments combined). Log-rank (Mantel-Cox) test to compare treated groups with the infected, non-treated group. ****P<0.0001.


C . Virus neutralizing antibodies detected in the serum of surviving mice from different experimental groups .

C. Virus neutralizing antibodies detected in the serum of surviving mice from different experimental groups.


D . Residual human monoclonal antibodies in the serum of representative surviving mice at 100 dpi ( LOD : limit of detection = 0 . 02 µg / mL ) .

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D. Residual human monoclonal antibodies in the serum of representative surviving mice at 100 dpi (LOD: limit of detection = 0.02 µg/mL).


E . Viral load in the brain of mice from different experimental groups . The samples were either collected at the time of death , or at 100 dpi for the survivors .

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E. Viral load in the brain of mice from different experimental groups. The samples were either collected at the time of death, or at 100 dpi for the survivors.


Cytokines and innate immune mediators ' profile in the brain of mice from different experimental groups : ( F ) IFN - β1 , ( G ) ISG15 , ( H ) Mx1 , detected in one brain hemisphere of mice from different experimental groups . The expression of the genes of interest was normalized to the GAPDH housekeeping gene .

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Cytokines and innate immune mediators' profile in the brain of mice from different experimental groups: (F) IFN-β1, (G) ISG15, (H) Mx1, detected in one brain hemisphere of mice from different experimental groups. The expression of the genes of interest was normalized to the GAPDH housekeeping gene.


Cytokines and innate immune mediators ' profile in the brain of mice from different experimental groups : , ( I ) IFN - γ , ( J ) CXCL10 , ( K ) CCL5 , ( L ) IL - 6 , ( M ) TNF - α and ( N ) IL - 1β , detected in one brain hemisphere of mice from different experimental groups . The expression of the genes of interest was normalized to the GAPDH housekeeping gene .

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Cytokines and innate immune mediators' profile in the brain of mice from different experimental groups: , (I) IFN-γ, (J) CXCL10, (K) CCL5, (L) IL-6, (M) TNF-α and (N) IL-1β, detected in one brain hemisphere of mice from different experimental groups. The expression of the genes of interest was normalized to the GAPDH housekeeping gene.


A - R . Histopathological analysis of the brain in Tha - RABV - infected non - treated mice ( at 11 dpi ) , versus surviving mice ( at 100 dpi ) treated with intramuscular and intracerebroventricular administration of the RVC20 and RVC58 monoclonal antibodies cocktail . The detection of Tha - RABV , using immunohistochemistry ( IHC ) on brain sections , revealed a heterogeneous invasion between the non - treated mice , ranging from extensive ( A ) to multifocal ( B ) . Higher magnification of extensive invasion in the cortex ( F ) , hippocampus ( G ) , brain stem ( H ) and cerebellum ( I ) . ( C , J , K , L , M ) In contrast , even if a non - specific IHC signal was sometimes observed , no virus was detected on the brain sections of treated mice at 100 days post - infection . Only rare signs of neuro - inflammation were observed , as perivascular cuffing ( D ) ( 2 / 7 mice ) and minimal meningitis ( E ) ( 3 / 7 mice ) . Data information : A - C and F - M : Tha - RABV IHC D - E : HE staining . F - I are high magnifications of A ; J - M are high magnifications of

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A-R. Histopathological analysis of the brain in Tha-RABV-infected non-treated mice (at 11 dpi), versus surviving mice (at 100 dpi) treated with intramuscular and intracerebroventricular administration of the RVC20 and RVC58 monoclonal antibodies cocktail. The detection of Tha-RABV, using immunohistochemistry (IHC) on brain sections, revealed a heterogeneous invasion between the non-treated mice, ranging from extensive (A) to multifocal (B). Higher magnification of extensive invasion in the cortex (F), hippocampus (G), brain stem (H) and cerebellum (I). (C,J,K,L,M) In contrast, even if a non-specific IHC signal was sometimes observed, no virus was detected on the brain sections of treated mice at 100 days post-infection. Only rare signs of neuro-inflammation were observed, as perivascular cuffing (D) (2/7 mice) and minimal meningitis (E) (3/7 mice). Data information: A-C and F-M: Tha-RABV IHC D-E: HE staining. F-I are high magnifications of A; J-M are high magnifications of C


( N , O ) Microglial cells were in general not reactive , but ( P ) small foci of reactive microglia ( hyperplasia with thicker cell processes ) could randomly be detected . As for control non - treated mice , reactive microglial cells ( Q ) ( 2 / 7 mice ) or resting microglia ( R ) were observed in treated mice . Representative images . Data information : N - R : Iba1 IHC to detect microglial cells O - P are high magnifications of N .

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(N,O) Microglial cells were in general not reactive, but (P) small foci of reactive microglia (hyperplasia with thicker cell processes) could randomly be detected. As for control non-treated mice, reactive microglial cells (Q) (2/7 mice) or resting microglia (R) were observed in treated mice. Representative images. Data information: N-R: Iba1 IHC to detect microglial cells O-P are high magnifications of N.


A Western blot analysis of LC3 in SH - SY5Y cells after IU1 treatment ( 100 μM , 24h ) . Graphs represent mean ± SEM . N = 8

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A Western blot analysis of LC3 in SH-SY5Y cells after IU1 treatment (100 μM, 24h). Graphs represent mean± SEM. N= 8


B - D Electron microscope images were taken to quantify the number of autophagosomes and autolysosome present in control ( DMSO ) or IU1 treated cells . At least 55 cells were analysed from 3 different experiments . Graphs represent mean ± SEM

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B-D Electron microscope images were taken to quantify the number of autophagosomes and autolysosome present in control (DMSO) or IU1 treated cells. At least 55 cells were analysed from 3 different experiments. Graphs represent mean± SEM


E Western blot analysis of mitochondrial marker proteins ATP5a , HSP60 and normalised by actin after 24h of DMSO ( C ) or IU1 ( 100 μM ) treatment . N = 3 . Graphs represent mean ± SEM

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E Western blot analysis of mitochondrial marker proteins ATP5a, HSP60 and normalised by actin after 24h of DMSO (C) or IU1 (100 μM) treatment. N=3. Graphs represent mean± SEM


F - I Confocal images of cells transfected with mito - YFP and treated with DMSO ( F , control ) or IU1 ( G ) for 24h . Fragmentation index ( H ) and total mitochondrial volume / cell were measured and represented in ( I ) , mean ± SEM . At least 35 cells were analysed from 3 different experimental conditions . Scale bar 10 μm

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F-I Confocal images of cells transfected with mito-YFP and treated with DMSO (F, control) or IU1 (G) for 24h. Fragmentation index (H) and total mitochondrial volume / cell were measured and represented in (I), mean ±SEM. At least 35 cells were analysed from 3 different experimental conditions. Scale bar 10 μm


J - L Mito - YFP transfected cells were treated with IU1 ( 100 μM , 24h ) , fixed and immuno labelled for LC3 . Number of LC3 dots / cell ( K ) and LC3 dot overlapping mitochondria ( L ) were counted . At least 35 cells from 3 independent experiments were analysed . Scale bar : 10 μm

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J-L Mito-YFP transfected cells were treated with IU1 (100 μM, 24h), fixed and immuno labelled for LC3. Number of LC3 dots / cell (K) and LC3 dot overlapping mitochondria (L) were counted. At least 35 cells from 3 independent experiments were analysed. Scale bar: 10 μm


M - N Cells were transfected with mito - keima and treated with DMSO or IU1 for 24h and subjected to FACS analysis , counting 10 , 000 cells per experiment . ( M ) represents the shift of the signal intensity in IU1 treated cells when excited at 560 nm ( false colour ) . ( N ) represents the average intensity of the mito - keima protein when excited at 560 nm and the ratio between 560 : 405 nm . Graphs represent mean ± SEM from 3 different sets of

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M-N Cells were transfected with mito-keima and treated with DMSO or IU1 for 24h and subjected to FACS analysis, counting 10,000 cells per experiment. (M) represents the shift of the signal intensity in IU1 treated cells when excited at 560 nm (false colour). (N) represents the average intensity of the mito-keima protein when excited at 560 nm and the ratio between 560: 405 nm. Graphs represent mean ± SEM from 3 different sets of experiments


A - B Western blot analysis of the indicated proteins in cell lysates from SH - SY5Y cells , treated with different concentrations of IU1 ( 1 - 100 μM ) for 24h . Bar graphs represents mean ± SEM . Blots are representative of three independent experiments . ANOVA followed by Dunnets test

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A-B Western blot analysis of the indicated proteins in cell lysates from SH-SY5Y cells, treated with different concentrations of IU1 (1-100 μM) for 24h. Bar graphs represents mean ± SEM. Blots are representative of three independent experiments. ANOVA followed by Dunnets test


C - D Representative images of MEF cells knocked out for the indicated mitochondrial dynamics protein and transfected with mito - YFP ( C ) with / without IU1 treatment ( 100 μM , 48h ) . Scale bar 10 μm . ( D ) Mitochondrial volume / cell was measured as described and represented as mean ± SEM . At least 35 cells were

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C-D Representative images of MEF cells knocked out for the indicated mitochondrial dynamics protein and transfected with mito-YFP (C) with / without IU1 treatment (100 μM, 48h). Scale bar 10 μm. (D) Mitochondrial volume / cell was measured as described and represented as mean ±SEM. At least 35 cells were evaluated


E - F Western blot analysis of HSP60 and ATP5a , as mitochondrial marker in the indicated knock out MEF cells ( with / without the indicated DRP1 variant ) after 48h of IU1 treatment . Bar graphs represent mean ± SEM , n = at least 3 . Student ' s t test ;

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E-F Western blot analysis of HSP60 and ATP5a, as mitochondrial marker in the indicated knock out MEF cells (with / without the indicated DRP1 variant) after 48h of IU1 treatment. Bar graphs represent mean ± SEM, n= at least 3. Student's t test;


A - D Wild type MEF ( A ) , Hela ( B ) and PINK1 KO MEF ( C ) cells were treated with IU1 ( 48h , 100 μM ) and electron microscope images were evaluated for autophagic vesicle formation . ( D ) bar graphs represents mean ± SEM number of autophagosomes and autolysosome / cell from 3 different set of experiments ; at least 40 cells were analysed from each

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A-D Wild type MEF (A), Hela (B) and PINK1 KO MEF (C) cells were treated with IU1 (48h, 100 μM) and electron microscope images were evaluated for autophagic vesicle formation. (D) bar graphs represents mean± SEM number of autophagosomes and autolysosome / cell from 3 different set of experiments; at least 40 cells were analysed from each group


E Western blot analysis of HSP60 protein with or without IU1 treatment in the indicated cell type . Bar graphs represent mean ± SEM . N = at least 3 independent experiments . Student ' s t test . * P ≤ 0 . 05 , * * P ≤ 0 . 01

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E Western blot analysis of HSP60 protein with or without IU1 treatment in the indicated cell type. Bar graphs represent mean ± SEM. N= at least 3 independent experiments. Student's t test. *P ≤ 0.05, **P ≤ 0.01


A - B SH - SY5Y cells were immunostained for ATP5a ( green ) and 20S proteasome core ( red ) after IU1 treatment ( 6h - 24h ) . Scale bar 10 μm . Colour mapping of the co - localisation of HSP60 and 20S proteasome from the selected regions were generated by image j and represented in the bottom panel . Intensity of the co - localisation is represented in the colour map scale at the bottom of the panel . ( B ) 20S proteasome subunit and mitochondria co - localisation was quantified by measuring Mander ' s coefficient . Bar graphs represent mean fold change ( mean ± SEM ) from 3 different experiments . At least 30 cells were evaluated for the calculations . ANOVA followed by Dunnet ' s test

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A-B SH-SY5Y cells were immunostained for ATP5a (green) and 20S proteasome core (red) after IU1 treatment (6h-24h). Scale bar 10 μm. Colour mapping of the co-localisation of HSP60 and 20S proteasome from the selected regions were generated by image j and represented in the bottom panel. Intensity of the co-localisation is represented in the colour map scale at the bottom of the panel. (B) 20S proteasome subunit and mitochondria co-localisation was quantified by measuring Mander's coefficient. Bar graphs represent mean fold change (mean ± SEM) from 3 different experiments. At least 30 cells were evaluated for the calculations. ANOVA followed by Dunnet's test


C Electron microscopy images showing subcellular localization of immuno - gold labelled 20S subunit of proteasome complex after 12h or 24h of IU1 treatment . Experiments were repeated twice in two biological replicates with similar results . The contrast in the magnified region is enhanced from the original image to highlight the immunogold labelled signals

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C Electron microscopy images showing subcellular localization of immuno- gold labelled 20S subunit of proteasome complex after 12h or 24h of IU1 treatment. Experiments were repeated twice in two biological replicates with similar results. The contrast in the magnified region is enhanced from the original image to highlight the immunogold labelled signals


D - E Protein proximity ligation assay for Prohibitin2 ( PHB2 ) and LC3 after 24h IU1 treatment . Images showing signal are merged with bright field images to count the signal dots ( cyan false colour ) as depicted in the figure . In the inset only fluorescence signal is shown , without merging with the bright field from the selected regions . Scale bar 10 μm . ( E ) Bar graphs represent mean ± SEM of the number of signal dots per cell from 3 different experiments . Student ' s t test ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001

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D-E Protein proximity ligation assay for Prohibitin2 (PHB2) and LC3 after 24h IU1 treatment. Images showing signal are merged with bright field images to count the signal dots (cyan false colour) as depicted in the figure. In the inset only fluorescence signal is shown, without merging with the bright field from the selected regions. Scale bar 10 μm. (E) Bar graphs represent mean ±SEM of the number of signal dots per cell from 3 different experiments. Student's t test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001


Electron microscope images of mitochondria from SH - SY5Y e , as indicated in the figure , with / without USP14 inhibition / knock down . Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point . Bar graphs represent mean ( ± SEM ) of percentage ruptured mitochondria from at least 3 different experiments . For cells , total 150 - 300 mitochondria for the control group and 150 - 190 mitochondria from the IU1 treated groups were

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Electron microscope images of mitochondria from SH-SY5Y e, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were counted


Representative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60 , ATP5a and TOM20 . Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins ( with : without trypsin ) from at least 3 independent

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Representative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60, ATP5a and TOM20. Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins (with : without trypsin) from at least 3 independent experiments


Electron microscope images of mitochondria fro MEF WT , MEF PINK1 K as indicated in the figure , with / without USP14 inhibition / knock down . Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point . Bar graphs represent mean ( ± SEM ) of percentage ruptured mitochondria from at least 3 different experiments . For cells , total 150 - 300 mitochondria for the control group and 150 - 190 mitochondria from the IU1 treated groups were

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Electron microscope images of mitochondria fro MEF WT, MEF PINK1 K as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were counted


Electron microscope images of mitochondria fro Hela an e , as indicated in the figure , with / without USP14 inhibition / knock down . Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point . Bar graphs represent mean ( ± SEM ) of percentage ruptured mitochondria from at least 3 different experiments . For cells , total 150 - 300 mitochondria for the control group and 150 - 190 mitochondria from the IU1 treated groups were

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Electron microscope images of mitochondria fro Hela an e, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were counted


Electron microscope images of mitochondria fro Drosophila thoracic muscle , as indicated in the figure , with / without USP14 inhibition / knock down . Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point . Bar graphs represent mean ( ± SEM ) of percentage ruptured mitochondria from at least 3 different experiments For flies , more than 200 mitochondria from each group were counted from 5 different

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Electron microscope images of mitochondria fro Drosophila thoracic muscle, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments For flies, more than 200 mitochondria from each group were counted from 5 different experiments


A Life span analysis of flies of the indicated genetic background . At least 50 flies were monitored from each group . Log - rank test ( Mantel - Cox test , Mantel - Haenszel test , p < 0 . 0001 )

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A Life span analysis of flies of the indicated genetic background. At least 50 flies were monitored from each group. Log-rank test (Mantel-Cox test, Mantel-Haenszel test, p < 0.0001)


B Graph bar shows mean ± SEM of the climbing performance of flies of the indicated genotypes . n = 4 - 7 . ANOVA followed by Newman Keuls test

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B Graph bar shows mean± SEM of the climbing performance of flies of the indicated genotypes. n=4-7. ANOVA followed by Newman Keuls test


C Quantitative analysis of respiratory fitness of isolated mitochondria extracted from flies of the indicated genotypes . Graph shows respiratory control rate ( RCR ) calculated as described in materials and methods . Graphs represent mean ± SEM . N = 4 - 5 , ANOVA followed by Newman Keuls test

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C Quantitative analysis of respiratory fitness of isolated mitochondria extracted from flies of the indicated genotypes. Graph shows respiratory control rate (RCR) calculated as described in materials and methods. Graphs represent mean± SEM. N=4-5, ANOVA followed by Newman Keuls test


D Dopamine content in the fly heads ( 15 day old ) was measured by HPLC and the individual fold change values are represented in the scatter graph . Graphs represent mean ± SEM . N = 14 , 11 and 11 fly heads for control , PINK1 and PINK1 + USP14 KD fly respectively . ANOVA followed by Newman Keuls test

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D Dopamine content in the fly heads (15 day old) was measured by HPLC and the individual fold change values are represented in the scatter graph. Graphs represent mean± SEM. N= 14, 11 and 11 fly heads for control, PINK1 and PINK1+USP14 KD fly respectively. ANOVA followed by Newman Keuls test


E Enlarged TEM representative images of flight muscle mitochondria of the indicated genotypes . The experiment was repeated 3 times in replicates . Bar graphs represent quantification of electron dense mitochondria ( represented as grey bar ) and depolarised mitochondrial with ruptured cristae ( represented as white bars ) from the indicated genotypes

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E Enlarged TEM representative images of flight muscle mitochondria of the indicated genotypes. The experiment was repeated 3 times in replicates. Bar graphs represent quantification of electron dense mitochondria (represented as grey bar) and depolarised mitochondrial with ruptured cristae (represented as white bars) from the indicated genotypes


( A ) Life span analysis of flies of the indicated genetic background . At least 50 flies were analysed for each group . Log - rank test ( Mantel - Cox test , Mantel - Haenszel test , p < 0 . 0001 )

O O O O O O O O O O O O O O O O O O O O O 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) Life span analysis of flies of the indicated genetic background. At least 50 flies were analysed for each group. Log-rank test (Mantel-Cox test, Mantel-Haenszel test, p < 0.0001)


( B ) Graph bar shows mean ± SEM of the climbing performance of flies of the indicated genotypes . One way ANOVA , Newman Keuls test , n = 3

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(B) Graph bar shows mean± SEM of the climbing performance of flies of the indicated genotypes. One way ANOVA, Newman Keuls test, n=3


( C ) Quantitative analysis of respiratory fitness of isolated mitochondria extracted from flies of the indicated genotypes . Graph shows ( mean ± SEM , n = 3 independent experiments ) respiratory control rate ( RCR ) calculated as described . One way ANOVA , Newman Keuls test

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(C) Quantitative analysis of respiratory fitness of isolated mitochondria extracted from flies of the indicated genotypes. Graph shows (mean± SEM, n=3 independent experiments) respiratory control rate (RCR) calculated as described. One way ANOVA, Newman Keuls test


( D ) Enlarged TEM representative images of flight muscle mitochondria of the indicated genotypes . The experiment was repeated three times in replicates . Bar graphs represent quantification of electron dense mitochondria ( represented as grey bar ) and mitochondrial with ruptured cristae ( represented as white bars ) from the indicated genotypes

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Enlarged TEM representative images of flight muscle mitochondria of the indicated genotypes. The experiment was repeated three times in replicates. Bar graphs represent quantification of electron dense mitochondria (represented as grey bar) and mitochondrial with ruptured cristae (represented as white bars) from the indicated genotypes


( a ) Identification of Beclin1 as a substrate of ROCK1 via affinity purification . Left panel : HeLa cells growing in control ( C : nutrient - rich media ) or starved in HBSS ( H ) for 4 h were lysed and subjected to IP with ROCK1 cross - linked beads . Proteins bound were eluted and detected by SDS - PAGE , followed by silver staining that presents precipitated proteins identified by LC - MS / MS . Right panel : HeLa cells grown in control media or HBSS ( 4 h ) were collected , whole cell lysates prepared and immunoprecipitated with ROCK1 cross - linked beads . The resulting immune complexes were divided into two , and were subjected to SDS - PAGE and analysed for Beclin1 ( MES buffer ) and ROCK1 ( MOPS buffer ) . Inputs and IP were analysed by western blotting . HBSS - treated cell extracts were used for IgG IP controls . Whole cell lysates ( 10 % ) were run for inputs . Total cell lysates were rerun and analysed for LC3 and β - actin .

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(a) Identification of Beclin1 as a substrate of ROCK1 via affinity purification. Left panel: HeLa cells growing in control (C: nutrient-rich media) or starved in HBSS (H) for 4 h were lysed and subjected to IP with ROCK1 cross-linked beads. Proteins bound were eluted and detected by SDS-PAGE, followed by silver staining that presents precipitated proteins identified by LC-MS/MS. Right panel: HeLa cells grown in control media or HBSS (4 h) were collected, whole cell lysates prepared and immunoprecipitated with ROCK1 cross-linked beads. The resulting immune complexes were divided into two, and were subjected to SDS-PAGE and analysed for Beclin1 (MES buffer) and ROCK1 (MOPS buffer). Inputs and IP were analysed by western blotting. HBSS-treated cell extracts were used for IgG IP controls. Whole cell lysates (10%) were run for inputs. Total cell lysates were rerun and analysed for LC3 and β-actin.


( b ) Co - IP was performed with 293T whole cell extracts by using ROCK1 - crosslinked agarose , and the resulting IP was blotted with indicated antibodies . 293T cells transfected with Flag - Beclin1 for 36 h , cultured in control media or starved ( 4 h ) , were immunoprecipitated with anti - Flag beads . Inputs and immune complexes were resolved and detected . Total cell lysates were rerun and examined for LC3 and β - actin expression .

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(b) Co-IP was performed with 293T whole cell extracts by using ROCK1-crosslinked agarose, and the resulting IP was blotted with indicated antibodies. 293T cells transfected with Flag-Beclin1 for 36 h, cultured in control media or starved (4 h), were immunoprecipitated with anti-Flag beads. Inputs and immune complexes were resolved and detected. Total cell lysates were rerun and examined for LC3 and β-actin expression.


( c ) Interaction of YFP - ROCK1 and CFP - Beclin1 was examined by using live cell spinning disk microscopy and FRET in live cells . Graph represents FRETAverage ratio changes ( YFP - ROCK1 / CFP - Beclin1 ) upon starvation . Data represents mean ± s . e . m . ; n = 6 . Three different experiments were performed . For the control , the right top graph represents CFP intensity over time ( corrected for photobleach ) , and the right bottom right represents YFP intensity ( corrected for photobleach ) . In both control FRET experiments , no significant change was observed in fluorescence , in the presence or absence of HBSS medium . P values were calculated using Student ' s t - test .

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(c) Interaction of YFP-ROCK1 and CFP-Beclin1 was examined by using live cell spinning disk microscopy and FRET in live cells. Graph represents FRETAverage ratio changes (YFP-ROCK1/CFP-Beclin1) upon starvation. Data represents mean±s.e.m.; n=6. Three different experiments were performed. For the control, the right top graph represents CFP intensity over time (corrected for photobleach), and the right bottom right represents YFP intensity (corrected for photobleach). In both control FRET experiments, no significant change was observed in fluorescence, in the presence or absence of HBSS medium. P values were calculated using Student's t-test.


( a ) Inhibition of ROCK1 activity ablates the interaction between Beclin1 and ROCK1 . HeLa cells were untreated ( H2O ) or treated with 10 μM ROCK1 inhibitor , Y27632 , for 8 h and then cultured in control media ( + ) or HBSS ( − ) for 4 h . Cells were collected , lysed and immunoprecipitated with Beclin1 ( left panel ) or ROCK1 ( right panel ) antibody . Left panel : immune complexes were divided into two ( ROCK1 - MOPS buffer and Beclin1 - MES buffer ) and probed for ROCK1 and Beclin1 . Relative values of ROCK1 versus Beclin1 are shown .

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(a) Inhibition of ROCK1 activity ablates the interaction between Beclin1 and ROCK1. HeLa cells were untreated (H2O) or treated with 10 μM ROCK1 inhibitor, Y27632, for 8 h and then cultured in control media (+) or HBSS (−) for 4 h. Cells were collected, lysed and immunoprecipitated with Beclin1 (left panel) or ROCK1 (right panel) antibody. Left panel: immune complexes were divided into two (ROCK1-MOPS buffer and Beclin1-MES buffer) and probed for ROCK1 and Beclin1. Relative values of ROCK1 versus Beclin1 are shown.


( b ) An increase in ROCK1 - mediated phosphorylation of MYPT1 upon starvation . HeLa and EJ cells were starved in HBSS for indicated time points and endogenous ROCK1 was immunoprecipitated . An in vitro kinase assay was performed using MYPT1 as substrate and immune - complexed ROCK1 . Proteins were resolved and visualized with Coomassie staining ( top ) , western blot analysis ( middle ) and autoradiography ( bottom ) . Inputs were examined for ROCK1 and β - actin .

(b) An increase in ROCK1-mediated phosphorylation of MYPT1 upon starvation. HeLa and EJ cells were starved in HBSS for indicated time points and endogenous ROCK1 was immunoprecipitated. An in vitro kinase assay was performed using MYPT1 as substrate and immune-complexed ROCK1. Proteins were resolved and visualized with Coomassie staining (top), western blot analysis (middle) and autoradiography (bottom). Inputs were examined for ROCK1 and β-actin.


( c ) HeLa cells incubated in HBSS were collected and endogenous phosphorylation of MYPT1 was determined by western blotting . The same blot was then washed in TBST for 30 min , blocked in 5 % milk and was analysed for total MYPT1 as a control . Extracts from this experiment were rerun on 7 . 5 % gel and analysed for ROCK1 and β - actin .

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(c) HeLa cells incubated in HBSS were collected and endogenous phosphorylation of MYPT1 was determined by western blotting. The same blot was then washed in TBST for 30 min, blocked in 5% milk and was analysed for total MYPT1 as a control. Extracts from this experiment were rerun on 7.5% gel and analysed for ROCK1 and β-actin.


( d ) ROCK activity is increased upon starvation . Cell lines ( HeLa and EJ ) grown in control ( + Glu ) or starved with HBSS for 1 and 3 h were lysed , extracts normalized and ROCK kinase activity was measured by performing enzyme - linked immunosorbent assay . Graphs represent ROCK activity in starved cells plotted as percent of ROCK1 activity in control ( glucose - rich ) cells . Data were obtained from triplicates performed at the same time ( mean ± s . d . ) . P - value was calculated using Student ' s t - test .

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(d) ROCK activity is increased upon starvation. Cell lines (HeLa and EJ) grown in control (+Glu) or starved with HBSS for 1 and 3 h were lysed, extracts normalized and ROCK kinase activity was measured by performing enzyme-linked immunosorbent assay. Graphs represent ROCK activity in starved cells plotted as percent of ROCK1 activity in control (glucose-rich) cells. Data were obtained from triplicates performed at the same time (mean±s.d.). P-value was calculated using Student's t-test.


( e ) ROCK1 activity is not dependent on Beclin1 . siControl ( siCont . ) and siBeclin1 knockdown cells were incubated in starvation media ( HBSS ) for indicated time . Resulting cell extracts were used to confirm Beclin1 knockdown and ROCK1 expression ( left panel ) . Cell extracts from the same experiment were used for enzyme - linked immunosorbent assay measuring ROCK activity ( right panel ) . Graph represents mean ± s . d . of duplicate samples read at the same time ( Student ' s t - test ; NS , not significant ) .

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(e) ROCK1 activity is not dependent on Beclin1. siControl (siCont.) and siBeclin1 knockdown cells were incubated in starvation media (HBSS) for indicated time. Resulting cell extracts were used to confirm Beclin1 knockdown and ROCK1 expression (left panel). Cell extracts from the same experiment were used for enzyme-linked immunosorbent assay measuring ROCK activity (right panel). Graph represents mean±s.d. of duplicate samples read at the same time (Student's t-test; NS, not significant).


( a ) The effect of ROCK1 depletion on autophagy marker , LC3II . MEF ROCK1flox / flox cells were generated and treated with Con or Ad - Cre for 72 h . Cells were then incubated in nutrient - rich ( + Glucose ) or HBSS media for 2 h . Cell extracts were resolved by SDS - PAGE and ROCK1 KO was confirmed by western blotting ( left panel ) . The same blot was used for LC3 and β - actin expression . Cells from the same experiment were fixed with chilled acetone and immunofluroscence was performed against endogenous LC3 ( red ) . Blue represents nuclear DAPI staining . Scale bar , 20 μm .

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(a) The effect of ROCK1 depletion on autophagy marker, LC3II. MEF ROCK1flox/flox cells were generated and treated with Con or Ad-Cre for 72 h. Cells were then incubated in nutrient-rich (+Glucose) or HBSS media for 2 h. Cell extracts were resolved by SDS-PAGE and ROCK1 KO was confirmed by western blotting (left panel). The same blot was used for LC3 and β-actin expression. Cells from the same experiment were fixed with chilled acetone and immunofluroscence was performed against endogenous LC3 (red). Blue represents nuclear DAPI staining. Scale bar, 20 μm.


( b ) EJ cells ( left ) with a stable knockdown using shCont . or shROCK1 # 1 were incubated in control ( + glucose ) or nutrient - free ( HBSS ) medium ( − glucose ) for 24 h and lysed . Lysates were subjected to western blotting with antibodies indicated . HeLa cells ( right ) with a transient knockdown using siCont . and siROCK1 # 2 for 30 h were further incubated in control or HBSS media for 8 h . Whole cell extracts were prepared , resolved by SDS - PAGE and analysed for ROCK1 , LC3 and β - actin . Relative ratios of LC3II / LC3I are shown .

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(b) EJ cells (left) with a stable knockdown using shCont. or shROCK1#1 were incubated in control (+glucose) or nutrient-free (HBSS) medium (−glucose) for 24 h and lysed. Lysates were subjected to western blotting with antibodies indicated. HeLa cells (right) with a transient knockdown using siCont. and siROCK1#2 for 30 h were further incubated in control or HBSS media for 8 h. Whole cell extracts were prepared, resolved by SDS-PAGE and analysed for ROCK1, LC3 and β-actin. Relative ratios of LC3II/LC3I are shown.


( c ) HeLa cells were treated with H2O or 10 μM ROCK1 inhibitor , Y27632 , for 8 h . Cells were then cultured in high glucose ( 0 h ) or starved for in HBSS nutrient - free media for 6 h . One hour before collection , cells were incubated in dimethylsulphoxide ( control ) or 0 . 1 μM bafilomycin - A1 . Whole cell lysates were resolved by SDS - PAGE and were analysed by western blotting with indicated antibodies .

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(c) HeLa cells were treated with H2O or 10 μM ROCK1 inhibitor, Y27632, for 8 h. Cells were then cultured in high glucose (0 h) or starved for in HBSS nutrient-free media for 6 h. One hour before collection, cells were incubated in dimethylsulphoxide (control) or 0.1 μM bafilomycin-A1. Whole cell lysates were resolved by SDS-PAGE and were analysed by western blotting with indicated antibodies.


( d ) HeLa cells incubated with H2O or 10 μM Y27632 for 8 h were washed with PBS and cultured in control ( + Glu ) or HBSS media ( − Glu ) for an additional 8 h . Cells were fixed in acetone and immunofluorescence was performed against endogenous LC3 antibody ( green ) . Scale bar , 20 μm . Graph represents % of LC3 punctae / cell ( mean ± s . d . ) , n = 25 cells ; ( Student ' s t - test ) * * P = 0 . 00041 .

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(d) HeLa cells incubated with H2O or 10 μM Y27632 for 8 h were washed with PBS and cultured in control (+Glu) or HBSS media (−Glu) for an additional 8 h. Cells were fixed in acetone and immunofluorescence was performed against endogenous LC3 antibody (green). Scale bar, 20 μm. Graph represents % of LC3 punctae/cell (mean±s.d.), n=25 cells; (Student's t-test) **P=0.00041.


( e ) Transmission electron micrographs of EJ cells with stable shCont . or shROCK1 # 1 knockdown , cultured in control or HBSS medium for 16 h . Arrow bars indicate autophagic vacuoles . Scale bar , 500 nm .

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(e) Transmission electron micrographs of EJ cells with stable shCont. or shROCK1#1 knockdown, cultured in control or HBSS medium for 16 h. Arrow bars indicate autophagic vacuoles. Scale bar, 500 nm.