( 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 ) .

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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 the drinking water for 1 week , either or not pretreated with antibiotics ( AB ) for three weeks . ( 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 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 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 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

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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 ( 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

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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 levels of Defa6 and Lyz2 , two Paneth cell markers , and Ripk3 , a TNF - induced cell - death gene , in ileum of the 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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 + / + 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 .

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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 ) and 6h after challenge , ileum was taken and RT - qPCR performed to study gene expression of cell death ISRE genes Mlkl and

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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 ) and 6h after challenge , ileum was taken and RT - qPCR performed to study gene expression of cell death ISRE genes Stat1 .

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 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 ) .

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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 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 ) .

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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 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 ) .

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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 , and 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 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

(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 and fatty acids metabolism in the gastrocnemius muscle from the indicated genotypes ( n = 5 mice per group ) . * p < 0 . 05 .

(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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 the indicated mice stained for SDH activity ( Top ) as well as MHC1 immunofluorescence ( IF ) ( Bottom ) , Representative images were shown . Scale bar : 500 μm .

(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 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 .

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(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 ) 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 ) .

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

(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 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 ) .

(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 analysis of Fnip1 and Fnip2mRNA levels in the gastrocnemius muscle of the indicated genotypes ( n = 5 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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O 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) (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 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 .

(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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (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 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 ) .

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

(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 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 ) .

(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 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 ) .

(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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 ) .

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

(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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (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 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 ) .

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

(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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ratio ( RER ) during a graded exercise regimen on a motorized treadmill . mdx , n = 8 ; mdx / 499Tg , n = 6 . ‡ 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

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

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

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


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

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

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


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 ) .

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

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 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 .

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

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.


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

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

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


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 ) .

O O O O O O O O O O O O O O O O O O O O O O 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 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).


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

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

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


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

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

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


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 ) .

O O O O O O O O O O O O O O O O O O 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 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).


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 ) .

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).


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

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


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 .

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

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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

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

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 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 .

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

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

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


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 .

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

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 . The asterisks denote the targeted region of homology on the donor dsDNA molecules .

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

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 ) , 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 .

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

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 the 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.


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 .

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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 - tailed duplex PSCs in reactions with Rad54 ( N = 117 ) , Rdh54NRad54 ( N = 150 ) , Rad54NRdh54 ( N = 0 ) and 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 - tailed duplex PSCs in reactions with Rad54 ( N = 86 ) and Rdh54NRad54 ( N = 18 ) . The error bars represent the s . d . of the data .

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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 and PLK1 in MLS cell 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 nuclear expression of YAP1 , FOXM1 , and PLK1 in MLS patient 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]).


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 .

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

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 - test . LS , non - myxoid liposarcoma .

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 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 .

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 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 .

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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.


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 .

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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 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 .

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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 ) 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 ) .

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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 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 .

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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 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 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 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 .

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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 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 .

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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 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 .

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 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 ) .

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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 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 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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.


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 .

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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.


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

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) and Atg7flox / flox ; nestin - Cre ( n = 26 ) mice over 28 weeks .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 .

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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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 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 .

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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 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 .

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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 , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 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 .

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

m, 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 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 .

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 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 .

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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 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 .

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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.


c , ATP - dependent degradation of [ 35S ] - labelled ODC . Degradation of [ 35S ] - labelled ODC was assayed using crude extracts from P28 Atg7flox / + ; nestin - Cre and Atg7flox / flox ; nestin - Cre brains . The experiment was repeated three times , and values represent mean ± s . d . In the above assays , there were no significant differences between Atg7flox / + ; nestin - Cre and Atg7flox / flox ; nestin - Cre mice .

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c, ATP-dependent degradation of [35S]-labelled ODC. Degradation of [35S]-labelled ODC was assayed using crude extracts from P28 Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre brains. The experiment was repeated three times, and values represent mean ± s.d. In the above assays, there were no significant differences between Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre mice.


d , Immunoblot analysis of 26S proteasome components . Homogenates from P28 Atg7flox / flox , Atg7flox / + ; nestin - Cre and Atg7flox / flox ; nestin - Cre brains were immunoblotted with antibodies against the indicated proteins . Data shown are representative of three separate experiments . There was no change in proteasome status for the different 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 O O O O O O O O

d, Immunoblot analysis of 26S proteasome components. Homogenates from P28 Atg7flox/flox, Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre brains were immunoblotted with antibodies against the indicated proteins. Data shown are representative of three separate experiments. There was no change in proteasome status for the different genotypes.


A - C . Immunodetection of BES1 - CFP from heat - treated plants . Plants pre - grown at 21 ° C were ( A ) exposed for 60 minutes to the indicated temperatures , ( B ) to 45 ° C for the indicated period of time , or ( C ) to 45 ° C and , following sampling after 30 minutes ( lane 2 ) and 60 minutes ( lane 3 ) , returned to 21 ° C for 3 hours ( lane 4 ) . CBB , Coomassie Brilliant Blue stained gel .

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A-C. Immunodetection of BES1-CFP from heat-treated plants. Plants pre-grown at 21°C were (A) exposed for 60 minutes to the indicated temperatures, (B) to 45°C for the indicated period of time, or (C) to 45°C and, following sampling after 30 minutes (lane 2) and 60 minutes (lane 3), returned to 21°C for 3 hours (lane 4). CBB, Coomassie Brilliant Blue stained gel.


D . Subcellular localization of BES1 - CFP in roots of 7 - day - old plants after heat shock . 35S : BES1 - CFP seedlings were grown at 21 ° C and either left untreated or exposed for 60 minutes to 45 ° C . Left : representative photos . Right : quantification of the nuclear / cytoplasmic signal . Data show the mean ± SD . n = 25 . The asterisks indicate significant differences to the untreated plants by Student ' s t - test ( * * * P ≤ 0 . 001 ) .

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D. Subcellular localization of BES1-CFP in roots of 7-day-old plants after heat shock. 35S:BES1-CFP seedlings were grown at 21°C and either left untreated or exposed for 60 minutes to 45°C. Left: representative photos. Right: quantification of the nuclear/cytoplasmic signal. Data show the mean ±SD. n=25. The asterisks indicate significant differences to the untreated plants by Student's t-test (***P≤0.001).


F . Heat stress resistance of bes1 - 1 , bes1 - 2 and bes1 - D as compared to wild - type . For basal resistance assays ( top charts ) plants were directly exposed to 45 ° C for the indicated time . For acquired resistance ( bottom charts ) , plants were primed with the respective temperatures for 120 minutes before transfer to 45 ° C for 150 minutes . Data show the mean ± SE . n = 7 . The asterisks indicate significant differences compared to wild - type by Student ' s t - test ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ) .

F. Heat stress resistance of bes1-1, bes1-2 and bes1-D as compared to wild-type. For basal resistance assays (top charts) plants were directly exposed to 45°C for the indicated time. For acquired resistance (bottom charts), plants were primed with the respective temperatures for 120 minutes before transfer to 45°C for 150 minutes. Data show the mean ±SE. n=7. The asterisks indicate significant differences compared to wild-type by Student's t-test (*P≤0.05; **P≤0.01; ***P≤0.001).


B . Immunodetection of BES1 - CFP from heat - stressed plants , treated with epiBL or BRZ . 35S : BES1 - CFP plants were treated for 24 hours with DMSO ( control ) , 1 μM 24 - epiBL or 5 μM BRZ and then heat - stressed as follows : lane 1 : untreated control ; lane 2 : 45 ° C for 30 minutes ; lane 3 : 45 ° C for 60 minutes ; lane 4 : 45 ° C for 60 minutes and recovery at 21 ° C for 180 minutes .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

B. Immunodetection of BES1-CFP from heat-stressed plants, treated with epiBL or BRZ. 35S:BES1-CFP plants were treated for 24 hours with DMSO (control), 1 μM 24-epiBL or 5 μM BRZ and then heat-stressed as follows: lane 1: untreated control; lane 2: 45°C for 30 minutes; lane 3: 45°C for 60 minutes; lane 4: 45°C for 60 minutes and recovery at 21°C for 180 minutes.


C , D . Immunodetection of BES1 - CFP from heat - treated bri1 - 1 or det2 - 1 plants . Left : phenotypes of 10 - day - old plants of all lines as compared to wild - type Col - 0 ( wt ) . Right : Immunoblots from the lines grown and treated as in B .

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C, D. Immunodetection of BES1-CFP from heat-treated bri1-1 or det2-1 plants. Left: phenotypes of 10-day-old plants of all lines as compared to wild-type Col-0 (wt). Right: Immunoblots from the lines grown and treated as in B.


F . Immunodetection of BES1 - CFP from heat - treated bin2 - 1 plants .

F. Immunodetection of BES1-CFP from heat-treated bin2-1 plants.


A . Immunodetection of BES1 - CFP from heat - stressed plants , treated with ABA or Fluridone ( Flu ) . 35S : BES1 - CFP plants were treated for 24 hours with DMSO ( control ) , 5 μM ABA or 10 μM Fluridone and heat - stressed as follows : lane 1 : untreated control ; lane 2 : 45 ° C for 30 minutes ; lane 3 : 45 ° C for 60 minutes . The non - phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue .

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 B-CONTROLLED_VAR 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

A. Immunodetection of BES1-CFP from heat-stressed plants, treated with ABA or Fluridone (Flu). 35S:BES1-CFP plants were treated for 24 hours with DMSO (control), 5 μM ABA or 10 μM Fluridone and heat-stressed as follows: lane 1: untreated control; lane 2: 45°C for 30 minutes; lane 3: 45°C for 60 minutes. The non-phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue.


C . Immunodetection of native BES1 from heat - stressed and epiBL - treated abitM plants . abitM and wild - type plants were grown on ½ MS medium either containing DMSO as a control or 1 μM epiBL and either left untreated or exposed to 45 ° C for 60 minutes . The non - phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue .

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

C. Immunodetection of native BES1 from heat-stressed and epiBL-treated abitM plants. abitM and wild-type plants were grown on ½ MS medium either containing DMSO as a control or 1 μM epiBL and either left untreated or exposed to 45°C for 60 minutes. The non-phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue.


C . ChIP to determine BES1 - CFP enrichment on the promoters of HSP70 . 3 , HSP70 . 4 and HSP90 . 1 following heat stress . 35S : BES1 - CFP and wild - type plants grown at 21 ° C were either left untreated or treated with 45 ° C for 60 minutes . BES1 - CFP was immuno - precipitated with α - GFP beads . Fragments were quantified in the precipitates with qPCR and the ratios of samples with antibody to without antibody were calculated . TA3 ( 70 . 3p and 90 . 1p ) or 5s rRNA ( 70 . 4p ) were used for normalization . UBQ5 served as an internal control . Data show the mean ± SE . n = 3 biological replicates measured in 3 technical repeats . Significant differences as compared to wild - type at 21 ° C , calculated by Student ' s t - test , are shown ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ) .

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C. ChIP to determine BES1-CFP enrichment on the promoters of HSP70.3, HSP70.4 and HSP90.1 following heat stress. 35S:BES1-CFP and wild-type plants grown at 21°C were either left untreated or treated with 45°C for 60 minutes. BES1-CFP was immuno-precipitated with α-GFP beads. Fragments were quantified in the precipitates with qPCR and the ratios of samples with antibody to without antibody were calculated. TA3 (70.3p and 90.1p) or 5s rRNA (70.4p) were used for normalization. UBQ5 served as an internal control. Data show the mean ±SE. n=3 biological replicates measured in 3 technical repeats. Significant differences as compared to wild-type at 21°C, calculated by Student's t-test, are shown (*P≤0.05; **P≤0.01; ***P≤0.001).


A . EMSAs showing in vitro DNA interaction of recombinant BES1 and / or HSFA1 with HSE - containing fragments of the HSP70 . 3 and HSP90 . 1 promoters . Unlabeled competitor fragments with functional HSEs ( C1 + HSE ) or mutated versions ( C2 - HSE ) were used for competition studies in 100x molar access ( lanes 6 and 7 ) .

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A. EMSAs showing in vitro DNA interaction of recombinant BES1 and/or HSFA1 with HSE-containing fragments of the HSP70.3 and HSP90.1 promoters. Unlabeled competitor fragments with functional HSEs (C1 +HSE) or mutated versions (C2 -HSE) were used for competition studies in 100x molar access (lanes 6 and 7).


B . BiFC assays showing cells co - transformed with nYFP - BES1 and cYFP - HSFA1a . The fusion proteins were co - expressed in N . benthamiana leaves and fluorescence was visualized . Two nuclei are highlighted and shown in a 6 - fold magnification . Scale bar = 20 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

B. BiFC assays showing cells co-transformed with nYFP-BES1 and cYFP-HSFA1a. The fusion proteins were co-expressed in N. benthamiana leaves and fluorescence was visualized. Two nuclei are highlighted and shown in a 6-fold magnification. Scale bar=20 μm.