C . Mitochondrial fraction from 293T cells ectopically expressing GASZ , MitoPLD , AIFM1 , or SIRT3 were treated in the presence or absence of Triton X - 100 and / or Proteinase K , and examined by Western blots .

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C. Mitochondrial fraction from 293T cells ectopically expressing GASZ, MitoPLD, AIFM1, or SIRT3 were treated in the presence or absence of Triton X-100 and/or Proteinase K, and examined by Western blots.


D . GST pull - down assay of GST - tagged GASZ with FLAG - GASZ produced from 293T cells .

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D. GST pull-down assay of GST-tagged GASZ with FLAG-GASZ produced from 293T cells.


E . Co - IP assays of FLAG or GFP - tagged GASZ in 293T cells . Antibodies for Co - IP ( IP ) or Western blots ( WB ) were listed on the left .

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E. Co-IP assays of FLAG or GFP-tagged GASZ in 293T cells. Antibodies for Co-IP (IP) or Western blots (WB) were listed on the left.


F . BiFC assays in which YFP was detected by flow cytometry in control 293T cells , or 293T cells co - expressed with GASZ fused with N - or C - terminal half of YFP . Ctrl : empty vector control .

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F. BiFC assays in which YFP was detected by flow cytometry in control 293T cells, or 293T cells co-expressed with GASZ fused with N- or C-terminal half of YFP. Ctrl: empty vector control.


G . Co - IP assays of GFP - GASZ with various FLAG - tagged wild - type GASZ or its deletion mutants in 293T

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G. Co-IP assays of GFP-GASZ with various FLAG-tagged wild-type GASZ or its deletion mutants in 293T cells


A . Mitochondria visualized by MitoTracker ( Red ) when wild - type GASZ or GASZ with MLS mutation was expressed in HeLa cells . Scale bar : 15 m .

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A. Mitochondria visualized by MitoTracker (Red) when wild-type GASZ or GASZ with MLS mutation was expressed in HeLa cells. Scale bar: 15 m.


B . EM on 293T cells with empty vector control ( Ctrl ) , wild - type GASZ , or GASZ with F to P mutation in MLS . Scale bar : 1 m . N : nucleus .

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B. EM on 293T cells with empty vector control (Ctrl), wild-type GASZ, or GASZ with F to P mutation in MLS. Scale bar: 1 m. N: nucleus.


C . Averaged percentage of HeLa cells ( determined from 100 cells in each experiment ) with different types of mitochondria determined by MitoTracker ( Red ) staining when transfected with GASZ , its SAM / ZIP deletion mutant , or GASZ with MLS mutation .

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C. Averaged percentage of HeLa cells (determined from 100 cells in each experiment) with different types of mitochondria determined by MitoTracker (Red) staining when transfected with GASZ, its SAM/ZIP deletion mutant, or GASZ with MLS mutation.


D . HeLa cells transfected with COX4 - DsRed , mito - PAGFP , and empty vector control , GASZ or GASZ with MLS mutation , followed by photoactivation of PAGFP . Representative images of PAGFP and DsRed were taken at 0 , 10 , and 20 min post photoactivation . Scale Bar : 10 m . Insets show images with higher magnification . The averaged exponential decay constant ( ± s . e . m . ) of normalized PAGFP fluorescence within the designated region ( white circle ) at photoactivation area over a period of 10 min was shown as a bar graph below . n > 20 cells for each group .

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D. HeLa cells transfected with COX4-DsRed, mito-PAGFP, and empty vector control, GASZ or GASZ with MLS mutation, followed by photoactivation of PAGFP. Representative images of PAGFP and DsRed were taken at 0, 10, and 20 min post photoactivation. Scale Bar: 10 m. Insets show images with higher magnification. The averaged exponential decay constant (±s.e.m.) of normalized PAGFP fluorescence within the designated region (white circle) at photoactivation area over a period of 10 min was shown as a bar graph below. n>20 cells for each group.


E . Averaged percentage of C18 - 4 cells ( determined from 100 cells in experiment ) with different types of mitochondria determined by MitoTracker ( Red ) staining when transfected with Su9 - GFP or GFP - GASZ .

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E. Averaged percentage of C18-4 cells (determined from 100 cells in experiment) with different types of mitochondria determined by MitoTracker (Red) staining when transfected with Su9-GFP or GFP-GASZ.


F . Real - time PCR analyses of mtDNA copy numbers on testes at different time points postnatal , relative to a single copy gene , PECAM , encoded by nuclear DNA . n = 5 per genotype .

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F. Real-time PCR analyses of mtDNA copy numbers on testes at different time points postnatal, relative to a single copy gene, PECAM, encoded by nuclear DNA. n=5 per genotype.


G . Histochemistry of respiratory enzymes , COX , NADH , and SDH on testical sections from mice 10 days postnatal . Scale bar : 100 m .

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G. Histochemistry of respiratory enzymes, COX, NADH, and SDH on testical sections from mice 10 days postnatal. Scale bar: 100 m.


A - B . Co - IP assays of ectopically expressed FLAG - GASZ and MFN - HA in 293T cells ( A )

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A-B. Co-IP assays of ectopically expressed FLAG-GASZ and MFN-HA in 293T cells (A)


A - B . Co - IP assays of endogenous proteins of GASZ and MFNs from adult testes ( B ) .

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A-B. Co-IP assays of endogenous proteins of GASZ and MFNs from adult testes (B).


C . Co - IP assays of HA - tagged MFN1 with various FLAG - tagged wild - type GASZ , its domain deletion mutants , or GASZ with MLS mutations in 293T cells .

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C. Co-IP assays of HA-tagged MFN1 with various FLAG-tagged wild-type GASZ, its domain deletion mutants, or GASZ with MLS mutations in 293T cells.


D . Averaged percentage of MEFs with different types of mitochondria were determined according to the mitochondrial morphology by MitoTracker ( Red ) staining in wild - type MEFs , or MEFs containing MFN null mutations transfected with empty vector control ( Ctrl ) or a GASZ expressing plasmid .

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D. Averaged percentage of MEFs with different types of mitochondria were determined according to the mitochondrial morphology by MitoTracker (Red) staining in wild-type MEFs, or MEFs containing MFN null mutations transfected with empty vector control (Ctrl) or a GASZ expressing plasmid.


E . PEG fusion assays of Su9 - GFP and COX4 - DsRed labeled wild - type MEFs , or MEFs containing MFN null mutations transfected with empty vector control ( Ctrl ) or a GASZ expressing plasmid . Bar graph represents averaged percentage of areas with different levels of mitochondrial fusion .

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E. PEG fusion assays of Su9-GFP and COX4-DsRed labeled wild-type MEFs, or MEFs containing MFN null mutations transfected with empty vector control (Ctrl) or a GASZ expressing plasmid. Bar graph represents averaged percentage of areas with different levels of mitochondrial fusion.


A . Gross morphology of testes from MFN1f / f - Cre compared to their wild - type littermates .

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A. Gross morphology of testes from MFN1f/f-Cre compared to their wild-type littermates.


B . Histological studies of testis sections from wild - type mice or MFN1f / f - Cre littermates at different time points postnatal . Red stars : tubules with spermatocytes .

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B. Histological studies of testis sections from wild-type mice or MFN1f/f-Cre littermates at different time points postnatal. Red stars: tubules with spermatocytes.


C . IHF of PLZF ( day 14 postnatal ) , MILI , MVH , and GASZ / CytC ( day 7 ) of testes sections . Scale bars for PLZF are 50 m and 10 m for others .

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C. IHF of PLZF (day 14 postnatal), MILI, MVH, and GASZ/CytC (day 7) of testes sections. Scale bars for PLZF are 50 m and 10 m for others.


D . EM of testical sections from wild - type neonatal mice or MFN1f / f - Cre littermates . N : nucleus .

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D. EM of testical sections from wild-type neonatal mice or MFN1f/f-Cre littermates. N: nucleus.


E . Histochemistry of respiratory enzymes , COX , NADH , and SDH on testical sections from mice 10 days postnatal . Scale bar : 100 m .

E. Histochemistry of respiratory enzymes, COX, NADH, and SDH on testical sections from mice 10 days postnatal. Scale bar: 100 m.


A . Tryptophan - fluorescence time course associated to Na + ( blue ) and K + ( black ) binding , respectively , to detergent - purified apo EAAT1CRYST at pH 7 . 4 . Horizontal and vertical scale bars represent time and ∆ F / F0 , respectively . B . Tryptophan - fluorescence time course associated to L - asp binding in presence of 1 mM ( blue ) and absence ( black ) of Na + , respectively , at pH 9 .

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A. Tryptophan-fluorescence time course associated to Na+ (blue) and K+ (black) binding, respectively, to detergent-purified apo EAAT1CRYST at pH 7.4. Horizontal and vertical scale bars represent time and ∆F/F0, respectively. B. Tryptophan-fluorescence time course associated to L-asp binding in presence of 1 mM (blue) and absence (black) of Na+, respectively, at pH 9. C


C . Na + binding isotherms of EAAT1CRYST at pH 7 . 4 in the presence ( pink ) and absence ( blue ) of UPCH101 . Mutation D400380N at Na3 strongly impairs Na + binding ( green ) . Solid lines represent fits of Hill equation .

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C. Na+ binding isotherms of EAAT1CRYST at pH 7.4 in the presence (pink) and absence (blue) of UPCH101. Mutation D400380N at Na3 strongly impairs Na+ binding (green). Solid lines represent fits of Hill equation.


A . Na + - induced L - glutamate uptake by purified EAAT1CRYST reconstituted in liposomes loaded with K + . Choline ( Ch + ) condition was subtracted to the Na + condition .

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A. Na+-induced L-glutamate uptake by purified EAAT1CRYST reconstituted in liposomes loaded with K+. Choline (Ch+) condition was subtracted to the Na+ condition.


B . Log - log plots of Na + ( red , n = 19 ) and L - asp ( blue , n = 27 ) KD , as a function of pH at [ Na + ] = 0 . 5 mM . Na + ( n = 10 ) and L - asp ( n = 7 ) titration data at pH 7 . 4 and 9 . 0 , respectively , are also reported in Fig . 1 . Solid lines are fits of straight lines .

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B. Log-log plots of Na+ (red, n=19) and L-asp (blue, n=27) KD, as a function of pH at [Na+]=0.5 mM. Na+ (n=10) and L-asp (n=7) titration data at pH 7.4 and 9.0, respectively, are also reported in Fig. 1. Solid lines are fits of straight lines.


C . Log - log plots of L - asp KD for EAAT1CRYST - E406386Q ( light blue , n = 24 ) and EAAT1CRYST - Y405385F ( dark blue , n = 18 ) , as a function of pH at [ Na + ] = 0 . 5 mM . .

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C. Log-log plots of L-asp KD for EAAT1CRYST-E406386Q (light blue, n=24) and EAAT1CRYST-Y405385F (dark blue, n=18), as a function of pH at [Na+]=0.5 mM..


A . Purified EAAT1CRYST takes up L - glutamate in liposomes loaded with K + or Rb + , but not with choline ( Ch + ) , or with Ch + and Ca2 + . Plots depict data as in Fig . 3A .

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A. Purified EAAT1CRYST takes up L-glutamate in liposomes loaded with K+ or Rb+, but not with choline (Ch+), or with Ch+ and Ca2+. Plots depict data as in Fig. 3A.


B . Ca2 + binding isotherms of EAAT1CRYST at pH = 7 . 4 in the presence ( pink ) and absence ( blue ) of UPCH101 . Mutation D400380N at Na3 strongly impairs Ca2 + binding ( green ) . Symbols represent average and s . e . m . values of at least three independent experiments . Solid lines represent fits of Hill equation and dashed line is the fit in Fig . 1C ( blue line ) corresponding to Na + binding .

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B. Ca2+ binding isotherms of EAAT1CRYST at pH=7.4 in the presence (pink) and absence (blue) of UPCH101. Mutation D400380N at Na3 strongly impairs Ca2+ binding (green). Symbols represent average and s.e.m. values of at least three independent experiments. Solid lines represent fits of Hill equation and dashed line is the fit in Fig. 1C (blue line) corresponding to Na+ binding.


C . Log - log plot of L - asp KD as a function of Ca2 + ( n = 13 ) .

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C. Log-log plot of L-asp KD as a function of Ca2+ (n=13).


. C - J Bar Chart with data points showing the abundance of indicated bacterial families in the indicated group of mice . Data are expressed as CSS - normalized bacterial

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. C-J Bar Chart with data points showing the abundance of indicated bacterial families in the indicated group of mice. Data are expressed as CSS-normalized bacterial counts


A - C Bar chart with individual points showing the levels of propionate acetate and butyrate in wt and mdx mice receiving DFZ or vehicle , measured by NMR or GC - MS . Data are expressed as μg / ml or bin intensity ( arbitrary unit - a . u . ) D - F Bar chart with individual points showing the levels of pyruvate , succinate and lactate in wt and mdx mice receiving vehicle or DFZ , measured by NMR .

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A-C Bar chart with individual points showing the levels of propionate acetate and butyrate in wt and mdx mice receiving DFZ or vehicle, measured by NMR or GC-MS. Data are expressed as μg/ml or bin intensity (arbitrary unit - a.u.) D-F Bar chart with individual points showing the levels of pyruvate, succinate and lactate in wt and mdx mice receiving vehicle or DFZ, measured by NMR. G


A - B Muscle coordination and strength were measured in 19 weeks old control and mdx mice treated with vehicle ( DMSO ) , NaB ( 100 mg kg − 1 / daily ) or DFZ ( 1 . 2 mg kg − 1 / daily ) for 3 weeks using the rotarod and weight test . Bar charts show the latency to fall or drop the weight of wt and dystrophic mice .

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A-B Muscle coordination and strength were measured in 19 weeks old control and mdx mice treated with vehicle (DMSO), NaB (100 mg kg−1/daily) or DFZ (1.2 mg kg−1/daily) for 3 weeks using the rotarod and weight test. Bar charts show the latency to fall or drop the weight of wt and dystrophic mice.


A - I Bar chart with individual points showing the mRNA expression levels of the indicated genes measured in the gastrocnemius of control and mdx mice treated with or without NaB and DFZ .

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A-I Bar chart with individual points showing the mRNA expression levels of the indicated genes measured in the gastrocnemius of control and mdx mice treated with or without NaB and DFZ.


J - K Representative blotting and bar chart with individual points showing the expression and / or phosphorylation of pAKT / AKT and COX2 in the gastrocnemius of the indicated six groups of mice .

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J-K Representative blotting and bar chart with individual points showing the expression and/or phosphorylation of pAKT/AKT and COX2 in the gastrocnemius of the indicated six groups of mice.


A - B Levels of AEA and 2 - AG in plasma samples of wt and mdx mice expressed as pmol mg − 1 of wet tissue weight .

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A-B Levels of AEA and 2-AG in plasma samples of wt and mdx mice expressed as pmol mg−1 of wet tissue weight.


C - D Bar charts with individual points showing the mRNA expression levels of CB1 and CB2 measured in the gastrocnemius of the indicated groups of mice .

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C-D Bar charts with individual points showing the mRNA expression levels of CB1 and CB2 measured in the gastrocnemius of the indicated groups of mice.


E Representative blots showing the expression levels of CB1 and CB2 proteins in the gastrocnemius of the indicated groups of mice . F Quantification of CB1 and CB2 proteins to the housekeeping protein GAPDH .

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E Representative blots showing the expression levels of CB1 and CB2 proteins in the gastrocnemius of the indicated groups of mice. F Quantification of CB1 and CB2 proteins to the housekeeping protein GAPDH. D


A - C Bar charts with individual points showing the mRNA expression levels of Ulk , Pink and Becn1 measured in control and mdx mice treated with rimonabant ( 0 . 5 mg kg - 1 ) .

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A-C Bar charts with individual points showing the mRNA expression levels of Ulk, Pink and Becn1 measured in control and mdx mice treated with rimonabant (0.5 mg kg-1).


D Representative blots showing the expression levels of LC3I and LC3II proteins in the gastrocnemius of the indicated groups of mice . E , F Quantification of LC3I and LC3II proteins to the housekeeping protein GAPDH .

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D Representative blots showing the expression levels of LC3I and LC3II proteins in the gastrocnemius of the indicated groups of mice. E, F Quantification of LC3I and LC3II proteins to the housekeeping protein GAPDH. D


A - B Bar chart with individual points showing the mRNA expression levels of Il6 and Cox2 in control ( vehicle , DMSO ) and / or GPR109A - silenced C2C12 myoblasts exposed to LPS ( 1 μg / ml ) in the presence or absence of either MK1903 ( 1 μM ) , rosiglitazone ( 1 μM ) or T007 ( 1 μM ) .

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A-B Bar chart with individual points showing the mRNA expression levels of Il6 and Cox2 in control (vehicle, DMSO) and/or GPR109A-silenced C2C12 myoblasts exposed to LPS (1 μg/ml) in the presence or absence of either MK1903 (1 μM), rosiglitazone (1 μM) or T007 (1 μM).


A - F Bar chart with individual points showing the mRNA expression levels of Cb1 , Daglα , Daglβ , Magl , Napepld , and Faah in control ( vehicle , DMSO ) and / or GPR109A - silenced C2C12 myoblasts exposed to LPS ( 1 μg / ml ) in the presence or absence of either MK1903 ( 1 μM ) , rosiglitazone ( 1 μM ) or T007 ( 1 μM ) .

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A-F Bar chart with individual points showing the mRNA expression levels of Cb1, Daglα, Daglβ, Magl, Napepld, and Faah in control (vehicle, DMSO) and/or GPR109A-silenced C2C12 myoblasts exposed to LPS (1 μg/ml) in the presence or absence of either MK1903 (1 μM), rosiglitazone (1 μM) or T007 (1 μM).


G - H Levels of AEA and 2 - AG were measured in C2C12 cells exposed to LPS ( 1 μg / ml ) or NaB ( 3 mM ) for 24 h . I Effect of ACEA ( 1 μM ) and rimonabant ( 1 μM ) on autophagosome formation measured in C2C12 cells .

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G-H Levels of AEA and 2-AG were measured in C2C12 cells exposed to LPS (1 μg/ml) or NaB (3 mM) for 24 h. I Effect of ACEA (1 μM) and rimonabant (1 μM) on autophagosome formation measured in C2C12 cells. D


C - J Bar chart with individual points showing the expression of selected miRNAs in control and Gpr109A - silenced C2C12 myoblasts exposed to LPS ( 1 μg / ml ) in the presence or absence of either NaB ( 3 mM ) , MK1903 ( 1 μM ) , rosiglitazone ( 1 μM ) . NaB was also tested in the presence or absence of either rosiglitazone ( 1 μM ) or T007 ( 1 μM ) .

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C-J Bar chart with individual points showing the expression of selected miRNAs in control and Gpr109A-silenced C2C12 myoblasts exposed to LPS (1 μg/ml) in the presence or absence of either NaB (3 mM), MK1903 (1 μM), rosiglitazone (1 μM). NaB was also tested in the presence or absence of either rosiglitazone (1 μM) or T007 (1 μM).


A - E Bar chart showing the mRNA expression levels of IL6 , COX2 , ULK 1 , ATG13 and ATG4 mRNA in primary human myoblasts isolated from one healthy donor ( HD ) and five DMD donors ( D1 - D5 ) .

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A-E Bar chart showing the mRNA expression levels of IL6, COX2, ULK 1, ATG13 and ATG4 mRNA in primary human myoblasts isolated from one healthy donor (HD) and five DMD donors (D1- D5).


( A - B ) Hemoglobin ( A ) and hematocrit ( B ) levels of healthy subjects and cachectic cancer patients presenting a body weight loss superior to 10 % of initial body weight ( 19 healthy subjects , 17 cachectic patients ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(A-B) Hemoglobin (A) and hematocrit (B) levels of healthy subjects and cachectic cancer patients presenting a body weight loss superior to 10% of initial body weight (19 healthy subjects, 17 cachectic patients). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( C ) TFR1 mRNA levels in muscle biopsies from cancer - patients of late stage cachexia ( 19 healthy subjects , 17 cachectic patients with at least 10 % total body weight loss ) Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(C) TFR1 mRNA levels in muscle biopsies from cancer-patients of late stage cachexia (19 healthy subjects, 17 cachectic patients with at least 10% total body weight loss) Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( F ) TFR1 mRNA levels in the gastrocnemius of mice subjected to iron deprivation by feeding with an iron deficient diet ( IDD ) combined to a phlebotomy ( PHL ) ( n = 5 - 6 ) Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(F) TFR1 mRNA levels in the gastrocnemius of mice subjected to iron deprivation by feeding with an iron deficient diet (IDD) combined to a phlebotomy (PHL) (n=5-6) Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( G ) Cross - sectional area of skeletal muscle fiber transfected with shSCR ( scramble ) and shTFR1 ( n = 3 - 4 ) and representative picture of a shTFR1 transfected fiber . Scale bar = 50µm . ( H ) Cross - sectional area of skeletal muscle fibers transfected with TFR - pHuji ( n = 4 ) and representative picture of a TFR - pHuji transfected fiber . Scale bar = 50µm . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(G) Cross-sectional area of skeletal muscle fiber transfected with shSCR (scramble) and shTFR1 (n=3-4) and representative picture of a shTFR1 transfected fiber. Scale bar=50µm. (H) Cross-sectional area of skeletal muscle fibers transfected with TFR-pHuji (n=4) and representative picture of a TFR-pHuji transfected fiber. Scale bar=50µm. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( L ) Reprensentative pictures and diameter measurements of human myoblast - derived myotubes after 48h treatment with DFO 100µM ( n = 3 ) . Scale bar = 50µm . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(L) Reprensentative pictures and diameter measurements of human myoblast-derived myotubes after 48h treatment with DFO 100µM (n=3). Scale bar=50µm. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( C ) TFR1 protein expression and densitometric quantification in mouse gastrocnemius ( n = 5 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(C) TFR1 protein expression and densitometric quantification in mouse gastrocnemius (n=5). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( E ) Representative Western blot of IRP2 in mouse gastrocnemius and densitometric quantification ( n = 5 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(E) Representative Western blot of IRP2 in mouse gastrocnemius and densitometric quantification (n=5). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( H ) Representative Western blot of ferritin Heavy and Light chains in mouse gastrocnemius and densitometric quantification ( n = 4 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(H) Representative Western blot of ferritin Heavy and Light chains in mouse gastrocnemius and densitometric quantification (n=4). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( I - J ) ICP - MS quantification of total ( I ) and protein - bound ( J ) iron in mouse quadriceps ( n = 4 - 5 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(I-J) ICP-MS quantification of total (I) and protein-bound (J) iron in mouse quadriceps (n=4-5). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( A ) ICP - MS quantification of mitochondrial iron in mouse quadriceps ( n = 6 - 8 ) . ( B ) Gastrocnemius heme content quantified by fluorescent heme assay ( n = 4 - 6 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ICP-MS quantification of mitochondrial iron in mouse quadriceps (n=6-8). (B) Gastrocnemius heme content quantified by fluorescent heme assay (n=4-6). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( C - D ) mRNA levels of mitochondrial iron importer MFRN2 ( C ) and the rate limiting enzyme of heme synthesis ALAS2 ( D ) normalized to 18s in mouse gastrocnemius ( n = 4 - 5 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(C-D) mRNA levels of mitochondrial iron importer MFRN2 (C) and the rate limiting enzyme of heme synthesis ALAS2 (D) normalized to 18s in mouse gastrocnemius (n=4-5). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( F ) Succinate Dehydrogenase activity staining in transversal sections of mouse gastrocnemius and corresponding intensity quantification ( n = 3 - 4 ) . Scale bar = 50µm . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Succinate Dehydrogenase activity staining in transversal sections of mouse gastrocnemius and corresponding intensity quantification (n=3-4). Scale bar= 50µm. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( B ) Western blot of mitochondrial OXPHOS respiratory complexes in C2C12 myotubes treated for 48H with C26CM and ferric citrate . ( n = 3 )

O O O 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) Western blot of mitochondrial OXPHOS respiratory complexes in C2C12 myotubes treated for 48H with C26CM and ferric citrate. (n=3)


( G - H ) Representative microscopic pictures and diameter of C2C12 myotubes stained for myosin heavy chain ( G ) or human myoblast - derived myotubes ( H ) after 48h treatment with C26 CM and ferric citrate ( n = 3 per condition ) . Scale bars = 50 and 100 µm , respectively . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by one - way Anova with Bonferroni ' s correction Data are mean ± SEM . ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 compared to control and # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 CM - treated group .

O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) Representative microscopic pictures and diameter of C2C12 myotubes stained for myosin heavy chain (G) or human myoblast-derived myotubes (H)after 48h treatment with C26 CM and ferric citrate (n=3 per condition). Scale bars =50 and 100 µm, respectively. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by one-way Anova with Bonferroni's correction Data are mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and ##p < 0.01, ###p < 0.001 compared to C26 CM-treated group.


( B ) Kaplan - Meier depicting the survival of C26 - tumor bearing mice after I . V . injection of saline or iron every 5 days post C26 - injection ( 3 - month - old Balb / C , n = 8 - 11 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by chi - square test for the survival curves Data are mean ± SEM . ∗ p < 0 . 05 and ∗ ∗ ∗ p < 0 . 001 compared to control and # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 untreated group .

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

(B) Kaplan-Meier depicting the survival of C26-tumor bearing mice after I.V. injection of saline or iron every 5 days post C26-injection (3-month-old Balb/C, n=8-11). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by chi-square test for the survival curves Data are mean ± SEM. ∗p < 0.05 and ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.


Immunofluorescent staining of myosin heavy chain fast ( green ) and slow ( red ) of transversal sections of gastrocnemius ( midbelly ) with corresponding frequency distribution Scale bar = 100µm . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by one - way Anova with Bonferroni ' s correction Data are mean ± SEM . ∗ p < 0 . 05 and ∗ ∗ ∗ p < 0 . 001 compared to control and # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 untreated group .

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

Immunofluorescent staining of myosin heavy chain fast (green) and slow (red) of transversal sections of gastrocnemius (midbelly) with corresponding frequency distribution Scale bar=100µm. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by one-way Anova with Bonferroni's correction Data are mean ± SEM. ∗p < 0.05 and ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.


( I - K ) mRNA levels of Murf 1 ( I ) , Atrogin 1 ( J ) , and REDD1 ( K ) normalized to GAPDH in gastrocnemius ( n = 5 - 14 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by one - way Anova with Bonferroni ' s correction , Data are mean ± SEM . ∗ p < 0 . 05 and ∗ ∗ ∗ p < 0 . 001 compared to control and # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 untreated group .

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

(I-K) mRNA levels of Murf 1 (I), Atrogin 1 (J), and REDD1 (K) normalized to GAPDH in gastrocnemius (n=5-14). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by one-way Anova with Bonferroni's correction, Data are mean ± SEM. ∗p < 0.05 and ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.


( B ) Aconitase activity of quadriceps lysates normalized to protein content ( n = 3 - 7 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 compared to control and # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 untreated group .

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

(B) Aconitase activity of quadriceps lysates normalized to protein content (n=3-7). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.


( E ) Representative Western blot and densitometric quantification of phospho - AMPK and total AMPK ( stripped and re - blotted ) in the gastrocnemius of C26 - tumor bearing mice after iron carboxymaltose supplementation ( n = 6 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 compared to control and # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 untreated group .

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

(E) Representative Western blot and densitometric quantification of phospho-AMPK and total AMPK (stripped and re-blotted) in the gastrocnemius of C26-tumor bearing mice after iron carboxymaltose supplementation (n=6). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.


( F - H ) mRNA levels of ACOT 1 ( F ) , MCD ( G ) , and CPT1B ( H ) normalized to GAPDH in gastrocnemius ( n = 5 - 10 ) . Data information : For all data , n represents the number of biological replicates . Statistical significance was calculated by unpaired , two - tailed Student ' s T - test . Data are mean ± SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 compared to control and # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 untreated group .

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

(F-H) mRNA levels of ACOT 1 (F), MCD (G), and CPT1B (H) normalized to GAPDH in gastrocnemius (n=5-10). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.


( A - B ) Grip force of dominant or non - dominant arm in iron - deficient cancer patients , expressed in percentage of baseline force ( A ) and absolute values normalized to height ( B ) before / after single dose of iron carboxymaltose ( 15mg / kg , 7 subjects ) . Data information : For all data , n represents In graph ( A ) , the boxes represent the range of values with the median value being the central band and whiskers the SEM .

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

(A-B) Grip force of dominant or non-dominant arm in iron-deficient cancer patients, expressed in percentage of baseline force (A) and absolute values normalized to height (B) before/after single dose of iron carboxymaltose (15mg/kg, 7 subjects). Data information: For all data, n represents In graph (A), the boxes represent the range of values with the median value being the central band and whiskers the SEM.


Overall structure of the RBD - ACE2 - B0AT1 complex . ( A ) Cryo - EM map of the RBD - ACE2 - B0AT1 complex . Left : Overall reconstruction of the ternary complex at 2 . 9 Å . Inset : focused refined map of RBD .

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

Overall structure of the RBD-ACE2-B0AT1 complex.(A) Cryo-EM map of the RBD-ACE2-B0AT1 complex. Left: Overall reconstruction of the ternary complex at 2.9 Å. Inset: focused refined map of RBD.


Overall structure of the RBD - ACE2 - B0AT1 complex . The complex is colored by subunits , with the protease domain ( PD ) and the Collectrin - like domain ( CLD ) colored cyan and blue in one of the ACE2 protomers , respectively . The glycosylation moieties are shown as sticks .

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

Overall structure of the RBD-ACE2-B0AT1 complex. The complex is colored by subunits, with the protease domain (PD) and the Collectrin-like domain (CLD) colored cyan and blue in one of the ACE2 protomers, respectively. The glycosylation moieties are shown as sticks.


Cryo - EM density of the interface between RBD and ACE2 . The density , shown as pink meshes , is contoured at 12 σ .

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Cryo-EM density of the interface between RBD and ACE2.The density, shown as pink meshes, is contoured at 12 σ.


A , Misreading ( A ) were measured by dual luciferase gain - of - function assays using wild - type mitohybrid ribosomes ( n = 8 misreading , mutant A1555G mitohybrid ribosomes ( n = 8 misreading , E . coli wild - type ribosomes ( n = 9 ) , E . coli mutant A127Y ribosomes ( n = 9 ) , M . smegmatis merodiploid wild - type ribosomes ( n = 9 ) , and M . smegmatis merodiploid mutant S152Y ribosomes ( n = 9 ) . Mistranslation was determined by assessing the misreading at His245 ( CAC codon ) of the F - luc gene that was replaced by the near - cognate codon CGC or the non - cognate codon AGA , both encoding for Arg . Results are derived by calculating mutant hFluc / hRluc activity related to wild - type hFluc / hRluc activity .

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A, Misreading (A) were measured by dual luciferase gain-of-function assays using wild-type mitohybrid ribosomes (n=8 misreading, mutant A1555G mitohybrid ribosomes (n=8 misreading, E. coli wild-type ribosomes (n=9), E.coli mutant A127Y ribosomes (n=9), M. smegmatis merodiploid wild-type ribosomes (n=9), and M. smegmatis merodiploid mutant S152Y ribosomes (n=9). Mistranslation was determined by assessing the misreading at His245 (CAC codon) of the F-luc gene that was replaced by the near-cognate codon CGC or the non-cognate codon AGA, both encoding for Arg. Results are derived by calculating mutant hFluc/hRluc activity related to wild-type hFluc/hRluc activity.


read - through ( B ) were measured by dual luciferase gain - of - function assays using wild - type mitohybrid ribosomes n = 4 read - through ) , mutant A1555G mitohybrid ribosomes n = 4 read - through ) , E . coli wild - type ribosomes ( n = 9 ) , E . coli mutant A127Y ribosomes ( n = 9 ) , M . smegmatis merodiploid wild - type ribosomes ( n = 9 ) , and M . smegmatis merodiploid mutant S152Y ribosomes ( n = 9 ) . To determine read - through the Asp357 ( GAC codon ) of the F - luc gene was replaced by the nonsense TGA stop codon . Results are derived by calculating mutant hFluc / hRluc activity related to wild - type hFluc / hRluc activity .

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read-through (B) were measured by dual luciferase gain-of-function assays using wild-type mitohybrid ribosomes n=4 read-through), mutant A1555G mitohybrid ribosomes n=4 read-through), E. coli wild-type ribosomes (n=9), E.coli mutant A127Y ribosomes (n=9), M. smegmatis merodiploid wild-type ribosomes (n=9), and M. smegmatis merodiploid mutant S152Y ribosomes (n=9). To determine read-through the Asp357 (GAC codon) of the F-luc gene was replaced by the nonsense TGA stop codon. Results are derived by calculating mutant hFluc/hRluc activity related to wild-type hFluc/hRluc activity.


C Ratio of 35S - Cysteine and 35S - Methionine incorporation by in - vitro translation using wild - type mitohybrid ribosomes , mutant A1555G mitohybrid ribosomes , and MT - CO1 mRNA . Following immunoprecipitation , the MT - CO1 band was quantified by autoradiography , wild - type mitohybrid ribosomes were set as 1 ( n = 3 ) , error bars indicate SEM ; * * * * p < 0 . 001 ( Student ' s t - test ) .

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C Ratio of 35S-Cysteine and 35S-Methionine incorporation by in-vitro translation using wild-type mitohybrid ribosomes, mutant A1555G mitohybrid ribosomes, and MT-CO1 mRNA. Following immunoprecipitation, the MT-CO1 band was quantified by autoradiography, wild-type mitohybrid ribosomes were set as 1 (n=3), error bars indicate SEM; ****p<0.001 (Student's t-test).


D Ratio of 35S - Cysteine and 35S - Methionine incorporation by in - vitro translation in the presence of tobramycin using wild - type mitohybrid ribosomes , mutant A1555G mitohybrid ribosomes , and MT - CO1 mRNA ; the MT - CO1 band was quantified by autoradiography .

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D Ratio of 35S-Cysteine and 35S-Methionine incorporation by in-vitro translation in the presence of tobramycin using wild-type mitohybrid ribosomes, mutant A1555G mitohybrid ribosomes, and MT-CO1 mRNA; the MT-CO1 band was quantified by autoradiography.


E Misreading of wild - type mitohybrid ribosomes and mutant A1555G mitohybrid ribosomes in the presence of tobramycin . Misreading was assessed by dual luciferase assay , and results derived by calculating mutant hFluc / hRluc activity related to wild - type hFluc / hRluc activity .

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E Misreading of wild-type mitohybrid ribosomes and mutant A1555G mitohybrid ribosomes in the presence of tobramycin. Misreading was assessed by dual luciferase assay, and results derived by calculating mutant hFluc/hRluc activity related to wild-type hFluc/hRluc activity.


F In - vitro translation of MT - CO1 - TGA mRNA and MT - CO1 - AGA mRNA using mutant A1555G mitohybrid ribosomes in the presence of tobramycin ( 0 - 20 µM ) . Autoradiography of immunoprecipitated MT - CO1 proteins ; top : 35S - Met labelling , bottom : 14C - Lys labelling . In - organello translated 35S - Met labelled MT - CO1 was used as a marker . * MT - CO1 , * * MT - CO1 extended by read - through , MT - CO1 AGA - polyA and MT - CO1 TGA - polyA constructs used for in - vitro translation are schematized .

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F In-vitro translation of MT-CO1-TGA mRNA and MT-CO1-AGA mRNA using mutant A1555G mitohybrid ribosomes in the presence of tobramycin (0 - 20 µM). Autoradiography of immunoprecipitated MT-CO1 proteins; top: 35S-Met labelling, bottom: 14C-Lys labelling. In-organello translated 35S-Met labelled MT-CO1 was used as a marker. * MT-CO1, ** MT-CO1 extended by read-through, MT-CO1 AGA-polyA and MT-CO1 TGA-polyA constructs used for in-vitro translation are schematized.


G Ratio of 14C - Lys and 35S - Met labelled immunoprecipitated MT - CO1 proteins . In - vitro translation using MT - CO1 - TGA mRNA , MT - CO1 - AGA mRNA , and mutant A1555G mitohybrid ribosomes in the presence of tobramycin ( 0 - 20 µM ) . The corresponding MT - CO1 band was quantified by autoradiography .

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G Ratio of 14C-Lys and 35S-Met labelled immunoprecipitated MT-CO1 proteins. In-vitro translation using MT-CO1-TGA mRNA, MT-CO1-AGA mRNA, and mutant A1555G mitohybrid ribosomes in the presence of tobramycin (0 - 20 µM). The corresponding MT-CO1 band was quantified by autoradiography.


A Autoradiography of in - organello mitochondrial translation products derived from HEK293 wild - type cells in the presence of tobramycin , incorporation of 35S - Met and 35S - Cys in MT - CO1 protein . Following in - organello translation proteins were immunoprecipitated and analyzed by autoradiography . Lane 1 , 3 : controls ; lanes 2 , 4 : in - organello translation in the presence of 1000 µM tobramycin . MT - CO1 bands were quantified and ratio of 35S - Cys / 35S - Met calculated , the 35S - Cys / 35S - Met ratio in the absence of tobramycin was set as 1 ( n = 3 ) , error bars indicate SEM .

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A Autoradiography of in-organello mitochondrial translation products derived from HEK293 wild-type cells in the presence of tobramycin, incorporation of 35S-Met and 35S-Cys in MT-CO1 protein. Following in-organello translation proteins were immunoprecipitated and analyzed by autoradiography. Lane 1, 3: controls; lanes 2, 4: in-organello translation in the presence of 1000 µM tobramycin. MT-CO1 bands were quantified and ratio of 35S-Cys / 35S-Met calculated, the 35S-Cys / 35S-Met ratio in the absence of tobramycin was set as 1 (n=3), error bars indicate SEM.


B Ratios of 35S - Cys / 35S - Met labelled MT - CO1 and MT - CO2 proteins synthesized in in - organello translation and effect of tobramycin . Following in - organello translation in the presence of tobramycin ( 1000 µM , 2000 µM ) proteins were immunoprecipitated and analyzed by autoradiography . MT - CO1 and MT - CO2 bands were quantified and ratio of 35S - Cys / 35S - Met incorporation calculated ( n = 3 ) , error bars indicate SEM ; * p < 0 . 05 , ns , not significant ( Student ' s t - test ) .

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B Ratios of 35S-Cys / 35S-Met labelled MT-CO1 and MT-CO2 proteins synthesized in in-organello translation and effect of tobramycin. Following in-organello translation in the presence of tobramycin (1000 µM, 2000 µM) proteins were immunoprecipitated and analyzed by autoradiography. MT-CO1 and MT-CO2 bands were quantified and ratio of 35S-Cys / 35S-Met incorporation calculated (n=3), error bars indicate SEM; *p<0.05, ns, not significant (Student's t-test).


Ratio of 35S - Cys and 35S - Met labelled MT - CO1 protein synthesized in in - organello translation using mitochondria from HEK293 MRPS5 wild - type cells and HEK MRPS5 mutant V336Y cells . Following in - organello translation , proteins were immunoprecipitated and analyzed by autoradiography . MT - CO1 bands quantified and ratios calculated . The ratio of the MRPS5 wild - type was set as 1 ( n = 8 clones ) , error bars indicate SD ; * * * p < 0 . 0001 ( Student ' s t - test ) .

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Ratio of 35S-Cys and 35S-Met labelled MT-CO1 protein synthesized in in-organello translation using mitochondria from HEK293 MRPS5 wild-type cells and HEK MRPS5 mutant V336Y cells. Following in-organello translation, proteins were immunoprecipitated and analyzed by autoradiography. MT-CO1 bands quantified and ratios calculated. The ratio of the MRPS5 wild-type was set as 1 (n=8 clones), error bars indicate SD; ***p<0.0001 (Student's t-test).


D Autoradiography of in - organello mitochondrial translation products derived from HEK293 MRPS5 wild - type cells in the presence of tobramycin ( 0 - 750 µM ) . Proteins were 35S - Met labeled and loaded either directly on SDS - PAGE ( total , top ) or following poly - lysine immunoprecipitation ( bottom ) . * MT - CO1 and * * MT - CO1 with poly - lysine extension .

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D Autoradiography of in-organello mitochondrial translation products derived from HEK293 MRPS5 wild-type cells in the presence of tobramycin (0-750 µM). Proteins were 35S-Met labeled and loaded either directly on SDS-PAGE (total, top) or following poly-lysine immunoprecipitation (bottom). * MT-CO1 and ** MT-CO1 with poly-lysine extension.


E Quantification of MT - CO1 proteins in HEK293 MRPS5 wild - type cells and HEK293 MRPS5 mutant V336Y cells . In - organello mitochondrial translation in the presence of 35S - Met and 750 µM tobramycin . Proteins were loaded either directly on SDS - PAGE ( total ) or following poly - lysine immunoprecipitation and analyzed by autoradiography . For total protein samples the MT - CO1 band was quantified , for poly - lysine immunoprecipitated samples the extended MT - CO1 band was quantified and the ratio of extended MT - CO1 / total MT - CO1 calculated . The ratio of MRPS5 wild - type was set as 1 ( MRPS5 wild - type , n = 5 clones , MRPS5 V336Y , n = 7 clones ) , error bars indicate SD ; * * * p < 0 . 0001 ( Student ' s t - test ) .

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E Quantification of MT-CO1 proteins in HEK293 MRPS5 wild-type cells and HEK293 MRPS5 mutant V336Y cells. In-organello mitochondrial translation in the presence of 35S-Met and 750 µM tobramycin. Proteins were loaded either directly on SDS-PAGE (total) or following poly-lysine immunoprecipitation and analyzed by autoradiography. For total protein samples the MT-CO1 band was quantified, for poly-lysine immunoprecipitated samples the extended MT-CO1 band was quantified and the ratio of extended MT-CO1/total MT-CO1 calculated. The ratio of MRPS5 wild-type was set as 1 (MRPS5 wild-type, n=5 clones, MRPS5 V336Y, n=7 clones), error bars indicate SD; ***p<0.0001 (Student's t-test).


A Generation time ( n = 8 clones , ± SD ) .

A Generation time (n=8 clones, ± SD).


B Whole cell in - vivo mitochondrial translation as determined by 35S - Met incorporation ( n = 4 clones , ± SEM ) .

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B Whole cell in-vivo mitochondrial translation as determined by 35S-Met incorporation (n=4 clones, ± SEM).


C Autoradiography of in - organello translation using 35S - Met labelling .

C Autoradiography of in-organello translation using 35S-Met labelling.


Oxygen consumption rate ( OCR ) was measured in freshly isolated mitochondria from wild - type and mutant mouse cortex . A OCR in brain mitochondria from 19 - months mutant mice ( black circle ) compared with OCR from wild - type mice ( white circle ) . The sequential injection of mitochondrial substrates and inhibitors is indicated by dotted lines Values corresponding to the different respiratory states are shown .

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Oxygen consumption rate (OCR) was measured in freshly isolated mitochondria from wild-type and mutant mouse cortex. A OCR in brain mitochondria from 19-months mutant mice (black circle) compared with OCR from wild-type mice (white circle). The sequential injection of mitochondrial substrates and inhibitors is indicated by dotted lines Values corresponding to the different respiratory states are shown.


Oxygen consumption rate ( OCR ) was measured in freshly isolated mitochondria from wild - type and mutant mouse cortex . Baseline OCR , effect of age ( p < 0 . 0001 ) and genotype ( p < 0 . 05 ) .

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Oxygen consumption rate (OCR) was measured in freshly isolated mitochondria from wild-type and mutant mouse cortex. Baseline OCR, effect of age (p<0.0001) and genotype (p<0.05).


Oxygen consumption rate ( OCR ) was measured in freshly isolated mitochondria from wild - type and mutant mouse cortex . C State 3 OCR ( after addition of ADP ) , effect of age ( p < 0 . 001 ) and genotype ( p < 0 . 0001 ) .

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Oxygen consumption rate (OCR) was measured in freshly isolated mitochondria from wild-type and mutant mouse cortex. C State 3 OCR (after addition of ADP), effect of age (p<0.001) and genotype (p<0.0001).


Oxygen consumption rate ( OCR ) was measured in freshly isolated mitochondria from wild - type and mutant mouse cortex . D State 3 uncoupled OCR ( state 3u after addition of FCCP ) , effect of genotype ( p < 0 . 0001 ) and age - genotype interaction ( p < 0 . 0001 ) .

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Oxygen consumption rate (OCR) was measured in freshly isolated mitochondria from wild-type and mutant mouse cortex. D State 3 uncoupled OCR (state 3u after addition of FCCP), effect of genotype (p<0.0001) and age-genotype interaction (p<0.0001).


E Ratio of mitochondrial complex IV and complex II activities .

E Ratio of mitochondrial complex IV and complex II activities.


A Average distance to center as measure of anxiety in the open field ( ANOVA : genotype F1 , 24 = 16 . 24 p = 0 . 0005 , age F2 , 24 = 5 . 217 p = 0 . 0131 ) . Mutants show increased anxiety levels at 9 - and 14 - months .

A Average distance to center as measure of anxiety in the open field (ANOVA: genotype F1,24=16.24 p=0.0005, age F2,24=5.217 p=0.0131). Mutants show increased anxiety levels at 9- and 14-months.


B Estimated frequency of rearing activity as measure of exploratory drive in the open field ( ANOVA : genotype F1 , 24 = 39 . 40 p < 0 . 0001 , age x genotype F2 , 26 = 8 . 204 p = 0 . 0017 ) . Mutants showed little exploratory activity at 9 - and 14 - months .

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B Estimated frequency of rearing activity as measure of exploratory drive in the open field (ANOVA: genotype F1, 24=39.40 p<0.0001, age x genotype F2,26=8.204 p=0.0017). Mutants showed little exploratory activity at 9- and 14-months.


C Light - dark transition test , % time spent in the bright compartment ( ANOVA : genotype F1 , 16 = 7 . 393 p = 0 . 0152 ) . Reduced scores at 9 - months old animals indicate increased anxiety .

C Light-dark transition test, % time spent in the bright compartment (ANOVA: genotype F1,16=7.393 p=0.0152). Reduced scores at 9-months old animals indicate increased anxiety.


D Exploratory corner visits in dark and light phase during adaptation to the IntelliCage ( ANOVA : phase F1 , 18 = 617 . 2 p < 0 . 0001 ) . Corner visits during the dark phase as a correlate of exploratory activity showed premature reduction in 9 - months old animals .

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D Exploratory corner visits in dark and light phase during adaptation to the IntelliCage (ANOVA: phase F1,18=617.2 p<0.0001). Corner visits during the dark phase as a correlate of exploratory activity showed premature reduction in 9-months old animals.


E Learning in the hippocampus - dependent IntelliCage serial reversal task ( ANOVA : day F2 , 56 = 26 . 71 p < 0 . 0001 ) . Scores provided no evidence for a mutation effect on learning rate or performance .

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E Learning in the hippocampus-dependent IntelliCage serial reversal task (ANOVA: day F2,56=26.71 p<0.0001). Scores provided no evidence for a mutation effect on learning rate or performance.


F Learning in the hippocampus - dependent IntelliCage chaining task ( ANOVA : day F2 , 54 = 450 . 1 p < 0 . 0001 ) . Scores provided no evidence for a mutation effect on learning rate or performance .

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F Learning in the hippocampus-dependent IntelliCage chaining task (ANOVA: day F2,54=450.1 p<0.0001). Scores provided no evidence for a mutation effect on learning rate or performance.


G Water - maze place navigation , swim path during acquisition and reversal ( ANOVA : day F4 , 104 = 21 . 14 p < 0 . 0001 , genotype F1 , 26 = 13 . 11 p = 0 . 0013 , age x genotype F2 , 26 = 8 . 204 p = 0 . 0017 ) . All groups learned : controls showed reduced performance with increasing age , mutants showed poor performance already at 9 - and 14 - months .

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G Water-maze place navigation, swim path during acquisition and reversal (ANOVA: day F4,104=21.14 p<0.0001, genotype F1,26=13.11 p=0.0013, age x genotype F2,26=8.204 p=0.0017). All groups learned: controls showed reduced performance with increasing age, mutants showed poor performance already at 9- and 14-months.


H Number of wall approaches as indicator of poor coping with the test and non - spatial strategy choice ( ANOVA : genotype F1 , 26 = 18 . 31 p = 0 . 0002 , age F2 , 26 = 4 . 486 p = 0 . 0212 , age x genotype F2 , 26 = 7 . 068 p = 0 . 0035 ) . Number of wall approaches increased with age in controls and was prematurely increased in mutants at 9 - and 14 - months .

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H Number of wall approaches as indicator of poor coping with the test and non-spatial strategy choice (ANOVA: genotype F1,26=18.31 p=0.0002, age F2,26=4.486 p=0.0212, age x genotype F2,26=7.068 p=0.0035). Number of wall approaches increased with age in controls and was prematurely increased in mutants at 9- and 14-months.


Water - maze probe trial , spatial retention score for target zones 1 - 3 comprising 5 , 10 and 10 % of the pool surface versus remaining 75 % of surface ( ANOVA : zone F3 , 78 = 36 . 04 p < 0 . 0001 , genotype x zone F3 , 78 = 7 . 807 p = 0 . 0001 ) . Spatial selectivity declined with age in controls and was prematurely reduced in mutants at 9 - and 14 - months .

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Water-maze probe trial, spatial retention score for target zones 1-3 comprising 5, 10 and 10% of the pool surface versus remaining 75% of surface (ANOVA: zone F3,78=36.04 p<0.0001, genotype x zone F3,78=7.807 p=0.0001). Spatial selectivity declined with age in controls and was prematurely reduced in mutants at 9- and 14-months.


Noise exposure and ABR measurements . Exposure of mice to broadband noise ( 2 - 10 kHz ) of either 108 or 110 dB SPL resulted in elevations of auditory thresholds . Thresholds in Mrps5V338Y / V338Y mutant mice ( black bars ) were significantly higher after exposure than thresholds in littermates ( open bars ) , ( p < 0 . 0001 , two - way ANOVA for combined 108 and 110 dB data ) . Frequencies of 12 , 24 and 32 kHz were selected for measurement to cover the mid - frequency to high - frequency regions of the cochlea . n = 6 for baseline littermates ; n = 6 for baseline mutants ; n = 3 for all noise exposures .

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Noise exposure and ABR measurements. Exposure of mice to broadband noise (2-10 kHz) of either 108 or 110 dB SPL resulted in elevations of auditory thresholds. Thresholds in Mrps5V338Y/V338Y mutant mice (black bars) were significantly higher after exposure than thresholds in littermates (open bars), (p<0.0001, two-way ANOVA for combined 108 and 110 dB data). Frequencies of 12, 24 and 32 kHz were selected for measurement to cover the mid-frequency to high-frequency regions of the cochlea. n=6 for baseline littermates; n=6 for baseline mutants; n=3 for all noise exposures.


B . ITC binding assays of the UVR8 and HY5 VP - peptides versus the COP1 WD40 domain . The top panel represents the heats detected during each injection . The bottom panel represents the integrated heats of each injection , fitted to a one - site binding model ( solid line ) . The following concentrations were typically used ( titrant into cell ) : UVR8 - COP1 ( 2500 μM in 175 μM ) ; HY5 - COP1 ( 1500 μM in 175 μM ) . The insets show the dissociation constants ( Kd ) and stoichiometries of binding ( N ) ( ± standard deviation ) .

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

B. ITC binding assays of the UVR8 and HY5 VP-peptides versus the COP1 WD40 domain. The top panel represents the heats detected during each injection. The bottom panel represents the integrated heats of each injection, fitted to a one-site binding model (solid line). The following concentrations were typically used (titrant into cell): UVR8 - COP1 (2500 μM in 175 μM); HY5 - COP1 (1500 μM in 175 μM). The insets show the dissociation constants (Kd) and stoichiometries of binding (N) (± standard deviation).


E . ITC assays of the HY5 and UVR8 VP - peptides versus the different COP1 WD40 domain mutants The following concentrations were typically used ( titrant into cell ) : UVR8 - COP1Lys422Ala ( 1000 μM in 100 μM ) ; UVR8 - COP1Tyr441Ala ( 2500 μM in 90 μM ) ; UVR8 - COP1Trp467Ala ( 2500 μM in 90 μM ) ; HY5 - COP1Lys422Ala ( 1500 μM in 175 μM ) ; HY5 - COP1Tyr441Ala ( 1600 μM in 138 μM ) ; HY5 - COP1Trp467Ala ( 1600 μM in 112 μM ) . The insets show the dissociation constants ( Kd ) . The stoichiometries of binding ( N ) for UVR8 - COP1Lys422Ala = 0 . 87 ± 0 . 2 ; HY5 - COP1Lys422Ala = 0 . 83 ± 0 . 2 ; HY5 - COP1Tyr441Ala = 0 . 92 ± 0 . 3 ( All measurements ± standard deviation ; n . d . : no detectable binding ) .

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

E. ITC assays of the HY5 and UVR8 VP-peptides versus the different COP1 WD40 domain mutants The following concentrations were typically used (titrant into cell): UVR8 - COP1Lys422Ala (1000 μM in 100 μM); UVR8 - COP1Tyr441Ala (2500 μM in 90 μM); UVR8 - COP1Trp467Ala (2500 μM in 90 μM); HY5 - COP1Lys422Ala (1500 μM in 175 μM); HY5 - COP1Tyr441Ala (1600 μM in 138 μM); HY5 - COP1Trp467Ala (1600 μM in 112 μM). The insets show the dissociation constants (Kd). The stoichiometries of binding (N) for UVR8 - COP1Lys422Ala = 0.87 ± 0.2; HY5 - COP1Lys422Ala = 0.83 ± 0.2; HY5 - COP1Tyr441Ala = 0.92 ± 0.3 (All measurements ± standard deviation; n.d.: no detectable binding).


Images of representative individuals ( F ) of 4 - day - old seedlings grown with or without supplemental UV - B . The scale bar for all lines represent 5 mm except for cop1 - 5 where the scale bars represent 1 mm . Data Information , lines used : wild - type ( Ws ) , uvr8 - 7 , cop1 - 4 , cop1 - 5 / Pro35S : YFP - COP1 ( WT ) , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala , cop1 - 5 / Pro35S : YFP - COP1Trp467Ala and cop1 - 5 . # 1 and # 2 : independent transgenic lines .

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

Images of representative individuals (F) of 4-day-old seedlings grown with or without supplemental UV-B. The scale bar for all lines represent 5 mm except for cop1-5 where the scale bars represent 1 mm. Data Information , lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.


quantification of hypocotyl lengths ( G ) of 4 - day - old seedlings grown with or without supplemental UV - B . The scale bar for all lines represent 5 mm except for cop1 - 5 where the scale bars represent 1 mm . Violin and box plots are shown for n > 60 seedlings ; upper and lower hinges correspond to the first and third quartiles , the horizontal line in the interior of the box indicates the median . Data Information : lines used : wild - type ( Ws ) , uvr8 - 7 , cop1 - 4 , cop1 - 5 / Pro35S : YFP - COP1 ( WT ) , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala , cop1 - 5 / Pro35S : YFP - COP1Trp467Ala and cop1 - 5 . # 1 and # 2 : independent transgenic lines .

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

quantification of hypocotyl lengths (G) of 4-day-old seedlings grown with or without supplemental UV-B. The scale bar for all lines represent 5 mm except for cop1-5 where the scale bars represent 1 mm. Violin and box plots are shown for n > 60 seedlings; upper and lower hinges correspond to the first and third quartiles, the horizontal line in the interior of the box indicates the median. Data Information: lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.


Quantitative real - time PCR analysis of ( H ) HY5 expression . Four - day - old seedlings grown in white light were exposed to narrowband UV - B for 2 hours ( + UV - B ) , or not ( - UV - B ) . Error bars represent SEM of 3 biological replicates . Data Information : lines used : wild - type ( Ws ) , uvr8 - 7 , cop1 - 4 , cop1 - 5 / Pro35S : YFP - COP1 ( WT ) , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala , cop1 - 5 / Pro35S : YFP - COP1Trp467Ala and cop1 - 5 . # 1 and # 2 : independent transgenic lines .

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

Quantitative real-time PCR analysis of (H) HY5 expression. Four-day-old seedlings grown in white light were exposed to narrowband UV-B for 2 hours (+UV-B), or not (-UV-B). Error bars represent SEM of 3 biological replicates. Data Information: lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.


Quantitative real - time PCR analysis of ( I ) RUP2 expression . Four - day - old seedlings grown in white light were exposed to narrowband UV - B for 2 hours ( + UV - B ) , or not ( - UV - B ) . Error bars represent SEM of 3 biological replicates . Data Information lines used : wild - type ( Ws ) , uvr8 - 7 , cop1 - 4 , cop1 - 5 / Pro35S : YFP - COP1 ( WT ) , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala , cop1 - 5 / Pro35S : YFP - COP1Trp467Ala and cop1 - 5 . # 1 and # 2 : independent transgenic lines .

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

Quantitative real-time PCR analysis of (I) RUP2 expression. Four-day-old seedlings grown in white light were exposed to narrowband UV-B for 2 hours (+UV-B), or not (-UV-B). Error bars represent SEM of 3 biological replicates. Data Information lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.


A . ITC assay between the COP1 WD40 domain and full - length UVR8 pre - monomerized by UV - B light . Integrated heats are shown in solid , purple squares . For comparison , an ITC experiment between UVR8 VP peptide and the COP1 WD40 domain ( from Fig 1B ) is also shown in open , purple squares . The following concentrations were typically used ( titrant into cell ) : COP1 - UVR8 + UV - B ( 130 μM in 20 μM ) . The inset shows the dissociation constant ( Kd ) , stoichiometry of binding ( N ) ( ± standard deviation ) .

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

A. ITC assay between the COP1 WD40 domain and full-length UVR8 pre-monomerized by UV-B light. Integrated heats are shown in solid, purple squares. For comparison, an ITC experiment between UVR8 VP peptide and the COP1 WD40 domain (from Fig 1B) is also shown in open, purple squares. The following concentrations were typically used (titrant into cell): COP1 - UVR8 +UV-B (130 μM in 20 μM). The inset shows the dissociation constant (Kd), stoichiometry of binding (N) (± standard deviation).


B . Binding kinetics of UVR8 pre - monomerized by UV - B versus the COP1 WD40 domain obtained by grating - coupled interferometry ( GCI ) . Sensorgrams of UVR8 injected are shown in red , with their respective 1 : 1 binding model fits in black . The following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - UVR8 + UV - B ( highest concentration 2 μM ) . ka = association rate constant , kd = dissociation rate constant , Kd = dissociation constant .

O O O O O B-MEASURED_VAR 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 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 B-MEASURED_VAR 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

B. Binding kinetics of UVR8 pre-monomerized by UV-B versus the COP1 WD40 domain obtained by grating-coupled interferometry (GCI). Sensorgrams of UVR8 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8 +UV-B (highest concentration 2 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant.


D . Binding kinetics of UVR8ValPro / AlaAla pre - monomerized by UV - B versus the COP1 WD40 domain obtained by GCI experiments . Sensorgrams of UVR8 injected are shown in red , with their respective 1 : 1 binding model fits in black . The following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - UVR8ValPro / AlaAla + UV - B ( highest concentration 2 μM ) . ka = association rate constant , kd = disassociation rate constant , Kd = dissociation constant .

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

D. Binding kinetics of UVR8ValPro/AlaAla pre-monomerized by UV-B versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of UVR8 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8ValPro/AlaAla +UV-B (highest concentration 2 μM). ka = association rate constant, kd = disassociation rate constant, Kd = dissociation constant.


E . Binding kinetics of HY5 versus the COP1 WD40 domain obtained by GCI experiments . Sensorgrams of HY5 injected are shown in red , with their respective 1 : 1 binding model fits in black . The following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - HY5 ( highest concentration 2 μM ) . ka = association rate constant , kd = dissociation rate constant , Kd = dissociation constant .

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

E. Binding kinetics of HY5 versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of HY5 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - HY5 (highest concentration 2 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant.


F . Yeast 3 - hybrid analysis of the COP1 - HY5 interaction in the presence of UVR8 . ( Top ) Normalized Miller Units were calculated as a ratio of β - galactosidase activity in yeast grown under UV - B ( + UV - B ) versus yeast grown without UV - B ( - UV - B ) . Additionally , normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine , corresponding to induction ( - Met ) or repression ( + Met ) of Met25 promoter - driven UVR8 expression , respectively . Means and SEM for 3 biological repetitions are shown . ( Bottom ) representative filter lift assays . AD , activation domain ; BD , DNA binding domain ; Met , methionine .

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

F. Yeast 3-hybrid analysis of the COP1 - HY5 interaction in the presence of UVR8. (Top) Normalized Miller Units were calculated as a ratio of β-galactosidase activity in yeast grown under UV-B (+ UV-B) versus yeast grown without UV-B (- UV-B). Additionally, normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine, corresponding to induction (- Met) or repression (+ Met) of Met25 promoter-driven UVR8 expression, respectively. Means and SEM for 3 biological repetitions are shown. (Bottom) representative filter lift assays. AD, activation domain; BD, DNA binding domain; Met, methionine.


B . Representative image showing the phenotype of wild - type ( Ws ) , uvr8 - 7 and uvr8 - 7 / Pro35S : UVR8HY5C44 , uvr8 - 7 / Pro35S : UVR8HY5VP and uvr8 - 7 / Pro35S : UVR8TRIB1 seedlings grown for 4 days under white light ( - UV - B ) or white light supplemented with UV - B ( + UV - B ) . The scale bar represents 5 mm .

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

B. Representative image showing the phenotype of wild-type (Ws), uvr8-7 and uvr8-7/Pro35S:UVR8HY5C44, uvr8-7/Pro35S:UVR8HY5VP and uvr8-7/Pro35S:UVR8TRIB1 seedlings grown for 4 days under white light (- UV-B) or white light supplemented with UV-B (+ UV-B). The scale bar represents 5 mm.


C . Quantification of hypocotyl length data Violin and box plots are shown for n > 60 seedlings ; upper and lower hinges correspond to the first and third quartiles , the horizontal line in the interior of the box indicates the median .

O O O O O O O O O O O O O O O O O O O O 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. Quantification of hypocotyl length data Violin and box plots are shown for n > 60 seedlings; upper and lower hinges correspond to the first and third quartiles, the horizontal line in the interior of the box indicates the median.


D . Anthocyanin accumulation in seedlings Average and SEM are shown ( n = 3 ) .

D. Anthocyanin accumulation in seedlings Average and SEM are shown (n = 3).


E . Quantitative real - time PCR analysis of RUP2 , CHS , HY5 and ELIP2 expression in wild - type ( Ws ) , uvr8 - 7 and uvr8 - 7 / Pro35S : UVR8HY5C44 , uvr8 - 7 / Pro35S : UVR8HY5VP and uvr8 - 7 / Pro35S : UVR8TRIB1 4 - day old seedlings grown under white light in response to 2h of UV - B ( + UV - B vs . - UV - B ) . Error bars represent SEM of 3 biological replicates . Note : the primers used to detect the HY5 transcript abundance also bind to an identical region present in UVR8HY5C44 chimera .

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

E. Quantitative real-time PCR analysis of RUP2, CHS, HY5 and ELIP2 expression in wild-type (Ws), uvr8-7 and uvr8-7/Pro35S:UVR8HY5C44, uvr8-7/Pro35S:UVR8HY5VP and uvr8-7/Pro35S:UVR8TRIB1 4-day old seedlings grown under white light in response to 2h of UV-B (+UV-B vs. -UV-B). Error bars represent SEM of 3 biological replicates. Note: the primers used to detect the HY5 transcript abundance also bind to an identical region present in UVR8HY5C44 chimera.


F . Yeast 3 - hybrid analysis of the COP1 - HY5 interaction in the presence of the UVR8HY5C44 chimera . ( Left pane ) Normalized Miller Units were calculated as a ratio of β - galactosidase activity in yeast grown under UV - B ( + UV - B ) versus yeast grown without UV - B ( - UV - B ) . Additionally , normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine , corresponding to induction ( - Met ) or repression ( + Met ) of Met25 promoter - driven UVR8 expression , respectively . Means and SEM for 3 biological repetitions are shown . ( Right panel ) Representative filter lift assays of the yeast analyzed in left panel . AD , activation domain ; BD , DNA binding domain ; Met , methionine .

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

F. Yeast 3-hybrid analysis of the COP1-HY5 interaction in the presence of the UVR8HY5C44 chimera. (Left pane) Normalized Miller Units were calculated as a ratio of β-galactosidase activity in yeast grown under UV-B (+ UV-B) versus yeast grown without UV-B (- UV-B). Additionally, normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine, corresponding to induction (- Met) or repression (+ Met) of Met25 promoter-driven UVR8 expression, respectively. Means and SEM for 3 biological repetitions are shown. (Right panel) Representative filter lift assays of the yeast analyzed in left panel. AD, activation domain; BD, DNA binding domain; Met, methionine.


G . Binding kinetics of the UVR8HY5C44 chimera pre - monomerized by UV - B versus the COP1 WD40 domain obtained by GCI experiments . Sensorgrams of UVR8HY5C44 injected are shown in red , with their respective 1 : 1 binding model fits in black . The following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - UVR8HY5C44 + UV - B ( highest concentration 2 μM ) . ka = association rate constant , kd = disassociation rate constant , Kd = dissociation constant .

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

G. Binding kinetics of the UVR8HY5C44 chimera pre-monomerized by UV-B versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of UVR8HY5C44 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8HY5C44 +UV-B (highest concentration 2 μM). ka = association rate constant, kd = disassociation rate constant, Kd = dissociation constant.


H . Size - exclusion chromatography assay of purified , recombinant chimeric proteins expressed in Sf9 insect cells in the presence and absence of UV - B .

O O O O 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. Size-exclusion chromatography assay of purified, recombinant chimeric proteins expressed in Sf9 insect cells in the presence and absence of UV-B.


A . Table summarizing affinities ( Kd , dissociation constant ) of VP - containing peptides versus the COP1 WD40 domain ( WT ) and COP1Lys422Ala as determined by ITC . All values are μM ( ± standard deviation ) .

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A. Table summarizing affinities (Kd, dissociation constant) of VP-containing peptides versus the COP1 WD40 domain (WT) and COP1Lys422Ala as determined by ITC. All values are μM (± standard deviation).


D . ITC assays between the COL3 VP - peptide ( left ) and CRY1 VP - peptide ( right ) versus the COP1 WD40 and COP1Lys422Ala , with a table summarizing their corresponding affinities . The following concentrations were typically used ( titrant into cell ) : COL3 - COP1 ( 2240 μM in 195 μM ) ; COL3 - COP1Lys422Ala ( 1500 μM in 175 μM ) ; CRY1 - COP1 ( 750 μM in 75 μM ) ; CRY1 - COP1Lys422Ala ( 1500 μM in 175 μM ) . The inset shows the dissociation constant ( Kd ) . The stoichiometries of binding ( N ) for COL3 - COP1 = 0 . 75 ± 0 . 2 ; COL3 - COP1Lys422Ala = 0 . 90 ± 0 . 2 ; CRY1 - COP1 = 0 . 99 ± 0 . 3 ; CRY1 - COP1Lys422Ala = 0 . 98 ± 0 . 1 ( All measurements ± standard deviation ) .

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D. ITC assays between the COL3 VP-peptide (left) and CRY1 VP-peptide (right) versus the COP1 WD40 and COP1Lys422Ala, with a table summarizing their corresponding affinities. The following concentrations were typically used (titrant into cell): COL3 - COP1 (2240 μM in 195 μM); COL3 - COP1Lys422Ala (1500 μM in 175 μM); CRY1 - COP1 (750 μM in 75 μM); CRY1 - COP1Lys422Ala (1500 μM in 175 μM). The inset shows the dissociation constant (Kd). The stoichiometries of binding (N) for COL3 - COP1 = 0.75 ± 0.2; COL3 - COP1Lys422Ala = 0.90 ± 0.2; CRY1 - COP1 = 0.99 ± 0.3; CRY1 - COP1Lys422Ala = 0.98 ± 0.1 (All measurements ± standard deviation).


A . Representative image of wild - type ( Ws ) , cop1 - 5 / Pro35S : YFP - COP1 , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala and cop1 - 5 / Pro35S : YFP - COP1Trp467Ala transgenic lines grown for 39 days in long day conditions .

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A. Representative image of wild-type (Ws), cop1-5/Pro35S:YFP-COP1, cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala and cop1-5/Pro35S:YFP-COP1Trp467Ala transgenic lines grown for 39 days in long day conditions.