Representative Northern blot showing tRNA ( Gly ) and tRNA ( Asp ) fragmentation in bone marrow from 8 - day - old and adult mice . tRNA ( Ser ) was included as a Dnmt2 non - substrate tRNA , and 5S rRNA was included as a loading control .

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Representative Northern blot showing tRNA(Gly) and tRNA(Asp) fragmentation in bone marrow from 8-day-old and adult mice. tRNA(Ser) was included as a Dnmt2 non-substrate tRNA, and 5S rRNA was included as a loading control.


tRNA fragment coverage for the Dnmt2 substrates tRNA ( Gly ) and tRNA ( Asp ) , and the non - substrate tRNA ( Ser ) in bone marrow , as determined by deep sequencing . Wild - type coverage is represented in red , and Dnmt2 − / − coverage is indicated in blue . The plots illustrate the specific increase of 5 ′ - halves from tRNA - Gly and 3 ′ - halves from tRNA - Asp fragments in Dnmt2 mutants .

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tRNA fragment coverage for the Dnmt2 substrates tRNA(Gly) and tRNA(Asp), and the non-substrate tRNA(Ser) in bone marrow, as determined by deep sequencing. Wild-type coverage is represented in red, and Dnmt2−/− coverage is indicated in blue. The plots illustrate the specific increase of 5′-halves from tRNA-Gly and 3′-halves from tRNA-Asp fragments in Dnmt2 mutants.


Analysis of protein synthesis by dynamic SILAC . The top 10 % of proteins that are translated slower in Dnmt2 − / − versus wild - type cells are indicated in orange . The top 10 % of proteins that are translated faster in Dnmt2 − / − versus wild - type cells are shown in blue . A change of > twofold is indicated by the black lines .

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Analysis of protein synthesis by dynamic SILAC. The top 10% of proteins that are translated slower in Dnmt2−/− versus wild-type cells are indicated in orange. The top 10% of proteins that are translated faster in Dnmt2−/− versus wild-type cells are shown in blue. A change of >  twofold is indicated by the black lines.


Dimethyl - labeling analysis of 8 - day - old mouse bone marrow tissue . Proteins that were down - regulated ( < twofold change ) in Dnmt2 - deficient cells are indicated in red , and up - regulated proteins ( > twofold change ) are shown in green . Nestin and periostin are marked with arrows .

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Dimethyl-labeling analysis of 8-day-old mouse bone marrow tissue. Proteins that were down-regulated (< twofold change) in Dnmt2-deficient cells are indicated in red, and up-regulated proteins (> twofold change) are shown in green. Nestin and periostin are marked with arrows.


Correlation between protein and mRNA expression for differentially expressed proteins . Proteins that were down - regulated ( < twofold change ) in Dnmt2 - deficient cells are indicated in red , and up - regulated proteins ( > twofold change ) are shown in green . The Pearson correlation coefficient is − 0 . 03 .

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Correlation between protein and mRNA expression for differentially expressed proteins. Proteins that were down-regulated (< twofold change) in Dnmt2-deficient cells are indicated in red, and up-regulated proteins (> twofold change) are shown in green. The Pearson correlation coefficient is −0.03.


Representative staining of nestin and periostin in femur sections from 8 - day - old wild - type and Dnmt2 − / − mice . The anti - periostin staining is comparable in the periosteum of wild - type and Dnmt2 − / − mice , but is reduced in the bone marrow cells ( compare the highlighted areas ) . Scale bar : 100 μm . Nuclei are counterstained with DAPI ( blue ) .

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Representative staining of nestin and periostin in femur sections from 8-day-old wild-type and Dnmt2−/− mice. The anti-periostin staining is comparable in the periosteum of wild-type and Dnmt2−/− mice, but is reduced in the bone marrow cells (compare the highlighted areas). Scale bar: 100 μm. Nuclei are counterstained with DAPI (blue).


Quantitative differences in the length of the nestin - positive trabecular zone ( upper panel ) between wild - type ( 30 measurements , three mice ) and Dnmt2 − / − ( 21 measurements , two mice ) and in bone periostin - positive marrow areas ( lower panel ) between wild - type ( 26 measurements , three mice ) and Dnmt2 − / − ( 45 measurements , three mice ) in femur sections from 8 - day - old mice . Data are presented as mean ± SD .

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Quantitative differences in the length of the nestin-positive trabecular zone (upper panel) between wild-type (30 measurements, three mice) and Dnmt2−/− (21 measurements, two mice) and in bone periostin-positive marrow areas (lower panel) between wild-type (26 measurements, three mice) and Dnmt2−/− (45 measurements, three mice) in femur sections from 8-day-old mice. Data are presented as mean ± SD.


Frequency of amino acid substitutions in the dSILAC dataset . Differences between Asp > Glu and Glu > Asp transitions versus Asn > Lys and His > Gln were found to be highly significant ( P < 0 . 01 , Fisher ' s exact test ) .

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Frequency of amino acid substitutions in the dSILAC dataset. Differences between Asp > Glu and Glu > Asp transitions versus Asn > Lys and His > Gln were found to be highly significant (P < 0.01, Fisher's exact test).


B . Cell lines generated from WT , null , and mutant p53 esophagi were assessed by Western blot analysis for p53 protein expression . Relative intensity densitometry of Rab11 - FIP1 results are shown at the bottom .

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B. Cell lines generated from WT, null, and mutant p53 esophagi were assessed by Western blot analysis for p53 protein expression. Relative intensity densitometry of Rab11-FIP1 results are shown at the bottom.


C . Organoids generated from WT , mutant , and null p53 cell lines . Scale bar : 100μm . D . Quantification of the portion of organoids exhibiting a solid , intermediate or stratified morphology ( > 37 organoids from 2 - 3 cell lines per group ) .

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C. Organoids generated from WT, mutant, and null p53 cell lines. Scale bar: 100μm. D. Quantification of the portion of organoids exhibiting a solid, intermediate or stratified morphology (> 37 organoids from 2-3 cell lines per group).


E - G . Cells were subcutaneously injected in the lower flanks of nude mice ( n = 4 flanks / cell line . WT p53 2 cell lines , Mutant p53 - 3 cell lines , p53 null - 3 cell lines ) . E . The number of tumors formed in each group was quantified . F . Tumor growth was measured at the indicated time points post - implantation . Graph represents total tumor size per group . G . Average tumor weight following dissection ( n = 2 biological replicates for WT p53 cell lines , n = 3 biological replicates for mutant p53 cell lines , n = 3 biological replicates for p53 null cell lines , n = 4 technical replicates injected per cell line , calculated as average tumor weight per cell line ) . Error bars represent ± S . E . M . * p = 0 . 02 Mutant p53 vs WT p53 , * p = 0 . 02 Mutant p53 vs Null p53 ( One - way Anova ) .

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E-G. Cells were subcutaneously injected in the lower flanks of nude mice (n= 4 flanks/cell line. WT p53 2 cell lines, Mutant p53-3 cell lines, p53 null-3 cell lines). E. The number of tumors formed in each group was quantified. F. Tumor growth was measured at the indicated time points post-implantation. Graph represents total tumor size per group. G. Average tumor weight following dissection (n=2 biological replicates for WT p53 cell lines, n=3 biological replicates for mutant p53 cell lines, n=3 biological replicates for p53 null cell lines, n=4 technical replicates injected per cell line, calculated as average tumor weight per cell line). Error bars represent ±S.E.M. *p=0.02 Mutant p53 vs WT p53, *p=0.02 Mutant p53 vs Null p53 (One-way Anova).


B . Relative fold change in expression level of endocytic recycling genes in invasive vs . non - invasive cells , and in mutant vs . WT p53 cells based on microarray and RNA - seq analysis , respectively .

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B. Relative fold change in expression level of endocytic recycling genes in invasive vs. non-invasive cells, and in mutant vs. WT p53 cells based on microarray and RNA-seq analysis, respectively.


C . qPCR analysis of recycling - related gene expression in mutant p53 relative to WT p53 cells ( red and black bars , respectively ) . Graphs represent mean ± SEM ( n = 3 biological replicates for Rab11 - FIP1 , Rab25 , Myo5b and Clic3 ; n = 2 biological replicates for Rab5 ) . Rab11 - FIP1 * p = 0 . 0109 , Rab25 * * p = 0 . 0021 , Myo5b * * * p = 0 . 0001 , Clic3 * * p = 0 . 0051 , Rab5 no significant differences ( n . s . ) . ( Unpaired t - test ) .

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C. qPCR analysis of recycling-related gene expression in mutant p53 relative to WT p53 cells (red and black bars, respectively). Graphs represent mean ± SEM (n=3 biological replicates for Rab11-FIP1, Rab25, Myo5b and Clic3; n=2 biological replicates for Rab5). Rab11-FIP1 *p=0.0109, Rab25 **p=0.0021, Myo5b ***p=0.0001, Clic3 **p=0.0051, Rab5 no significant differences (n.s.). (Unpaired t-test).


A . Western blot confirming knockdown of Rab11 - FIP1 by shRNAs . Relative intensity by densitometry of Rab11 - FIP1 results are shown at the bottom .

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A. Western blot confirming knockdown of Rab11-FIP1 by shRNAs. Relative intensity by densitometry of Rab11-FIP1 results are shown at the bottom.


3D organoids were generated from WT and mutant p53 cells expressing non - targeting control ( NT ) or Rab11 - FIP1 shRNAs ( sh1 and sh2 ) . B . Diameter of organoids ( n = 80 - 130 organoids ) . Bar graph represents mean diameter ± SEM . * * * * p < 0 . 0001 WT or mutant p53 non - targeting control vs Rab11 - FIP1 shRNA1 , * * * p = 0 . 0006 WT non - targeting control vs Rab11 - FIP1 shRNA2 , * * * * p < 0 . 0001 mutant p53 non - targeting control vs Rab11 - FIP1 shRNA2 ( One - way Anova ) . * * * * p < 0 . 0001 WT vs mutant p53 non - targeting control ( Unpaired t - test ) .

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3D organoids were generated from WT and mutant p53 cells expressing non-targeting control (NT) or Rab11-FIP1 shRNAs (sh1 and sh2). B. Diameter of organoids (n= 80-130 organoids). Bar graph represents mean diameter ± SEM. ****p<0.0001 WT or mutant p53 non-targeting control vs Rab11-FIP1 shRNA1, ***p=0.0006 WT non-targeting control vs Rab11-FIP1 shRNA2, ****p<0.0001 mutant p53 non-targeting control vs Rab11-FIP1 shRNA2 (One-way Anova). ****p<0.0001 WT vs mutant p53 non-targeting control (Unpaired t-test).


3D organoids were generated from WT and mutant p53 cells expressing non - targeting control ( NT ) or Rab11 - FIP1 shRNAs ( sh1 and sh2 ) . C . Percent of WT p53 ( top panel ) and mutant ( bottom panel ) organoids exhibiting a solid , intermediate , or stratified morphology ( n > 50 organoids generated from three different cell passages quantified ) . Bar graph represents mean diameter ± SEM . n . s . WT p53 non - targeting control vs Rab11 - FIP1 shRNAs , * p = 0 . 046 mutant p53 non - targeting control vs Rab11 - FIP1 shRNA1 , * * * p = 0 . 0002 mutant p53 non - targeting control vs Rab11 - FIP1 shRNA2 ( One - way Anova ) .

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3D organoids were generated from WT and mutant p53 cells expressing non-targeting control (NT) or Rab11-FIP1 shRNAs (sh1 and sh2). C. Percent of WT p53 (top panel) and mutant (bottom panel) organoids exhibiting a solid, intermediate, or stratified morphology (n>50 organoids generated from three different cell passages quantified). Bar graph represents mean diameter ± SEM. n.s. WT p53 non-targeting control vs Rab11-FIP1 shRNAs, *p=0.046 mutant p53 non-targeting control vs Rab11-FIP1 shRNA1, ***p=0.0002 mutant p53 non-targeting control vs Rab11-FIP1 shRNA2 (One-way Anova).


3D organoids were generated from WT and mutant p53 cells expressing non - targeting control ( NT ) or Rab11 - FIP1 shRNAs ( sh1 and sh2 ) . D . Percent of WT p53 and mutant organoids exhibiting a basaloid - like morphology ( n > 50 organoids generated from three different cell passages quantified ) . Bar graph represents mean diameter ± SEM . n . s . WT p53 non - targeting control vs Rab11 - FIP1 shRNAs , * p = 0 . 046 mutant p53 non - targeting control vs Rab11 - FIP1 shRNA1 , * * * p = 0 . 0002 mutant p53 non - targeting control vs Rab11 - FIP1 shRNA2 ( One - way Anova ) .

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3D organoids were generated from WT and mutant p53 cells expressing non-targeting control (NT) or Rab11-FIP1 shRNAs (sh1 and sh2). D. Percent of WT p53 and mutant organoids exhibiting a basaloid-like morphology (n>50 organoids generated from three different cell passages quantified). Bar graph represents mean diameter ± SEM. n.s. WT p53 non-targeting control vs Rab11-FIP1 shRNAs, *p=0.046 mutant p53 non-targeting control vs Rab11-FIP1 shRNA1, ***p=0.0002 mutant p53 non-targeting control vs Rab11-FIP1 shRNA2 (One-way Anova).


3D organoids were generated from WT and mutant p53 cells expressing non - targeting control ( NT ) or Rab11 - FIP1 shRNAs ( sh1 and sh2 ) . E . WT p53 organoids stained for H & E ( left ) , Rab11 - FIP1 ( middle ) , and the basal cell marker p63 ( right ) by IHC . 40X . Scale bar = 100 μm . F . Mutant p53 organoids stained for H & E ( left ) , Rab11 - FIP1 ( middle ) , and the basal cell marker p63 ( right ) by IHC . 40X . Scale bar = 100 μm .

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3D organoids were generated from WT and mutant p53 cells expressing non-targeting control (NT) or Rab11-FIP1 shRNAs (sh1 and sh2). E. WT p53 organoids stained for H&E (left), Rab11-FIP1 (middle), and the basal cell marker p63 (right) by IHC. 40X. Scale bar = 100 μm. F. Mutant p53 organoids stained for H&E (left), Rab11-FIP1 (middle), and the basal cell marker p63 (right) by IHC. 40X. Scale bar = 100 μm.


A . EPC2 - hTERT - EGFR - p53R175H cells grown in OTC stained for RCP . Arrow indicates invasive region . ( scale bar = 100 μm ) .

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A. EPC2-hTERT-EGFR-p53R175H cells grown in OTC stained for RCP. Arrow indicates invasive region. (scale bar = 100 μm).


B . Transwell Boyden Chamber invasion assay of WT and mutant p53 cells expressing non - targeting control ( NT ) and Rab11 - FIP1 shRNAs ( sh1 and sh2 ) ( n = 9 technical replicates from 3 different passages ) . Error bars represent ± S . E . M . n . s . WT p53 NT vs sh1 , * * * p = 0 . 0005 WT p53 NT vs sh2 , n . s . Mutant p53 NT vs sh1 , * * p = 0 . 0026 Mutant p53 NT vs sh2 , ( One - way Anova ) * p = 0 . 0173 WT p53 NT vs Mutant p53 NT , ( unpaired t - test ) .

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B. Transwell Boyden Chamber invasion assay of WT and mutant p53 cells expressing non-targeting control (NT) and Rab11-FIP1 shRNAs (sh1 and sh2) (n=9 technical replicates from 3 different passages). Error bars represent ±S.E.M. n.s. WT p53 NT vs sh1, ***p=0.0005 WT p53 NT vs sh2, n.s. Mutant p53 NT vs sh1,** p=0.0026 Mutant p53 NT vs sh2, (One-way Anova) *p=0.0173 WT p53 NT vs Mutant p53 NT, (unpaired t-test).


C - D . WT and mutant p53 cells expressing non - target control and Rab11 - FIP1 shRNAs were grown in OTC . C . H & E staining of OTC with WT p53 ( top ) and mutant p53 ( bottom ) with invasive regions . Scale bar = 50 μm . D . Graph shows relative invasive area from OTC of WT p53 non - target control and Rab11 - FIP1 shRNAs . n = 5 technical replicates in WT p53 NT , n = 6 technical replicates in WT p53 sh1 , n = 6 technical replicates in WT p53 sh2 . Error bars represent ± S . E . M . * * * * p < 0 . 0001 WT p53 NT vs sh1 , n . s . WT p53 NT vs sh2 ( One - way Anova ) .

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C-D. WT and mutant p53 cells expressing non-target control and Rab11-FIP1 shRNAs were grown in OTC. C. H&E staining of OTC with WT p53 (top) and mutant p53 (bottom) with invasive regions. Scale bar = 50 μm. D. Graph shows relative invasive area from OTC of WT p53 non-target control and Rab11-FIP1 shRNAs. n=5 technical replicates in WT p53 NT, n=6 technical replicates in WT p53 sh1, n=6 technical replicates in WT p53 sh2. Error bars represent ±S.E.M. **** p<0.0001 WT p53 NT vs sh1, n.s. WT p53 NT vs sh2(One-way Anova).


A . Immunofluorescent staining for Rab11 - FIP1 in TE1 and TE2 ESCC cell lines expressing a non - targeting control shRNA or shRNA against Rab11 - FIP1 . Scale bar = 50 μm B . Immunofluorescent staining for f - actin in TE1 and TE2 ESCC cell lines expressing a non - targeting control shRNA or shRNA against Rab11 - FIP1 . Scale bar = 50 μm C . Immunofluorescent staining for E - cadherin , in TE1 and TE2 ESCC cell lines expressing a non - targeting control shRNA or shRNA against Rab11 - FIP1 . Scale bar = 50 μm

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A. Immunofluorescent staining for Rab11-FIP1 in TE1 and TE2 ESCC cell lines expressing a non-targeting control shRNA or shRNA against Rab11-FIP1. Scale bar= 50 μm B. Immunofluorescent staining for f-actin in TE1 and TE2 ESCC cell lines expressing a non-targeting control shRNA or shRNA against Rab11-FIP1. Scale bar= 50 μm C. Immunofluorescent staining for E-cadherin, in TE1 and TE2 ESCC cell lines expressing a non-targeting control shRNA or shRNA against Rab11-FIP1. Scale bar= 50 μm


D . qPCR analysis of epithelial and mesenchymal gene expression in TE1 ( left ) and TE2 ( right ) cells expressing Rab11 - FIP1 shRNA relative to control cells ( dotted line ) . Graph error bars represent mean SEM ( n = 3 technical replicates from three different cell passages ) . n . s . TE2 SNAI2 , * p < 0 . 05 , ( Unpaired t - test ) .

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D. qPCR analysis of epithelial and mesenchymal gene expression in TE1 (left) and TE2 (right) cells expressing Rab11-FIP1 shRNA relative to control cells (dotted line). Graph error bars represent mean SEM (n=3 technical replicates from three different cell passages). n.s. TE2 SNAI2, *p<0.05,(Unpaired t-test).


E . qPCR analysis of epithelial and mesenchymal gene expression following ZEB1 depletion in TE1 ( left ) and TE2 ( right ) cells expressing Rab11 - FIP1 shRNA relative to control cells ( dotted line ) . Graph error bars represent mean SEM ( n = 3 technical replicates from three different cell passages ) . * * p < 0 . 01 , * p < 0 . 05 , n . s . TE1 CDH1 , VIM . SNAI1 , SNAI2 , TE2 VIM , SNAI1 ( Unpaired t - test ) .

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E. qPCR analysis of epithelial and mesenchymal gene expression following ZEB1 depletion in TE1 (left) and TE2 (right) cells expressing Rab11-FIP1 shRNA relative to control cells (dotted line). Graph error bars represent mean SEM (n=3 technical replicates from three different cell passages). **p<0.01, * p<0.05, n.s. TE1 CDH1, VIM. SNAI1, SNAI2, TE2 VIM, SNAI1 (Unpaired t-test).


( A ) Survival curves of C57BL / 6J naïve and adrenalectomized ( Adx ) mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged , i . p . , with 30 µg ( naïve ) or 2 µg ( Adx ) TNF , solved in sterile PBS , per 20 g body weight . No deaths occurred later than 72 h after TNF injection . Results represent combined data of 3 experiments .

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(A) Survival curves of C57BL/6J naïve and adrenalectomized (Adx) mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged, i.p., with 30 µg (naïve) or 2 µg (Adx) TNF, solved in sterile PBS, per 20 g body weight. No deaths occurred later than 72 h after TNF injection. Results represent combined data of 3 experiments.


( B ) Corticosterone levels in the serum of C57BL / 6J naïve and Adx mice after 7 days of 25 mM ZnSO4 supplementation to the drinking water ( N = 6 per group ) .

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(B) Corticosterone levels in the serum of C57BL/6J naïve and Adx mice after 7 days of 25 mM ZnSO4 supplementation to the drinking water (N=6 per group).


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


( A - B ) Expression 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 ) .

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

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

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


( D ) Expression of genes ( RT - qPCR ) involved in muscle glucose 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 .

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


( E ) Respiratory exchange 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 ) .

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


( B ) Luciferase reporters containing 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 ) .

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


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

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


( D ) ( Top ) Fnip1 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 .

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


( A ) ( Left ) Representative western blot 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 .

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


( B , C ) Results of RT - qPCR 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 ) .

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


( D ) ( Top ) Representative western blot 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 .

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


( E ) Oxygen consumption rates ( OCR ) in 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 ) .

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


( F ) Results of RT - qPCR 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 ) .

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


( G ) Oxygen consumption rates ( OCR ) in 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 ) .

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


( A ) Mean expression 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 ) .

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


( B ) Mitochondrial respiration rates were determined from mitochondria isolated from 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 ) .

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


( C ) ( Left ) Representative western blot 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 ) .

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


( D ) Representative western blot 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 ) .

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


( E ) ( Top ) Representative MHC1 immunofluorescence ( IF ) in 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 .

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


( F ) 5 - week - old 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 ) .

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


( G ) 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 ) .

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


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

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


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

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


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

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


Kymograph illustrating 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 ) .

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


Kymograph showing 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 .

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


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

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


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 ) .

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


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

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


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

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


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 ) .

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


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 .

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


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 .

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


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 .

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


Comparison of fold binding inhibition for GFP - tagged Rad54 , Rdh54NRad54 , Rdh54 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 .

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


Widefield 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 .

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


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

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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 .

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


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

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


D - loop stimulation by Rad54 or Rdh54NRad54 in reactions with Rad51 ( 300 nM ) , 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 .

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


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

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


ATPase stimulation for GFP - tagged Rad54 , Rdh54NRad54 , Rdh54 or Rad54NRdh54 in 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 .

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


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

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


Competition assays with MLS 1765 - 92 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 .

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


Expression of total 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.