( C ) Survival curves of GRWT GRDim mice treated for 7 days with 25 mM ZnSO4 in drinking water challenged , i . p . , with 50 µg ( GRWT ) or 12 . 5 µg ( GRDim ) TNF , solved in sterile PBS , per 20 g bodyweight . No deaths occurred later than 50 h after TNF injection . Results represent combined data of 2 experiments .

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(C) Survival curves of GRWT and GRDim mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged, i.p., with 50 µg (GRWT) or 12.5 µg (GRDim) TNF, solved in sterile PBS, per 20 g bodyweight. No deaths occurred later than 50 h after TNF injection. Results represent combined data of 2 experiments.


Zinc in serum after 7 days of 25 mM ZnSO4 supplementation to drinking water of C57BL / 6J naïve Adx mice ( D , N = 3 - 6 ) ,

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Zinc levels in the serum after 7 days of 25 mM ZnSO4 supplementation to the drinking water of C57BL/6J naïve and Adx mice (D, N=3-6),


Zinc in serum after 7 days of 25 mM ZnSO4 supplementation to drinking water of GRWT GRDim mice ( E , N = 6 - 9 ) .

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Zinc levels in the serum after 7 days of 25 mM ZnSO4 supplementation to the drinking water of GRWT and GRDim mice (E, N=6-9).


( F ) Agtpbp1 gene - expression measured with RT - qPCR in ileum of GRWT GRDim mice treated for 7 days with 25 mM ZnSO4 in drinking water ( N = 3 per group ) .

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(F) Agtpbp1 gene-expression measured with RT-qPCR in the ileum of GRWT and GRDim mice treated for 7 days with 25 mM ZnSO4 in the drinking water (N=3 per group).


( G ) Survival curves of GRfl / fl GRVillKO mice treated for 7 days with 25 mM ZnSO4 in drinking water challenged , i . p . , with 35 µg TNF , solved in sterile PBS , per 20 g bodyweight . No deaths occurred later than 150 h after TNF injection . Results represent combined data of 6 experiments .

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(G) Survival curves of GRfl/fl and GRVillKO mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged, i.p., with 35 µg TNF, solved in sterile PBS, per 20 g bodyweight. No deaths occurred later than 150 h after TNF injection. Results represent combined data of 6 experiments.


( H ) Mouse Nr3c1 gene - expression measured with RT - qPCR in intestinal epithelial ( IECs ) of GRfl / fl GRVillKO mice ( N = 3 - 6 ) . Hprt Villin were used as house - keeping genes .

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(H) Mouse Nr3c1 gene-expression measured with RT-qPCR in the intestinal epithelial cells (IECs) of GRfl/fl and GRVillKO mice (N=3-6). Hprt and Villin were used as house-keeping genes.


( I ) Zinc in serum after 7 days of 25 mM ZnSO4 supplementation to drinking water of GRfl / fl GRVillKO mice ( N = 9 per group ) .

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(I) Zinc levels in the serum after 7 days of 25 mM ZnSO4 supplementation to the drinking water of GRfl/fl and GRVillKO mice (N=9 per group).


( D ) Regression curve plotting LFCs of genes induced by ZnSO4 in GRWT GRDim mice , as measured by - seq in ileum samples . genes , induced in ileum by DEX in GRWT with LFC > 0 . 8 , p < 0 . 05 are considered , while these genes with p < 0 . 05 in GRDim were considered . slope of correlation curve as well as Pearson correlation coefficient were calculated by Graphpad Prism .

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(D) Regression curve plotting LFCs of genes induced by ZnSO4 in GRWT and GRDim mice, as measured by RNA-seq in ileum samples. All genes, induced in ileum by DEX in GRWT with LFC>0.8, p<0.05 are considered, while these genes with p<0.05 in GRDim were considered. The slope of the correlation curve as well as Pearson correlation coefficient were calculated by Graphpad Prism.


( E ) C57BL / 6J mice received ( AB ) in drinking water for 3 , followed by 1 week 25 mM ZnSO4 in drinking water . During this week AB administration was continued by oral gavage . Mice were challenged i . v . with 12 . 5 µg TNF solved in sterile PBS survival recorded . Combined data of 2 experiments .

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(E) C57BL/6J mice received antibiotics (AB) in the drinking water for 3 weeks, followed by 1 week 25 mM ZnSO4 in the drinking water. During this week AB administration was continued by oral gavage. Mice were challenged i.v. with 12.5 µg TNF solved in sterile PBS and survival recorded. Combined data of 2 experiments.


( F ) Zinc measured in serum after ZnSO4 protocol ( N = 5 - 8 ) .

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(F) Zinc levels measured in serum after the antibiotics and ZnSO4 protocol (N=5-8).


Mice housed in germ - free ( GF ) or specific pathogen free ( SPF ) conditions received 25 mM ZnSO4 in drinking water for 1 week were then challenged i . p . with 35 µg TNF , dissolved in sterile PBS , per 20 g bodyweight . Combined data of 2 experiments .

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Mice housed in germ-free (GF) or specific pathogen free (SPF) conditions received 25 mM ZnSO4 in the drinking water for 1 week and were then challenged i.p. with 35 µg TNF, dissolved in sterile PBS, per 20 g bodyweight. Combined data of 2 experiments.


( H ) displays blood zinc in SPF GF mice treated for 1 week with water or ZnSO4 ( N = 3 / 4 per group ) .

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(H) displays the blood zinc levels in SPF and GF mice treated for 1 week with water or ZnSO4 (N=3/4 per group).


( I ) Ileum Slurry ( IS ) was isolated from C57BL / 6J mice put on 25 mM ZnSO4 or control water for 1 week 750 µL of this IS was injected i . p . in C57BL / 6J GF mice .

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(I) Ileum Slurry (IS) was isolated from C57BL/6J mice put on 25 mM ZnSO4 or control water for 1 week and 750 µL of this IS was injected i.p. in C57BL/6J GF mice.


Transcriptional downregulation of ISRE / IRF genes in intestinal epithelium by zinc part 1 . - seq experiment of GRWT GRDim mice which received normal drinking water or drinking water containing 25mM ZnSO4 for 1 week . Ileum was sampled - seq performed ( N = 5 - 6 per group ) . Genes downregulated by zinc compared to water control ( LFC < 0 . 8 p < 0 . 05 ) were considered . ( C ) Heat map of a selection of 48 genes of Fig 3B .

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Transcriptional downregulation of ISRE/IRF genes in the intestinal epithelium by zinc part 1. RNA-seq experiment of GRWT and GRDim mice which received normal drinking water or drinking water containing 25mM ZnSO4 for 1 week. Ileum was sampled and RNA-seq performed (N=5-6 per group). Genes downregulated by zinc compared to water control animals (LFC<0.8 and p<0.05) were considered. (C) Heat map of a selection of the 48 genes of Fig 3B.


( A - B ) Expression ( RT - qPCR data ) of Mlkl Zbp1 , from collection of 48 , in ileum biopsy samples , showing reducing effects of zinc in both GRWT GRDim mice , higher expression in GRDim compared to GRWT ( N = 4 - 6 / group ) .

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(A-B) Expression levels (RT-qPCR data) of Mlkl and Zbp1, from the collection of 48, in ileum biopsy samples, showing the reducing effects of zinc in both GRWT and GRDim mice, and the higher expression in GRDim compared to GRWT (N=4-6/group).


RT - qPCR data of Mlkl , Zbp1 in ileum biopsy samples of C57BL / 6J mice or different TLR full knockout mice . ( N = 4 / 23 per group )

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RT-qPCR data of Mlkl, Zbp1 in ileum biopsy samples of C57BL/6J mice or different TLR full knockout mice. (N=4/23 per group)


RT - qPCR data of Ripk3 , in ileum biopsy samples of C57BL / 6J mice or different TLR full knockout mice . ( N = 4 / 23 per group )

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RT-qPCR data of Ripk3, in ileum biopsy samples of C57BL/6J mice or different TLR full knockout mice. (N=4/23 per group)


RT - qPCR data of Mlkl , in ileum biopsy samples of C57BL / 6J mice treated with normal drinking water or 25 mM ZnSO4 in drinking water for 1 week , either or not pretreated with ( AB ) for three . ( N = 5 per group )

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RT-qPCR data of Mlkl , in ileum biopsy samples of C57BL/6J mice treated with normal drinking water or 25 mM ZnSO4 in the drinking water for 1 week, either or not pretreated with antibiotics (AB) for three weeks. (N=5 per group)


RT - qPCR data of Zbp1 , in ileum biopsy samples of C57BL / 6J mice treated with normal drinking water or 25 mM ZnSO4 in drinking water for 1 week , either or not pretreated with ( AB ) for three . ( N = 5 per group )

RT-qPCR data of Zbp1, in ileum biopsy samples of C57BL/6J mice treated with normal drinking water or 25 mM ZnSO4 in the drinking water for 1 week, either or not pretreated with antibiotics (AB) for three weeks. (N=5 per group)


( A - B ) C57BL / 6J mice received control or 25 mM ZnSO4 drinking water for 7 days were injected i . p . with 50 µg TNF , solved in 200 µL sterile PBS , per 20 g bodyweight or with PBS only . 6h later , sections through ileum were made stained with ( A ) Hematoxylin & Eosin MMP7 , a Paneth marker . Pictures at magnification of 20x were taken one representative picture of each condition is shown . Arrows point to crypts their ( ab ) normal occurrence ( see text ) . Scale bars : 50

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(A-B) C57BL/6J mice received control or 25 mM ZnSO4 drinking water for 7 days and were injected i.p. with 50 µg TNF, solved in 200 µL sterile PBS, per 20 g bodyweight or with PBS only. 6h later, sections through ileum were made and stained with (A) Hematoxylin & Eosin and MMP7, a Paneth cell marker. Pictures at magnification of 20x were taken and one representative picture of each condition is shown. Arrows point to crypts and their (ab)normal occurrence (see text). Scale bars: 50 μm


( C ) Transmission electron microscopy ( ) of ileum crypts show protective effects of zinc therapy on level of TNF induced effects on Paneth . C57BL / 6J mice ( N = 2 / group ) were put on normal drinking water or on 25 mM ZnSO4 water for 7 days . Mice were given 50 µG TNF or PBS 8h later , ileum samples were taken prepared for . Figure shows ileal crypts at a magnification of 1000x . In each of 4 conditions , 2 Paneth are selected in red

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(C) Transmission electron microscopy (TEM) images of ileum crypts show protective effects of zinc therapy on the level of TNF induced effects on Paneth cells. C57BL/6J mice (N=2 /group) were put on normal drinking water or on 25 mM ZnSO4 water for 7 days. Mice were given 50 µG TNF or PBS and 8h later, ileum samples were taken and prepared for TEM. Figure shows ileal crypts at a magnification of 1000x. In each of the 4 conditions, 2 Paneth cells are selected in red area


( D - F ) RT - qPCR measurement of mRNA of Defa6 Lyz2 , two Paneth markers , Ripk3 , a TNF - induced - gene , in ileum of experiment in A .

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(D-F) RT-qPCR measurement of mRNA levels of Defa6 and Lyz2, two Paneth cell markers, and Ripk3, a TNF-induced cell-death gene, in ileum of the experiment in A.


( A - B ) C57BL / 6J mice received control or 25 mM ZnSO4 drinking water for 7 days were injected i . p . with 50 µg TNF , solved in 200 µL sterile PBS , per 20 g bodyweight or with PBS only ( N = 5 ) . 18h after TNF challenge , spleen MLNs were isolated lysates prepared plated on TSA plates . Colonies were counted expressed as colonies per ml of lysate . 40 colonies per plate were prepared for Maldi Tof identification relative amounts of species found on plates are mentioned underneath figure . Data are shown as means + / - SD . p - value was analyzed by means of a Student ' s t - ( unpaired , two - ) .

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(A-B) C57BL/6J mice received control or 25 mM ZnSO4 drinking water for 7 days and were injected i.p. with 50 µg TNF, solved in 200 µL sterile PBS, per 20 g bodyweight or with PBS only (N=5). 18h after TNF challenge, spleen and MLNs were isolated and lysates prepared and plated on TSA plates. Colonies were counted and expressed as bacterial colonies per ml of lysate. 40 colonies per plate were prepared for Maldi Tof identification and the relative amounts of bacterial species found on the plates are mentioned underneath the figure. Data are shown as means +/- SD. p-value was analyzed by means of a Student's t-test (unpaired, two-tailed).


( E ) Impact of zinc in TSA plates on growth of colonies of 4 species commonly found in spleen MLNs of mice injected with TNF ( see A - B ) . Pure colonies of Staphylococcus nepalensis ( orange ) , Staphylococcus sciuri ( red ) , Eschericchia coli ( purple ) Enterococcus faecalis ( blue ) were grown in LB medium , then diluted plated on TSA plates containing increasing concentrations of ZnCL2 . 24h later , amounts of colonies were counted , amounts found in 0 mM ZnCl2 condition expressed as 100 % . Data are shown as mean + / - SD pooled from three independent experiments .

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(E) Impact of zinc concentration in bacterial TSA plates on the growth of colonies of 4 bacterial species commonly found in spleen and MLNs of mice injected with TNF (see A-B). Pure colonies of Staphylococcus nepalensis (orange), Staphylococcus sciuri (red), Eschericchia coli (purple) and Enterococcus faecalis (blue) were grown in LB medium, then diluted and plated on TSA plates containing increasing concentrations of ZnCL2. 24h later, the amounts of colonies were counted, and the amounts found in the 0 mM ZnCl2 condition expressed as 100%. Data are shown as mean +/- SD pooled from three independent experiments.


Monocolonization experiment . Mice ( N = 5 groups ) were treated with H20 or ZnSO4 for 7 days , after which ZnSO4 was removed replaced by H20 . 12h 24h later , mice were transplanted with PBS , 108 Staphylococcus sciuri or 108 S . nepalensis ( in 100 ul ) , 3h later a lethal dose of TNF ( 35 µg / mouse ) was injected , 24h later ( insert in F ) lethality ( F ) recorded estimated by one - way ANOVA Chi2 - resp . Survival significance was tested towards H20 control group .

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Monocolonization experiment. Mice (N=5 all groups) were treated with H20 or ZnSO4 for 7 days, after which the ZnSO4 was removed and replaced by H20. 12h and 24h later, the mice were transplanted with PBS, 108 Staphylococcus sciuri or 108 S. nepalensis (in 100 ul), and 3h later a lethal dose of TNF (35 µg/mouse) was injected, and 24h later body temperature (insert in F) and lethality (F) recorded and estimated by one-way ANOVA and Chi2-tests resp. Survival significance was tested towards the H20 control group.


Monocolonization experiment . Mice ( N = 5 groups ) were treated with H20 or ZnSO4 for 7 days , after which ZnSO4 was removed replaced by H20 . 12h 24h later , mice were transplanted with PBS , 108 Staphylococcus sciuri ( in 100 ul ) , 3h later a lethal dose of TNF ( 35 µg / mouse ) was injected In a second experiment ( G - I ) ( N = 4 groups ) with S . sciuri colonization , Zbp1 Stat1 , two key ISRE genes , Mt2 , a Zn - induced gene , were measured in ileum biopsies by RT - qPCR .

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Monocolonization experiment. Mice (N=5 all groups) were treated with H20 or ZnSO4 for 7 days, after which the ZnSO4 was removed and replaced by H20. 12h and 24h later, the mice were transplanted with PBS, 108 Staphylococcus sciuri (in 100 ul), and 3h later a lethal dose of TNF (35 µg/mouse) was injected In a second experiment (G-I) (N=4 all groups) with S. sciuri colonization, Zbp1 and Stat1, two key ISRE genes, and Mt2, a Zn-induced gene, were measured in ileum biopsies by RT-qPCR.


( A ) C57BL / 6J mice received 25 mM ZnSO4 or control water for 7 days . Mice were injected i . p . with 100 , 200 or 500 µg dexamethasone ( Dex , rapidexon ) , solved in sterile PBS , 30 min before a 50 µg TNF / 20 g bodyweight i . p . challenge . STAT1 + / + STAT1 - / - mice were challenged with equal TNF dose , as controls . No mice died later than 120 h after TNF injection . Data show combined results of 2 experiments .

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(A) C57BL/6J mice received 25 mM ZnSO4 or control water for 7 days. Mice were injected i.p. with 100, 200 or 500 µg dexamethasone (Dex, rapidexon), solved in sterile PBS, 30 min before a 50 µg TNF/20 g bodyweight i.p. challenge. STAT1+/+ and STAT1-/- mice were challenged with equal TNF dose, as controls. No mice died later than 120 h after TNF injection. Data show combined results of 2 experiments.


Mice ( N = 4 per group ) treated as in ( A ) 6h after challenge , ileum was taken RT - qPCR performed to study gene expression of ISRE genes Mlkl

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Mice (N=4 per group) treated as in (A) and 6h after challenge, ileum was taken and RT-qPCR performed to study gene expression of cell death ISRE genes Mlkl and Zbp1


Mice ( N = 4 per group ) treated as in ( A ) 6h after challenge , ileum was taken RT - qPCR performed to study gene expression of ISRE genes Stat1 .

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Mice (N=4 per group) treated as in (A) and 6h after challenge, ileum was taken and RT-qPCR performed to study gene expression of cell death ISRE genes Stat1.


( A ) RT - qPCR of Myh7b / miR - 499 Myh7 / miR - 208b during differentiation of myoblasts into mature myotubes ( n = 3 independent experiments ) . * p = 0 . 0005 ( Myh7b ) , * p < 0 . 0001 ( miR - 499 ) , * p = 0 . 0002 ( Myh7 ) , * p < 0 . 0001 ( miR - 208b ) .

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(A) RT-qPCR analysis of Myh7b/miR-499 and Myh7/miR-208b levels during differentiation of myoblasts into mature myotubes (n = 3 independent experiments). *p = 0.0005 (Myh7b), *p < 0.0001 (miR-499), *p = 0.0002 (Myh7), *p < 0.0001 (miR-208b).


( B ) Oxygen consumption rates ( OCR ) in myoblasts differentiated myotubes . Basal OCR was first measured , followed by administration of 10 mM sodium pyruvate , 2 μM oligomycin ( to inhibit ATP synthase ) , uncoupler FCCP ( 2 μM ) , or rotenone / antimycin ( Rot / A ) ( 1 μM ) as indicted . n = 3 separate experiments done with 10 biological replicates . * p < 0 . 0001 ( FCCP ) .

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(B) Oxygen consumption rates (OCR) in primary myoblasts and differentiated myotubes. Basal OCR was first measured, followed by administration of 10 mM sodium pyruvate, and 2 μM oligomycin (to inhibit ATP synthase), uncoupler FCCP (2 μM), or rotenone/antimycin (Rot/A) (1 μM) as indicted. n = 3 separate experiments done with 10 biological replicates. *p < 0.0001 (FCCP).


( C ) Mean expression ( RT - qPCR ) in vastus ( WV ) soleus ( Sol ) muscle from 8 - week - old wild type mice ( n = 5 mice per group ) . * p < 0 . 0001 ( Myh7b ) , * p < 0 . 0001 ( miR - 499 ) , * p < 0 . 0001 ( Myh7 ) , * p < 0 . 0001 ( miR - 208b ) .

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(C) Mean expression levels (RT-qPCR) in white vastus (WV) and soleus (Sol) muscle from 8-week-old male wild type mice (n = 5 mice per group). *p < 0.0001 (Myh7b), *p < 0.0001 (miR-499), *p < 0.0001 (Myh7), *p < 0.0001 (miR-208b).


( D ) Mitochondrial respiration rates were determined from mitochondria isolated from indicated muscle using pyruvate as substrate . ADP - dependent respiration , oligomycin - induced ( oligo ) , uncoupler FCCP , antimycin A ( AA ) are shown . n = 3 separate experiments done with 7 - 8 biological replicates . * p < 0 . 05 ( ADP ) .

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(D) Mitochondrial respiration rates were determined from mitochondria isolated from indicated muscle using pyruvate as substrate. ADP-dependent respiration, oligomycin-induced (oligo), uncoupler FCCP, and antimycin A (AA) are shown. n = 3 separate experiments done with 7-8 biological replicates. *p < 0.05 (ADP).


( E ) ( Left ) Schematic depicts increments of speed over time . ( Right ) Respiratory exchange ( RER ) during a graded exercise regimen as described in Materials Methods ( n = 5 mice per group ) . Notably , MCK - miR - 499 mice were able to exercise at a higher speed before exhaustion . * p < 0 . 05 .

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(E) (Left) Schematic depicts the increments of speed over time. (Right) Respiratory exchange ratio (RER) during a graded exercise regimen as described in Materials and Methods (n = 5 mice per group). Notably, MCK-miR-499 mice were able to exercise at a higher speed before exhaustion. *p < 0.05.


( F ) Bars represent mean blood lactate for 3 - month 15 - month old MCK - miR - 499 NTG mice following a 25 min run on a motorized treadmill . For 3 - month blood lactate , NTG , n = 9 ; MCK - miR - 499 , n = 10 . For 15 - month blood lactate , NTG , n = 7 ; MCK - miR - 499 , n = 5 . * p = 0 . 039 ( 3 - month ) , * p = 0 . 018 ( 15 - month ) .

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(F) Bars represent mean blood lactate levels for 3-month and 15-month old male MCK-miR-499 and NTG mice following a 25 min run on a motorized treadmill. For 3-month blood lactate, NTG, n = 9; MCK-miR-499, n = 10. For 15-month blood lactate, NTG, n = 7; MCK-miR-499, n = 5. *p = 0.039 (3-month), *p = 0.018 (15-month).


( G ) Mitochondrial respiration rates were determined from plantaris muscle of indicated genotypes using pyruvate / malate as substrate . Pyruvate / malate ( Py / M ) - stimulated , ADP - dependent respiration , oligomycin - induced ( oligo ) respiratory control ( RC ) are shown . NTG , n = 6 ; MCK - miR - 499 , n = 4 . * p = 0 . 015 ( Py / M ) , * p = 0 . 017 ( ADP ) , * p = 0 . 001 ( Oligo ) .

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(G) Mitochondrial respiration rates were determined from the plantaris muscle of the indicated genotypes using pyruvate/malate as substrate. Pyruvate/malate (Py/M)-stimulated, ADP-dependent respiration, oligomycin-induced (oligo) and the respiratory control ratio (RC) are shown. NTG, n = 6; MCK-miR-499, n = 4. *p = 0.015 (Py/M), *p = 0.017 (ADP), *p = 0.001 (Oligo).


( B ) Expression of - encoded mitochondrial - encoded genes ( RT - qPCR ) in gastrocnemius muscle from indicated genotypes ( n = 5 mice per group ) . * p < 0 . 05 .

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(B) Expression of the nuclear-encoded and mitochondrial-encoded genes (RT-qPCR) in the gastrocnemius muscle from the indicated genotypes (n = 5 mice per group). *p < 0.05.


( C ) Representative western blot performed with gastrocnemius muscle total extracts prepared from indicated mice using myoglobin , cytochrome c , β - actin ( control ) antibodies ( n = 8 mice per group ) .

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(C) Representative western blot analysis performed with gastrocnemius muscle total protein extracts prepared from the indicated mice using myoglobin, cytochrome c, and β-actin (control) antibodies (n = 8 mice per group).


( D ) Expression of genes ( RT - qPCR ) involved in muscle glucose fatty metabolism in gastrocnemius muscle from indicated genotypes ( n = 5 mice per group ) . * p < 0 . 05 .

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(D) Expression of genes (RT-qPCR) involved in muscle glucose and fatty acids metabolism in the gastrocnemius muscle from the indicated genotypes (n = 5 mice per group). *p < 0.05.


( E ) LDH isoenzymes were separated by polyacrylamide gel electrophoresis using whole extracts from NTG heart ( Ht , control ) gastrocnemius muscle from indicated mice . A representative gel showing 3 - 4 independent mice per group is shown . ( F ) of LDH isoenzyme activity gel electrophoresis in E . Values represent mean % ( + / - ) total LDH activity . * p < 0 . 05 .

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(E) LDH isoenzymes were separated by polyacrylamide gel electrophoresis using whole cell extracts from NTG heart (Ht, control) and gastrocnemius muscle from the indicated mice. A representative gel showing 3-4 independent mice per group is shown.(F) Quantification of LDH isoenzyme activity gel electrophoresis in E. Values represent the mean % (+/- SEM) total LDH activity. *p < 0.05.


( G ) Cross - section of gastrocnemius muscle from a 3 - month - old NTG MCK - miR - 499 stained for myosin I ATPase activity ( Top ) , SDH ( Middle ) , α - GPDH ( Bottom ) . Representative were shown . Scale bar : 500 μm .

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(G) Cross-section of the gastrocnemius muscle from a 3-month-old male NTG and MCK-miR-499 stained for myosin I ATPase activity (Top), SDH (Middle), and α-GPDH (Bottom). Representative images were shown. Scale bar: 500 μm.


( A ) ( Left ) Representative western blot of PGC - 1α expression in gastrocnemius muscle from indicated genotypes , heart ( Ht ) lysate from WT mice PGC - 1α mKO ( MCK - Cre ) mice as positive negative controls , respectively . ( Right ) of PGC - 1α / Tubulin signal ratios normalized ( = 1 . 0 ) to NTG control ( n = 5 mice per group ) . * p = 0 . 00017 .

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(A) (Left) Representative western blot analysis of PGC-1α protein expression in the gastrocnemius muscle from the indicated genotypes, heart (Ht) lysate from WT mice and PGC-1α mKO (MCK-Cre) mice as positive and negative controls, respectively. (Right) Quantification of the PGC-1α/Tubulin signal ratios normalized (=1.0) to the NTG control (n = 5 mice per group). *p = 0.00017.


( B ) Expression of Ldhb , Ldha , Mb ( myoglobin ) , Cox5a genes ( RT - qPCR ) in gastrocnemius muscle from indicated genotypes ( n = 5 mice per group ) . * p < 0 . 001 ( vs . NTG ) , ‡ p < 0 . 01 ( vs . 499Tg ) .

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(B) Expression of the Ldhb, Ldha, Mb (myoglobin), and Cox5a genes (RT-qPCR) in the gastrocnemius muscle from the indicated genotypes (n = 5 mice per group). *p < 0.001 (vs. NTG), ‡p < 0.01 (vs. 499Tg).


( C ) ( Left ) Representative western blot performed with gastrocnemius muscle total extracts prepared from indicated mice using cytochrome c , myoglobin , α - tubulin ( control ) antibodies . ( Right ) of Cyt c / Tubulin signal ratios normalized ( = 1 . 0 ) to NTG control . NTG , n = 5 ; 499Tg , n = 6 ; PGC - 1α mKO , n = 5 ; 499Tg / PGC - 1α mKO , n = 5 . * p < 0 . 01 ( vs . NTG ) , ‡ p = 0 . 00018 ( vs . 499Tg ) .

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(C) (Left) Representative western blot analysis performed with gastrocnemius muscle total protein extracts prepared from the indicated mice using cytochrome c, myoglobin, and α-tubulin (control) antibodies. (Right) Quantification of the Cyt c/Tubulin signal ratios normalized (=1.0) to the NTG control. NTG, n = 5; 499Tg, n = 6; PGC-1α mKO, n = 5; 499Tg/PGC-1α mKO, n = 5. *p < 0.01 (vs. NTG), ‡p = 0.00018 (vs. 499Tg).


( D ) Cross - section of gastrocnemius muscle from indicated mice stained for SDH activity ( Top ) as well as MHC1 immunofluorescence ( IF ) ( Bottom ) , Representative were shown . Scale bar : 500 μm .

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(D) Cross-section of gastrocnemius muscle from the indicated mice stained for SDH activity (Top) as well as MHC1 immunofluorescence (IF) (Bottom), Representative images were shown. Scale bar: 500 μm.


( E ) Respiratory exchange ( RER ) during a graded exercise regimen as described in Materials Methods . Notably , no significant increase in exhaust speed was observed in 499Tg / PGC - 1α mKO mice compared to PGC - 1α mKO mice . PGC - 1α mKO , n = 5 ; 499Tg / PGC - 1α mKO , n = 8 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Respiratory exchange ratio (RER) during a graded exercise regimen as described in Materials and Methods. Notably, no significant increase in exhaust speed was observed in 499Tg/PGC-1α mKO mice compared to PGC-1α mKO mice. PGC-1α mKO, n = 5; 499Tg/PGC-1α mKO, n = 8.


( F ) bars represent mean blood lactate from indicated mice following a 25 min run on a motorized treadmill . NTG , n = 9 ; 499Tg , n = 7 ; PGC - 1α mKO , n = 6 ; 499Tg / PGC - 1α mKO , n = 9 . * p = 0 . 0009 ( NTG vs . 499Tg ) , p = 0 . 316 ( NS , not significant ) .

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(F) The bars represent the mean blood lactate levels from the indicated mice following a 25 min run on a motorized treadmill. NTG, n = 9; 499Tg, n = 7; PGC-1α mKO, n = 6; 499Tg/PGC-1α mKO, n = 9. *p = 0.0009 (NTG vs. 499Tg), p = 0.316 (NS, not significant).


( B ) Luciferase reporters containing wild - type Fnip1 3 ' UTR or Fnip1 3 ' UTR mutated in predicted binding site of miR - 499 were used in cotransfection studies in HEK293T in presence or absence of plasmid expressing miR - 499 ( n = 3 independent experiments ) . * p < 0 . 0001 ( Fnip1 3 ' UTR ) ; * p = 0 . 023 ( Mutated ) .

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(B) Luciferase reporters containing the wild-type Fnip1 3'UTR or Fnip1 3'UTR mutated in the predicted binding site of miR-499 were used in cotransfection studies in HEK293T cells in the presence or absence of plasmid expressing miR-499 (n = 3 independent experiments). *p < 0.0001 (Fnip1 3'UTR); *p = 0.023 (Mutated).


( C ) RT - qPCR of Fnip1 Fnip2mRNA in gastrocnemius muscle of indicated genotypes ( n = 5 mice per group ) .

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(C) RT-qPCR analysis of Fnip1 and Fnip2mRNA levels in the gastrocnemius muscle of the indicated genotypes (n = 5 mice per group).


( D ) ( Top ) Fnip1 expression in gastrocnemius muscle from indicated mice . ( Bottom ) of Fnip1 / Tubulin signal ( n = 4 mice per group ) . * p = 0 . 014 .

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(D) (Top) Fnip1 protein expression in the gastrocnemius muscle from the indicated mice. (Bottom) Quantification of the Fnip1/Tubulin signal ratio (n = 4 mice per group). *p = 0.014.


( A ) ( Left ) Representative western blot performed on extracts of myotubes subjected to Fnip1 siRNA or control ( Con ) siRNA using phospho - AMPKα ( Thr172 ) AMPKα antibodies . ( Right ) of p - AMPKα / AMPKα signal ratios ( n = 3 independent experiments ) . * p = 0 . 037 .

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(A) (Left) Representative western blot analysis performed on extracts of myotubes subjected to Fnip1 siRNA or control (Con) siRNA using phospho-AMPKα (Thr172) and AMPKα antibodies. (Right) Quantification of the p-AMPKα/AMPKα signal ratios (n = 3 independent experiments). *p = 0.037.


( B , C ) Results of RT - qPCR for WT mouse myotubes after transfection with Fnip1 siRNAs or scrambled Con siRNAs as indicated . For C , 48 h post - siRNA transfection , myotubes were treated for 24 h with DMSO or 10 μm compound C before harvest ( n = 3 independent experiments ) . * p < 0 . 0001 ( Fnip1 in B ) , * p = 0 . 0001 ( PGC - 1α in B ) ; * p < 0 . 0001 ( vs . Con siRNA in C ) , ‡ p < 0 . 0001 ( vs . Fnip1 siRNA in C ) .

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(B, C) Results of RT-qPCR analysis for WT primary mouse myotubes after transfection with Fnip1 siRNAs or scrambled Con siRNAs as indicated. For C, 48 h post-siRNA transfection, myotubes were treated for 24 h with DMSO or 10 μm compound C before harvest (n = 3 independent experiments). *p < 0.0001 (Fnip1 in B), *p = 0.0001 (PGC-1α in B); *p < 0.0001 (vs. Con siRNA in C), ‡p < 0.0001 (vs. Fnip1 siRNA in C ).


( D ) ( Top ) Representative western blot performed on extracts of gastrocnemius muscle isolated from NTG or MCK - miR - 499 mice using phospho - AMPKα ( Thr172 ) , AMPKα , phospho - ACC ( Ser79 ) , or total ACC antibodies . ( Bottom ) of p - AMPKα / AMPKα p - ACC / ACC signal ratios normalized ( = 1 . 0 ) to NTG control ( n = 8 mice per group ) . * p < 0 . 01 .

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(D) (Top) Representative western blot analysis performed on extracts of the gastrocnemius muscle isolated from NTG or MCK-miR-499 mice using phospho-AMPKα (Thr172), AMPKα, phospho-ACC (Ser79), or total ACC antibodies. (Bottom) Quantification of the p-AMPKα/AMPKα and p-ACC/ACC signal ratios normalized (=1.0) to the NTG control (n = 8 mice per group). *p < 0.01.


( E ) Oxygen consumption rates ( OCR ) in mouse myotubes transfected with Fnip1 siRNA or Con siRNA . n = 7 separate experiments done with 5 biological replicates . * p < 0 . 05 ( Pyruvate ) , * p < 0 . 01 ( FCCP ) .

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(E) Oxygen consumption rates (OCR) in primary mouse myotubes transfected with Fnip1 siRNA or Con siRNA. n = 7 separate experiments done with 5 biological replicates. *p < 0.05 (Pyruvate), *p < 0.01 (FCCP).


( F ) Results of RT - qPCR for WT mouse myotubes subjected to inhibition of both miR - 499 miR - 208b ( Anti - miRs ) ( n = 4 independent experiments ) . * p = 0 . 037 ( Ppargc1a ) , * p = 0 . 0019 ( Fnip1 ) .

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(F) Results of RT-qPCR analysis for WT primary mouse myotubes subjected to inhibition of both miR-499 and miR-208b (Anti-miRs) (n = 4 independent experiments). *p = 0.037 (Ppargc1a), *p = 0.0019 (Fnip1).


( G ) Oxygen consumption rates ( OCR ) in mouse myotubes transfected with miR - 499 / miR - 208b inhibitors alone together with presence of Fnip1 siRNA . n = 4 separate experiments done with 5 biological replicates . * p < 0 . 01 ( Anti - miRs vs . Control ) .

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(G) Oxygen consumption rates (OCR) in primary mouse myotubes transfected with miR-499/miR-208b inhibitors alone and together with the presence of Fnip1 siRNA. n = 4 separate experiments done with 5 biological replicates. *p < 0.01 (Anti-miRs vs. Control).


( A ) Mean expression ( RT - qPCR ) in gastrocnemius muscle of 8 - week - old WT mdx mice ( n = 5 mice per group ) . * p = 0 . 009 ( Myh7b ) , * p = 0 . 0007 ( miR - 499 ) , * p = 0 . 0143 ( Myh7 ) , * p < 0 . 0011 ( miR - 208b ) .

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(A) Mean expression levels (RT-qPCR) in gastrocnemius muscle of 8-week-old male WT and mdx mice (n = 5 mice per group). *p = 0.009 (Myh7b), *p = 0.0007 (miR-499), *p = 0.0143 (Myh7), *p < 0.0011 (miR-208b).


( B ) Mitochondrial respiration rates were determined from mitochondria isolated from hindlimb muscle of indicated mice using pyruvate as substrate . n = 3 separate experiments done with 7 - 8 biological replicates . * p < 0 . 01 ( ADP ) .

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(B) Mitochondrial respiration rates were determined from mitochondria isolated from the hindlimb muscle of the indicated mice using pyruvate as substrate. n = 3 separate experiments done with 7-8 biological replicates. *p < 0.01 (ADP).


( C ) ( Left ) Representative western blot of PGC - 1α ( Top ) Fnip1 ( Bottom ) expression in gastrocnemius muscle from indicated genotypes with α - tubulin as loading control . ( Right ) of PGC - 1α / Tubulin Fnip1 / Tubulin signal ratios normalized ( = 1 . 0 ) to WT control . WT , n = 8 ; mdx , n = 5 ; mdx / 499Tg , n = 7 . * p < 0 . 0001 ( vs . WT ) , ‡ p < 0 . 0001 ( vs . mdx ) .

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(C) (Left) Representative western blot analysis of PGC-1α (Top) and Fnip1 (Bottom) protein expression in the gastrocnemius muscle from the indicated genotypes with α-tubulin as the loading control. (Right) Quantification of the PGC-1α/Tubulin and Fnip1/Tubulin signal ratios normalized (=1.0) to the WT control. WT, n = 8; mdx, n = 5; mdx/499Tg, n = 7. *p < 0.0001 (vs. WT), ‡p < 0.0001 (vs. mdx).


( D ) Representative western blot of myoglobin expression in gastrocnemius muscle from indicated genotypes with α - tubulin as loading control ( n = 5 mice per group ) .

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(D) Representative western blot analysis of myoglobin protein expression in the gastrocnemius muscle from the indicated genotypes with α-tubulin as the loading control (n = 5 mice per group).


( E ) ( Top ) Representative MHC1 immunofluorescence ( IF ) in soleus of indicated genotypes . Scale bar : 500 μm . ( Bottom ) Representative H & amp ; amp ; E of gastrocnemius muscle of indicated genotypes . Scale bar : 100 μm .

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(E) (Top) Representative MHC1 immunofluorescence (IF) in the soleus of the indicated genotypes. Scale bar: 500 μm. (Bottom) Representative H&amp;E staining of the gastrocnemius muscle of the indicated genotypes. Scale bar: 100 μm.


( F ) 5 - week - old WT , mdx , mdx / MCK - miR - 499 mice were euthanized serum creatine kinase activity was determined . WT , n = 10 ; mdx , n = 10 ; mdx / 499Tg , n = 14 . * p < 0 . 0001 ( vs . WT ) , ‡ p = 0 . 0003 ( vs . mdx ) .

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(F) 5-week-old male WT, mdx, and mdx/MCK-miR-499 mice were euthanized and serum creatine kinase activity was determined. WT, n = 10; mdx, n = 10; mdx/499Tg, n = 14. *p < 0.0001 (vs. WT), ‡p = 0.0003 (vs. mdx).


( G ) bars represent mean running time ( ± ) for 8 - week - old mice on a motorized treadmill . WT , n = 10 ; mdx , n = 13 ; mdx / 499Tg , n = 8 . * p = 0 . 01406 ( Running time , vs . WT ) , ‡ p = 0 . 005756 ( Running time , vs . mdx ) . * p = 0 . 004558 ( Running , vs . WT ) , ‡ p = 0 . 001668 ( Running , vs . mdx ) .

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(G) The bars represent the mean running time and distance (± SEM) for 8-week-old male mice on a motorized treadmill. WT, n = 10; mdx, n = 13; mdx/499Tg, n = 8. *p = 0.01406 (Running time, vs. WT), ‡p = 0.005756 (Running time, vs. mdx). *p = 0.004558 (Running distance, vs. WT), ‡p = 0.001668 (Running distance, vs. mdx).


( H ) Respiratory exchange ( RER ) during a graded exercise regimen on a motorized treadmill . mdx , n = 8 ; mdx / 499Tg , n = 6 . ‡ p < 0 . 05 .

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(H) Respiratory exchange ratio (RER) during a graded exercise regimen on a motorized treadmill. mdx, n = 8; mdx/499Tg, n = 6. ‡p < 0.05.


Widefield of RPA - mCherry - ssDNA molecules ( magenta ) in presence of GFP - Rdh54 ( green ; 1 , 3 , 10 30 nM ) .

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Widefield images of RPA-mCherry-ssDNA molecules (magenta) in the presence of GFP-Rdh54 (green; 1, 3, 10 and 30 nM).


Widefield of Rad51 - ssDNA molecules ( unlabeled ) in presence of GFP - Rdh54 ( green ; 1 , 3 , 10 30 nM ) .

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Widefield images of Rad51-ssDNA molecules (unlabeled) in the presence of GFP-Rdh54 (green; 1, 3, 10 and 30 nM).


Kymograph illustrating dissociation of GFP - Rdh54 from Rad51 - ssDNA ; arrowhead highlights time point at which free GFP - Rdh54 was flushed from sample chamber . Graph depicting dissociation rate of GFP - Rdh54 from Rad51 - ssDNA . Error bars represent s . d . of ssDNA molecules ( N = 25 ) .

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Kymograph illustrating the dissociation of GFP-Rdh54 from Rad51-ssDNA; arrowhead highlights the time point at which free GFP-Rdh54 was flushed from the sample chamber.   Graph depicting the dissociation rate of GFP-Rdh54 from Rad51-ssDNA. Error bars represent s.d. of individual ssDNA molecules (N=25).


Kymograph showing association of GFP - Rdh54 with a Rad51 - ssDNA molecule ; arrowhead highlights injection time point for GFP - Rdh54 . Association rates of GFP - Rdh54 on Rad51 PSCs ( N = 44 ) . Red lines represent mean s . d .

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Kymograph showing the association of GFP-Rdh54 with a Rad51-ssDNA molecule; arrowhead highlights the injection time point for GFP-Rdh54.   Association rates of GFP-Rdh54 on individual Rad51 PSCs (N=44). Red lines represent the mean and s.d.


of Rad51 - ssDNA molecules bound by both mCherry - Rdh54 ( 30 nM ; magenta ) GFP - Rad54 ( green ; 30 nM ) .

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Images of Rad51-ssDNA molecules bound by both mCherry-Rdh54 (30 nM; magenta) and GFP-Rad54 (green; 30 nM).


of bound GFP - Rad54 ( 10 nM ) signal for Rad51 - ssDNA molecules with without bound mCherry - Rdh54 ( 10 nM ) . Error bars represent s . d . of ssDNA molecules ( N = 21 ) .

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Quantification of bound GFP-Rad54 (10 nM) signal intensity for Rad51-ssDNA molecules with and without bound mCherry-Rdh54 (10 nM). Error bars represent s.d. of individual ssDNA molecules (N = 21).


showing increasing concentrations of GFP - Rad54 ( green ) only or mCherry - Rdh54 ( magenta ) only bound separately to Rad51 - ssDNA molecules .

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Images showing increasing concentrations of GFP-Rad54 (green) only or mCherry-Rdh54 (magenta) only bound separately to Rad51-ssDNA molecules.


of GFP - Rad54 ( green ) mCherry - Rdh54 ( magenta ) co - bound to same Rad51 - ssDNA molecules .

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Images of GFP-Rad54 (green) and mCherry-Rdh54 (magenta) co-bound to the same Rad51-ssDNA molecules.


of GFP - Rad54 binding in absence presence of mCherry - Rdh54 . data was fit by non - linear regression error bars represent s . d . of molecules ( N = 43 ) .

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Quantification of GFP-Rad54 binding in the absence and presence of mCherry-Rdh54. The data was fit by non-linear regression and error bars represent s.d. of individual molecules (N = 43).


of mCherry - Rdh54 binding in absence or presence of GFP - Rad54 . data was fit by non - linear regression error bars represent s . d . of molecules ( N = 43 ) .

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Quantification of mCherry-Rdh54 binding in the absence or presence of GFP-Rad54. The data was fit by non-linear regression and error bars represent s.d. of individual molecules (N = 43).


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Images of GFP-tagged Rad54, Rdh54NRad54, Rdh54 and Rad54NRdh54 (1, 3, 10 and 30 nM each) bound to Rad51-ssDNA molecules.


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Images of GFP-tagged Rad54, Rdh54NRad54, Rdh54 and Rad54NRdh54 (1, 3, 10 and 30 nM each) bound to Rad51-ssDNA molecules in the presence of 30 nM Hed1.


of integrated GFP signal for GFP - tagged Rad54 ( N = 75 ) , Rdh54NRad54 ( N = 46 ) , Rdh54 ( N = 50 ) Rad54NRdh54 ( N = 35 ; 1 , 3 , 10 30 nM each ) bound to Rad51 - ssDNA molecules . data was fit by non - linear regression error bars represent s . d . for molecules .

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Quantification of the integrated GFP signal intensity for GFP-tagged Rad54 (N = 75), Rdh54NRad54 (N = 46), Rdh54 (N = 50) and Rad54NRdh54 (N = 35; 1, 3, 10 and 30 nM each) bound to Rad51-ssDNA molecules. The data was fit by non-linear regression and error bars represent s.d. for individual DNA molecules.


Comparison of fold binding inhibition for GFP - tagged Rad54 , Rdh54NRad54 , Rdh54 Rad54NRdh54 in reactions conducted in presence of 30 nM Hed1 . dot represents mean error bars represent range for two independent experiments .

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Comparison of fold binding inhibition for GFP-tagged Rad54, Rdh54NRad54, Rdh54 and Rad54NRdh54 in reactions conducted in the presence of 30 nM Hed1. The dot represents the mean and the error bars represent the range for two independent experiments.


Widefield of Atto565 - labeled PSCs ( magenta ) bound to dsDNA curtains stained with YOYO1 ( green ) . PSCs were prepared with indicated combinations of Rad51 , Rdh54 GFP - Rad54 . Number of Atto565 - labeled PSC binding events per dsDNA molecule for PCS prepared with Rad54 Rdh54 only ( minus Rad51 ; N = 372 ) , Rad51 plus Rad54 only ( N = 117 ) , Rad51 plus Rdh54 only ( N = 258 ) , Rad51 plus Rdh54 Rad54 ( N = 303 ) . error bars represent s . d . for three independent experiments .

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Widefield images of Atto565-labeled PSCs (magenta) bound to dsDNA curtains stained with YOYO1 (green). The PSCs were prepared with the indicated combinations of Rad51, Rdh54 and GFP-Rad54.   Number of Atto565-labeled PSC binding events per dsDNA molecule for PCS prepared with Rad54 and Rdh54 only (minus Rad51 ; N = 372), Rad51 plus Rad54 only (N= 117), Rad51 plus Rdh54 only (N = 258), Rad51 plus Rdh54 and Rad54 (N = 303). The error bars represent the s.d. for three independent experiments.


Kymographs of Rad51 - duplex PSCs ( magenta ) containing indicated combinations of Rad54 Rdh54 .

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Kymographs of Rad51-tailed duplex PSCs (magenta) containing the indicated combinations of Rad54 and Rdh54 proteins.


Translocation velocities for duplex PSCs prepared with Rad54 only ( lane 1 ; N = 86 ) , Rad54 plus Rdh54 ( lane 2 ; N = 144 ) , Rad54 - K341R plus Rdh54 ( lane 3 ; N = 47 ) , Rad54 plus Rdh54 - K318R ( lane 4 ; N = 112 ) , or Rad54 - K341R plus Rdh54 - K318R ( lane 5 ; N = 64 ) . error bars represent s . d . Translocation distances for duplex PSCs prepared with Rad54 only ( lane 1 ; N = 86 ) , Rad54 plus Rdh54 ( lane 2 ; N = 144 ) , Rad54 - K341R plus Rdh54 ( lane 3 ; N = 47 ) , Rad54 plus Rdh54 - K318R ( lane 4 ; N = 112 ) , or Rad54 - K341R plus Rdh54 - K318R ( lane 5 ; N = 64 ) . Error bars were generated by bootstrapping . First passage recognition efficiency for duplex PSCs prepared with Rad54 only ( lane 1 ; N = 145 ) , Rad54 plus Rdh54 ( lane 2 ; N = 115 ) , Rad54 - K341R plus Rdh54 ( lane 3 ; N = 45 ) , Rad54 plus Rdh54 - K318R ( lane 4 ; N = 39 ) , or Rad54 - K341R plus Rdh54 - K318R ( lane 5 ; no translocation observed ) . Error bars ( s . d . ) were generated from three independent experiments .

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Translocation velocities for tailed duplex PSCs prepared with Rad54 only (lane 1; N = 86), Rad54 plus Rdh54 (lane 2; N = 144), Rad54-K341R plus Rdh54 (lane 3; N = 47), Rad54 plus Rdh54-K318R (lane 4; N = 112), or Rad54-K341R plus Rdh54-K318R (lane 5; N = 64). The error bars represent s.d.   Translocation distances for tailed duplex PSCs prepared with Rad54 only (lane 1; N = 86), Rad54 plus Rdh54 (lane 2; N = 144), Rad54-K341R plus Rdh54 (lane 3; N = 47), Rad54 plus Rdh54-K318R (lane 4; N = 112), or Rad54-K341R plus Rdh54-K318R (lane 5; N = 64). Error bars were generated by bootstrapping.   First passage recognition efficiency for tailed duplex PSCs prepared with Rad54 only (lane 1; N = 145), Rad54 plus Rdh54 (lane 2; N = 115), Rad54-K341R plus Rdh54 (lane 3; N = 45), Rad54 plus Rdh54-K318R (lane 4; N = 39), or Rad54-K341R plus Rdh54-K318R (lane 5; no translocation observed). Error bars (s.d.) were generated from three independent experiments.


PSC binding distributions following 10 min reactions for PSCs prepared with Rad54 plus Rdh54 ( N = 181 ) ; Rad54 plus Rdh54 - K318R ( N = 115 ) ; Rad54 - K341R plus Rdh54 ( N = 90 ) ; or Rad54 - K341R plus Rdh54 - K318R ( N = 54 ) , as indicated . Error bars were generated by bootstrapping . asterisks denote targeted region of homology on donor dsDNA molecules .

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PSC binding distributions following 10 min reactions for PSCs prepared with Rad54 plus Rdh54 (N= 181); Rad54 plus Rdh54-K318R (N= 115); Rad54-K341R plus Rdh54 (N = 90); or Rad54-K341R plus Rdh54-K318R (N = 54), as indicated. Error bars were generated by bootstrapping. The asterisks denote the targeted region of homology on the donor dsDNA molecules.


D - loop stimulation by Rad54 or Rdh54NRad54 in reactions with Rad51 ( 300 nM ) , duplex substrate ( 15 nM ) a pUC19 plasmid dsDNA ( 0 . 3 nM ) . of D - loop formation for Rad54 Rdh54NRad54 . Error bars represent s . d . of three independent experiments .

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D-loop stimulation by Rad54 or Rdh54NRad54 in reactions with Rad51 (300 nM), tailed duplex substrate (15 nM) and a pUC19 plasmid dsDNA (0.3 nM).   Quantification of D-loop formation for Rad54 and Rdh54NRad54. Error bars represent s.d. of three independent experiments.


D - loop stimulation by Rdh54 Rad54NRdh54 . of D - loop formation for Rad54 Rdh54NRad54 . Error bars represent s . d . of three independent experiments .

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D-loop stimulation by Rdh54 and Rad54NRdh54.   Quantification of D-loop formation for Rad54 and Rdh54NRad54. Error bars represent s.d. of three independent experiments.


ATPase stimulation for GFP - tagged Rad54 , Rdh54NRad54 , Rdh54 or Rad54NRdh54 in presence of Rad51 - ssDNA . Error bars represent s . d . of three independent experiments .

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ATPase stimulation for GFP-tagged Rad54, Rdh54NRad54, Rdh54 or Rad54NRdh54 in the presence of Rad51-ssDNA. Error bars represent s.d. of three independent experiments.


of Rad51 Atto565 - duplex PSCs bound per dsDNA molecule in dsDNA curtain homology search assays with Rad54 ( N = 117 ) , Rdh54NRad54 ( N = 150 ) or Rad54NRdh54 ( N = 175 ) . error bars represent s . d . for three independent experiments .

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Quantification of Rad51 Atto565-tailed duplex PSCs bound per dsDNA molecule in dsDNA curtain homology search assays with Rad54 (N = 117), Rdh54NRad54 (N = 150) or Rad54NRdh54 (N=175). The error bars represent the s.d. for three independent experiments.


Fraction translocating Rad51 Atto565 - duplex PSCs in reactions with Rad54 ( N = 117 ) , Rdh54NRad54 ( N = 150 ) , Rad54NRdh54 ( N = 0 ) Rdh54 ( N = 0 ) .

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Fraction translocating Rad51 Atto565-tailed duplex PSCs in reactions with Rad54 (N = 117), Rdh54NRad54 (N = 150), Rad54NRdh54 (N = 0) and Rdh54 (N = 0).


Translocation velocities of Rad51 Atto565 - duplex PSCs in reactions with Rad54 ( N = 86 ) Rdh54NRad54 ( N = 18 ) . error bars represent s . d . of data .

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Translocation velocities of Rad51 Atto565-tailed duplex PSCs in reactions with Rad54 (N = 86) and Rdh54NRad54 (N = 18). The error bars represent the s.d. of the data.


Yeast spot growth assays to monitor ability of Rad54 , Rdh54 , Rad54NRdh54 , Rdh54NRad54 to complement MMS sensitivity in rad54 ∆ yeast strains . Yeast spot growth assays to monitor ability of Rad54 , Rdh54 , Rad54NRdh54 , Rdh54NRad54 to complement MMS sensitivity in rdh54 ∆ yeast strains .

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Yeast spot growth assays to monitor the ability of Rad54, Rdh54, Rad54NRdh54, and Rdh54NRad54 to complement MMS sensitivity in rad54∆ yeast strains.   Yeast spot growth assays to monitor the ability of Rad54, Rdh54, Rad54NRdh54, and Rdh54NRad54 to complement MMS sensitivity in rdh54∆ yeast strains.


RIGER to identify genes that are preferentially essential in FUS - DDIT3 - expressing SCP - 1 . EV - transduced SCP - 1 20 FUS - DDIT3 - negative cancer lines screened with same shRNA library were used as reference set . Genes were ranked according to relative shRNA depletion , YAP1 was identified as top FUS ‑ DDIT3 ‑ specific essential gene . NES , normalized enrichment score .

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RIGER analysis to identify genes that are preferentially essential in FUS-DDIT3-expressing SCP-1 cells. EV-transduced SCP-1 cells and 20 FUS-DDIT3-negative cancer cell lines screened with the same shRNA library were used as reference set. Genes were ranked according to relative shRNA depletion, and YAP1 was identified as top FUS‑DDIT3‑specific essential gene. NES, normalized enrichment score.


Competition assays with SCP - 1 transduced with RFP - labeled NTC or YAP1 shRNAs . Flow cytometric of RFP - positive on day 9 relative to day 3 showed that YAP1 knockdown was preferentially toxic to FUS - DDIT3 ‑ expressing cultures .

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Competition assays with SCP-1 cells transduced with RFP-labeled NTC or YAP1 shRNAs. Flow cytometric quantification of RFP-positive cells on day 9 relative to day 3 showed that YAP1 knockdown was preferentially toxic to FUS-DDIT3‑expressing cultures.


Expression of YAP1 in SCP ‑ 1 transduced with FUS ‑ DDIT3 or EV liposarcoma lines . One of at least two independent experiments with similar results is shown . FUS ‑ DDIT3 ‑ expressing types are indicated in red .

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Expression of YAP1 in SCP‑1 cells transduced with FUS‑DDIT3 or EV and liposarcoma cell lines. One of at least two independent experiments with similar results is shown. FUS‑DDIT3‑expressing cell types are indicated in red.


Expression of YAP1 in ( yellow ) ( blue ) fractions from SCP - 1 transduced with FUS - DDIT3 or EV liposarcoma lines . One of at least two independent experiments with similar results is shown . FUS ‑ DDIT3 ‑ expressing types are indicated in red .

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Expression of YAP1 in cytoplasmic (yellow) and nuclear (blue) fractions from SCP-1 cells transduced with FUS-DDIT3 or EV and liposarcoma cell lines. One of at least two independent experiments with similar results is shown. FUS‑DDIT3‑expressing cell types are indicated in red.


Expression of FOXM1 PLK1 in MLS lines . One of at least two independent experiments with similar results is shown .

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Expression of FOXM1 and PLK1 in MLS cell lines. One of at least two independent experiments with similar results is shown.


Strong expression of YAP1 , FOXM1 , PLK1 in MLS samples ( original magnification , x10 [ inset , x20 ] ) .

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Strong nuclear expression of YAP1, FOXM1, and PLK1 in MLS patient samples (original magnification, x10 [inset, x20]).


of YAP1 expression in liposarcoma samples . Immunoreactivity was assessed using a semi ‑ quantitative score ( 0 , negative ; 1 , weak ; 2 , moderate ; 3 , strong ) defining in positive control ( hepatocellular carcinoma ) as strong . Only tumors with at least moderate ( semi ‑ quantitative score ≥ 2 ) ≥ 30 % YAP positive were considered positive for purposes of study .

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Intensity of nuclear YAP1 expression in liposarcoma patient samples. Immunoreactivity was assessed using a semi‑quantitative score (0, negative; 1, weak; 2, moderate; and 3, strong) defining the staining intensity in the positive control (hepatocellular carcinoma) as strong. Only tumors with at least moderate staining (semi‑quantitative score ≥2) and ≥30% YAP positive cells were considered positive for the purposes of the study.


Proportion of with YAP1 expression in liposarcoma samples . Boxes represent mean values lower upper quartiles . Whiskers represent minimum maximum values .

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Proportion of cells with nuclear YAP1 expression in liposarcoma patient samples. Boxes represent mean values and lower and upper quartiles. Whiskers represent minimum and maximum values.


Competition assays with liposarcoma lines transduced with RFP - labeled NTC or YAP1 shRNAs . Flow cytometric of RFP - positive on day 17 relative to day 3 showed that YAP1 knockdown was preferentially toxic to MLS . Error bars represent mean ± SD of two independent experiments .

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Competition assays with liposarcoma cell lines transduced with RFP-labeled NTC or YAP1 shRNAs. Flow cytometric quantification of RFP-positive cells on day 17 relative to day 3 showed that YAP1 knockdown was preferentially toxic to MLS cells. Error bars represent the mean ± SD of two independent experiments.


Aggregate data from competition assays shown in ( A ) . Statistical significance was assessed using an unpaired t - . LS , non - myxoid liposarcoma .

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Aggregate data from competition assays shown in (A). Statistical significance was assessed using an unpaired t-test. LS, non-myxoid liposarcoma.


Competition assays with MLS 1765 - 92 transduced with an RFP ‑ labeled NTC shRNA or an RFP - labeled shRNA against YAP1 3 ' UTR following transduction with EV or YAP1 coding . Flow cytometric of RFP ‑ positive on day 17 relative to day 3 showed that viability was rescued by expression of shRNA ‑ resistant YAP1 cDNA . Error bars represent mean ± SD of two independent experiments .

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Competition assays with MLS 1765-92 cells transduced with an RFP‑labeled NTC shRNA or an RFP-labeled shRNA against the YAP1 3' UTR following transduction with EV or the YAP1 coding sequence. Flow cytometric quantification of RFP‑positive cells on day 17 relative to day 3 showed that cell viability was rescued by expression of the shRNA‑resistant YAP1 cDNA. Error bars represent the mean ± SD of two independent experiments.


Expression of total exogenous V5 ‑ tagged YAP1 in MLS 1765 ‑ 92 shown in ( C ) . One of at least two independent experiments with similar results is shown .

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Expression of total and exogenous V5‑tagged YAP1 in MLS 1765‑92 cells shown in (C). One of at least two independent experiments with similar results is shown.


viability expression of FOXM1 PLK1 in MLS lines following siRNA - mediated YAP1 knockdown . Error bars represent mean ± SD of three independent experiments , unpaired t - . blots represent one of at least three independent experiments with similar results .

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Cell viability and expression of FOXM1 and PLK1 in MLS cell lines following siRNA-mediated YAP1 knockdown. Error bars represent the mean ± SD of three independent experiments, unpaired t-test. The blots represent one of at least three independent experiments with similar results.


Flow cytometric cycle of MLS lines following shRNA ‑ mediated YAP1 knockdown . Error bars represent mean ± SD of three independent experiments , two - way ANOVA ; ns , not significant .

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Flow cytometric cell cycle analysis of MLS cell lines following shRNA‑mediated YAP1 knockdown. Error bars represent the mean ± SD of three independent experiments, two-way ANOVA; ns, not significant.


Senescence ‑ associated β ‑ galactosidase ( SABG ) in MLS lines following shRNA - mediated YAP1 knockdown . Error bars represent mean ± SD of ten random microscopic fields , two - way ANOVA ) .

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Senescence‑associated β‑galactosidase (SABG) staining intensity in MLS cell lines following shRNA-mediated YAP1 knockdown. Error bars represent the mean ± SD of ten random microscopic fields, two-way ANOVA).


Expression of CDKN1A , CDKN2A , total phosphorylated RB1 , TP53 in MLS lines following shRNA ‑ mediated YAP1 knockdown . One of at least two independent experiments with similar results is shown .

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Expression of CDKN1A, CDKN2A, total and phosphorylated RB1, and TP53 in MLS cell lines following shRNA‑mediated YAP1 knockdown. One of at least two independent experiments with similar results is shown.


Expression of PARP caspase 3 / 8 in MLS lines following shRNA - mediated YAP1 knockdown . One of at least two independent experiments with similar results is shown .

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Expression of cleaved PARP and cleaved caspase 3/8 in MLS cell lines following shRNA-mediated YAP1 knockdown. One of at least two independent experiments with similar results is shown.


Expression of YAP1 downstream effectors in SCP - 1 transduced with FUS ‑ DDIT3 or EV . One of at least three independent experiments with similar results is shown .

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Expression of YAP1 and downstream effectors in SCP-1 cells transduced with FUS‑DDIT3 or EV. One of at least three independent experiments with similar results is shown.


Expression of YAP1 downstream effectors in ( yellow ) ( blue ) fractions from SCP - 1 transduced with FUS ‑ DDIT3 or EV . One of at least two independent experiments with similar results is shown .

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Expression of YAP1 and downstream effectors in cytoplasmic (yellow) and nuclear (blue) fractions from SCP-1 cells transduced with FUS‑DDIT3 or EV. One of at least two independent experiments with similar results is shown.


YAP1 - responsive luciferase activity in SCP - 1 transduced with FUS ‑ DDIT3 . Relative luciferase activity is displayed as fold change relative to control . Error bars represent mean ± SD of three independent experiments , unpaired t - .

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YAP1-responsive luciferase activity in SCP-1 cells transduced with FUS‑DDIT3. Relative luciferase activity is displayed as fold change relative to control. Error bars represent the mean ± SD of three independent experiments, unpaired t-test.


Localization of FUS - DDIT3 YAP1 in MLS 1765 - 92 . Nuclei were counterstained with DAPI . original magnification was x63 , were zoomed in four times for co - localization . correlation between red green was determined by Pearson coefficient ( square , for signal acquisition ) .

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Localization of FUS-DDIT3 and YAP1 in MLS 1765-92. Nuclei were counterstained with DAPI. The original magnification was x63, and images were zoomed in four times for co-localization analysis. The correlation between red and green fluorescence was determined by Pearson coefficient analysis (square, area for signal acquisition).


Co ‑ IP of transiently expressed FUS ‑ DDIT3 YAP1 from HEK293T . V5 - tagged YAP1 was pulled down using an anti - V5 antibody , interacting were detected by immunoblotting . One of at least two independent experiments with similar results is shown .

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Co‑IP of transiently expressed FUS‑DDIT3 and YAP1 from HEK293T cells. V5-tagged YAP1 was pulled down using an anti-V5 antibody, and interacting proteins were detected by immunoblotting. One of at least two independent experiments with similar results is shown.


Co - IP of endogenous FUS - DDIT3 YAP1 from MLS 1765 - 92 . FUS - DDIT3 was pulled down using an anti - DDIT3 antibody , interacting were detected by immunoblotting . One of at least two independent experiments with similar results is shown .

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Co-IP of endogenous FUS-DDIT3 and YAP1 from MLS 1765-92 cells. FUS-DDIT3 was pulled down using an anti-DDIT3 antibody, and interacting proteins were detected by immunoblotting. One of at least two independent experiments with similar results is shown.


Viability proliferation of MLS 402 ‑ 91 , MLS 1765 ‑ 92 , MLS 2645 - 94 cultured in presence of verteporfin . Error bars represent mean ± of one representative experiment performed in quintuplicate .

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Viability and proliferation of MLS 402‑91, MLS 1765‑92, and MLS 2645-94 cells cultured in the presence of verteporfin. Error bars represent the mean ± SEM of one representative experiment performed in quintuplicate.


Flow cytometric of apoptosis ( PARP ) mitotic fraction ( phosphorylated histone H3S10 ) in MLS lines cultured in presence of 0 . 25 µM Verteporfin . One of two independent experiments with similar results is shown .

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Flow cytometric analysis of apoptosis (cleaved PARP) and mitotic fraction (phosphorylated histone H3S10) in MLS cell lines cultured in the presence of 0.25 µM Verteporfin. One of two independent experiments with similar results is shown.


Expression of total YAP1 downstream effectors ( FOXM1 PLK1 ) in MLS lines treated with 0 . 5 or 1 µM verteporfin for 15 hours . One of at least three independent experiments with similar results is shown .

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Expression of total YAP1 and downstream effectors (FOXM1 and PLK1) in MLS cell lines treated with 0.5 or 1 µM verteporfin for 15 hours. One of at least three independent experiments with similar results is shown.


YAP1 - responsive luciferase activity in MLS lines transfected with a constitutively active YAP1S127A mutant treated with 1 µM verteporfin . Relative luciferase activity is displayed relative to control . Error bars represent mean ± SD of three independent experiments , unpaired t - .

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YAP1-responsive luciferase activity in MLS cell lines transfected with a constitutively active YAP1S127A mutant and treated with 1 µM verteporfin. Relative luciferase activity is displayed relative to control. Error bars represent the mean ± SD of three independent experiments, unpaired t-test.


formation on chicken CAM of MLS lines following shRNA - mediated YAP1 knockdown . number of tumors is given above each bar , Fisher exact ; ns , not significant .

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Tumor formation on chicken CAM of MLS cell lines following shRNA-mediated YAP1 knockdown. The number of tumors is given above each bar, Fisher exact test; ns, not significant.


growth on chicken CAM of MLS lines following treatment with 1 µM verteporfin . Shown are volumes representative of tumors .

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Tumor growth on chicken CAM of MLS cell lines following treatment with 1 µM verteporfin. Shown are tumor volumes and representative photographs of tumors.


C . cycle fractions inferred from Cycleflow in IFN - β - treated NSCs . n = 3 biological replicates .

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C. Cell cycle fractions inferred from Cycleflow in IFN-β-treated NSCs. n = 3 biological replicates.


C , D . Ribo - Seq results depicting translation efficiency as interaction of log2 fold changes ( LFC ) between footprints mapped to CDS , referred to as " CDS " , total after 2h or 14h IFN - β treatment . FDR < 10 % , LR - in DESeq2 . Genes with a p value < 0 . 1 after FDR correction are highlighted . n = 4 biological replicates .

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C,D. Ribo-Seq results depicting translation efficiency as the interaction of log2 fold changes (LFC) between footprints mapped to the CDS, referred to as "CDS counts", and total RNA after 2h or 14h IFN-β treatment. FDR < 10%, LR-Test in DESeq2. Genes with a p value<0.1 after FDR correction are highlighted. n = 4 biological replicates.


I . Representative WB phosphorylation ( log2FC ) of mTOR - related ribosomal S6 kinase ( S6K ) , ribosomal S6 ( S6 ) eIF4E - binding 1 ( 4E - BP1 ) from NSCs treated with IFN - β normalized to control ( t = 0h ) . Bars represent mean value . One - way ANOVA for biphasic response p - 4E - BP1S65 * ( p = 0 . 021 ) , p - S6S235 / 236 & p - S6S240 / 244 * * * ( p < 0 . 001 ) , p - S6KT389 * * ( p = 0 . 004 ) . n = biological replicates specified . p ≡ p - value .

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I. Representative WB and phosphorylation levels quantification (log2FC) of the mTOR-related proteins ribosomal protein S6 kinase (S6K), ribosomal protein S6 (S6) and eIF4E-binding protein 1 (4E-BP1) from NSCs treated with IFN-β and normalized to control (t=0h). Bars represent the mean value. One-way ANOVA for the biphasic response test p-4E-BP1S65 * (p=0.021), p-S6S235/236 & p-S6S240/244 ***(p<0.001), p-S6KT389 **(p=0.004). n = biological replicates specified. p ≡ p-value.


A . Representative WB image ( log2FC ) of p - TSC2Thr1462 & p - AktThr308 from IFNβ - treated NSCs normalized to control ( t = 0h ) . Bars represent mean value . One - way ANOVA for biphasic response p - TSC2T1462 * * * ( p < 0 . 001 ) , p - AktT308 * * ( p = 0 . 003 ) . n = 4 biological replicates . Data information : p ≡ p - value .

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A. Representative WB image and quantification (log2FC) of p-TSC2Thr1462 & p-AktThr308 from IFNβ-treated NSCs normalized to control (t=0h). Bars represent the mean value. One-way ANOVA for the biphasic response test p-TSC2T1462 ***(p<0.001), p-AktT308 **(p=0.003). n = 4 biological replicates. Data information: p ≡ p-value.


C . Representative WB quantifications ( log2FC ) of p - eIF2αS51 in IFN - β - treated NSCs normalized to control ( t = 0h ) . Bars represent mean value . Spearman ' s rank correlation . p - eIF2αS51 * * ( p = 0 . 003724 ) . n = 4 biological replicates . Data information : p ≡ p - value .

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C. Representative WB images and quantifications (log2FC) of p-eIF2αS51 in IFN-β-treated NSCs normalized to control (t=0h). Bars represent the mean value. Spearman's rank correlation test. p-eIF2αS51 **(p=0.003724). n = 4 biological replicates. Data information: p ≡ p-value.


E . Representative WB image quantifications ( log2FC ) of p - Rb1Ser780 from IFN - β - treated NSCs normalized to control ( t = 0h ) . Bars represent mean value . Spearman ' s rank correlation . p - Rb1S780 * * * ( p = 5 . 37e - 06 ) . n = 3 biological replicates . Data information : p ≡ p - value .

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E. Representative WB image and quantifications (log2FC) of p-Rb1Ser780 from IFN-β-treated NSCs normalized to control (t=0h). Bars represent the mean value. Spearman's rank correlation test. p-Rb1S780 ***(p=5.37e-06). n = 3 biological replicates. Data information: p ≡ p-value.


I , J . Luciferase activity controlled by upstream 5 ' UTR fragment from Sox2 ( WT mutant ) , Rps21 ( WT mutant ) Actb . Treatments were applied for 12h . Data is normalized to vehicle is represented as mean ± SD . n = 6 biological replicates . Two - way ANOVA with Tukey ' s multiple comparisons ( p - value specified ) . Data information : p ≡ p - value .

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I,J. Luciferase activity controlled by the upstream 5'UTR fragment from Sox2 (WT and mutant), Rps21 (WT and mutant) and Actb. Treatments were applied for 12h. Data is normalized to vehicle and is represented as mean ± SD. n = 6 biological replicates. Two-way ANOVA with Tukey's multiple comparisons test (p-value specified). Data information: p ≡ p-value.


B . vSVZ niche FACS quantifications of total NSCs ( Total NSCs ) & immunofluorescence of aNSCs ( Fraction of aNSCs ) in WT or IFNAGR KO mice at 2 , 7 22 months old ( MO ) . Data from mice is plotted . Inferred dynamics are depicted as a blue ( WT ) or red ( IFNAGR KO ) over time . Sample sizes : WT total at 60 days : 8 mice , WT total at 210 days : 3 mice , WT total at 660 days : 3 mice , WT fraction aNSCs at 60 days : 3 mice , WT fraction aNSCs at 660 days : 4 mice , IFNAGRKO total at 60 days : 8 mice , IFNAGRKO total at 180 days : 3 mice , IFNAGRKO total at 660 days : 3 mice , IFNAGRKO fraction aNSCs at 60 days : 3 mice , IFNAGRKO fraction aNSCs at 660 days : 3 mice .

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B. vSVZ niche FACS quantifications of total NSCs (Total NSCs) & immunofluorescence quantification of aNSCs (Fraction of aNSCs) in WT or IFNAGR KO mice at 2, 7 and 22 months old (MO). Data from individual mice is plotted. Inferred dynamics are depicted as a blue (WT) or red (IFNAGR KO) line over time. Sample sizes: WT total at 60 days: 8 mice, WT total at 210 days: 3 mice, WT total at 660 days: 3 mice, WT fraction aNSCs at 60 days: 3 mice, WT fraction aNSCs at 660 days: 4 mice, IFNAGRKO total at 60 days: 8 mice, IFNAGRKO total at 180 days: 3 mice, IFNAGRKO total at 660 days: 3 mice, IFNAGRKO fraction aNSCs at 60 days: 3 mice, IFNAGRKO fraction aNSCs at 660 days: 3 mice.


a , Impairment of two ATG - conjugation systems ( Atg12 LC3 ) in Atg7 - deficient brain . Brain homogenates from P28 mice were immunoblotted with antibodies against Atg7 , Atg5 LC3 . Actin was used as a loading control . Data shown are representative of three separate experiments .

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a, Impairment of two ATG-conjugation systems (Atg12 and LC3) in the Atg7-deficient brain. Brain homogenates from P28 mice were immunoblotted with antibodies against Atg7, Atg5 and LC3. Actin was used as a loading control. Data shown are representative of three separate experiments.


b , Kaplan - Meier survival curves of Atg7flox / + ; nestin - Cre ( n = 41 ) Atg7flox / flox ; nestin - Cre ( n = 26 ) mice over 28 .

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b, Kaplan-Meier survival curves of Atg7flox/+; nestin-Cre (n = 41) and Atg7flox/flox; nestin-Cre (n = 26) mice over 28 weeks.


c , Abnormal limb - clasping reflexes in Atg7flox / flox ; nestin - Cre mice at P28 . When lifted by tail , Atg7flox / + ; nestin - Cre mice behave normally , extending their hind limbs bodies . In contrast , Atg7flox / flox ; nestin - Cre mice bend their legs towards their trunk or tighten their back limbs to their bodies anterior limbs .

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c, Abnormal limb-clasping reflexes in Atg7flox/flox; nestin-Cre mice at P28. When lifted by the tail, Atg7flox/+; nestin-Cre mice behave normally, extending their hind limbs and bodies. In contrast, Atg7flox/flox; nestin-Cre mice bend their legs towards their trunk or tighten their back limbs to their bodies and anterior limbs.


d , Movement ataxia in Atg7flox / flox ; nestin - Cre mice at P28 . Motor coordination was tested using a rotarod . Atg7flox / + ; nestin - Cre ( n = 5 ) Atg7flox / flox ; nestin - Cre ( n = 5 ) mice were placed on a rod rotating at 20 r . p . m . , time spent on rod was counted . Data show mean ± s . d . * , P 0 . 01 ( Students t - ) . There was no significant sex difference in survival rate onset - stage of abnormal mice - clasping tremor in miceflox / flox ; nestin - Cre mice .

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d, Movement ataxia in Atg7flox/flox; nestin-Cre mice at P28. Motor coordination was tested using a rotarod assay. Atg7flox/+; nestin-Cre (n = 5) and Atg7flox/flox; nestin-Cre (n = 5) mice were placed on a rod rotating at 20 r.p.m., and the time spent on the rod was counted. Data show mean ± s.d. *, P 0.01 (Students t-test). There was no significant sex difference in survival rate and onset-stage of abnormal mice-clasping and tremor in miceflox/flox; nestin-Cre mice.


a - f , Histological analyses of Atg7flox / + ; nestin - Cre ( left ) Atg7flox / flox ; nestin - Cre ( right ) cerebral cortex at P56 . Cryosections were stained with HE ( a - d ) or immunostained for glial marker GFAP ( e , f ) . Boxed areas in a b are magnified in c d , respectively . Arrows in c point to large pyramidal neurons in cerebral cortex .

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a-f, Histological analyses of Atg7flox/+; nestin-Cre (left) and Atg7flox/flox; nestin-Cre (right) cerebral cortex at P56. Cryosections were stained with HE (a-d) or immunostained for the glial marker GFAP (e, f). Boxed areas in a and b are magnified in c and d, respectively. Arrows in c point to large pyramidal neurons in the cerebral cortex.


g - j , Histological of Atg7flox / + ; nestin - Cre ( left ) Atg7flox / flox ; nestin - Cre ( right ) cerebellum at P56 . Cryosections were stained with HE ( g , h ) or immunostained for Purkinje marker calbindin ( i , j ) . Arrows in g h indicate cerebellar Purkinje .

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g-j, Histological analysis of Atg7flox/+; nestin-Cre (left) and Atg7flox/flox; nestin-Cre (right) cerebellum at P56. Cryosections were stained with HE (g, h) or immunostained for the Purkinje marker calbindin (i, j). Arrows in g and h indicate cerebellar Purkinje cells.


k - p , TUNEL of cerebral cortex ( k , l ) cerebellum ( n , o ) at P56 in Atg7flox / + ; nestin - Cre ( k , n ) Atg7flox / flox ; nestin - Cre ( l , o ) sections . TUNEL - positive are indicated with arrows shown as higher magnification beneath panels l , n o . Histograms show average number ( ± s . d . ) of TUNEL - positive in ten sections for three of each genotype ( m , p ) . * , P 0 . 05 ( t - ) . Scale bars , 1 mm ( a , b ) , 100 µm ( c - j ) , 250 µm ( k , l , n , o ) . We observed no sex difference in brain or neuronal loss in Atg7flox / flox ; nestin - Cre mice .

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k-p, TUNEL staining of the cerebral cortex (k, l) and cerebellum (n, o) at P56 in Atg7flox/+; nestin-Cre (k, n) and Atg7flox/flox; nestin-Cre (l, o) sections. TUNEL-positive cells are indicated with arrows and shown as higher magnification images beneath panels l, n and o. Histograms show the average number (± s.d.) of TUNEL-positive cells in ten sections for three animals of each genotype (m, p). *, P 0.05 (t-test). Scale bars, 1 mm (a, b), 100 µm (c-j), 250 µm (k, l, n, o). We observed no sex difference in brain morphology or neuronal loss in Atg7flox/flox; nestin-Cre mice.


a - l , presence of ubiquitin - positive dots was examined by immunohistochemistry in several regions including cerebral cortex ( a , b ) , cerebellum ( c , d ) , hippocampus ( e , f ) , hypothalamus ( g , h ) , amygdala ( i , j ) pontine nuclei ( k , l ) of Atg7flox / + ; nestin - Cre Atg7flox / flox ; nestin - Cre mice . Note presence of numerous ubiquitin dots in amygdala hypothalamus of representative mutants . Scale bars , 50 µm .

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a-l, The presence of ubiquitin-positive dots was examined by immunohistochemistry in several regions including cerebral cortex (a, b), cerebellum (c, d), hippocampus (e, f), hypothalamus (g, h), amygdala (i, j) and pontine nuclei (k, l) of Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre mice. Note the presence of numerous ubiquitin dots in the amygdala and hypothalamus of the representative mutants. Scale bars, 50 µm.


m , n , Electron of brain of Atg7flox / flox ; nestin - Cre mice . Inclusion bodies ( arrows ) were often observed in Atg7flox / flox ; nestin - Cre hypothalamus . boxed region in m is shown in n . Inclusion bodies were not detected in Atg7flox / + ; nestin - Cre brain ( data not shown ) . Scale bars , 5 µm ( m ) , 1 µm ( n ) . o , Immunoelectron micrograph of ubiquitin in a representative Atg7flox / flox ; nestin - Cre hypothalamus . N , nucleus . Scale bar , 1 µm .

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m, n, Electron micrographs of the brain of Atg7flox/flox; nestin-Cre mice. Inclusion bodies (arrows) were often observed in Atg7flox/flox; nestin-Cre hypothalamus. The boxed region in m is shown in n. Inclusion bodies were not detected in Atg7flox/+; nestin-Cre brain (data not shown). Scale bars, 5 µm (m), 1 µm (n). o, Immunoelectron micrograph of ubiquitin in a representative Atg7flox/flox; nestin-Cre hypothalamus. N, nucleus. Scale bar, 1 µm.


p , q , Immunohistochemical detection of ubiquitin - positive inclusions in cerebral cortex ( p ) hypothalamus ( q ) at P9 , P18 P56 . Brain sections of each genotype at indicated ages were immunostained with an anti - ubiquitin antibody . Ubiquitin - positive inclusions appeared at P18 became larger with ageing in brain of Atg7flox / flox ; nestin - Cre mice . Scale bars , 50 µm .

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

p, q, Immunohistochemical detection of ubiquitin-positive inclusions in the cerebral cortex (p) and hypothalamus (q) at P9, P18 and P56. Brain sections of each genotype at the indicated ages were immunostained with an anti-ubiquitin antibody. Ubiquitin-positive inclusions appeared at P18 and became larger with ageing in the brain of Atg7flox/flox; nestin-Cre mice. Scale bars, 50 µm.


a , Increase in ubiquitinated in Atg7flox / flox ; nestin - Cre mouse brain over time . Homogenates of P9 , P18 P56 brains from Atg7flox / + ; nestin - Cre Atg7flox / flox ; nestin - Cre mice were immunoblotted with an anti - ubiquitin antibody . An anti - actin antibody was used as a loading control . Data shown are representative of three separate experiments . We observed no sex difference in accumulation of ubiquitin in Atg7flox / flox ; nestin - Cre mice .

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

a, Increase in ubiquitinated proteins in Atg7flox/flox; nestin-Cre mouse brain over time. Homogenates of P9, P18 and P56 brains from Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre mice were immunoblotted with an anti-ubiquitin antibody. An anti-actin antibody was used as a loading control. Data shown are representative of three separate experiments. We observed no sex difference in the accumulation of ubiquitin in Atg7flox/flox; nestin-Cre mice.


b , Peptide hydrolysis activity of 20S 26Sproteasomes . Homogenates from P28Atg7flox / + ; nestin - Cre ( blue ) Atg7flox / flox ; nestin - Cre ( pink ) brains were fractionated by glyceroldensity gradient centrifugation ( 10 - 40 % glycerol from fraction 1 to fraction 30 ) . Aliquots from each fraction were used for of chymotryptic activity of proteasomes using Suc - LLVY - AMC as a substrate in absence ( top ) or presence ( bottom ) of 0 . 05 % SDS . sedimenting positions of 20S 26Sproteasomes are indicated with arrowheads . Note that whereas 26Sproteasomes exist in active forms in tissues , 20Sproteasomes are latent are activated artificially by a low of SDS .

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

b, Peptide hydrolysis activity of 20S and 26Sproteasomes. Homogenates from P28Atg7flox/+; nestin-Cre (blue) and Atg7flox/flox; nestin-Cre (pink) brains were fractionated by glyceroldensity gradient centrifugation (10-40% glycerol from fraction 1 to fraction 30). Aliquots from each fraction were used for the assay of chymotryptic activity of proteasomes using Suc-LLVY-AMC as a substrate in the absence (top) or presence (bottom) of 0.05% SDS. The sedimenting positions of 20S and 26Sproteasomes are indicated with arrowheads. Note that whereas 26Sproteasomes exist in active forms in tissues, 20Sproteasomes are latent and are activated artificially by a low concentration of SDS.