( 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 .

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(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 .

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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 .

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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 .

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

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 .

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

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

Images of 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

quantification of 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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

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


B . Representative image of individual wild - type ( Ws ) , co - 11 , cop1 - 5 / Pro35S : YFP - COP1 and cop1 - 5 / Pro35S : YFP - COP1Lys422Ala plants grown for 39 days in long days conditions .

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B. Representative image of individual wild-type (Ws), co-11, cop1-5/Pro35S:YFP-COP1 and cop1-5/Pro35S:YFP-COP1Lys422Ala plants grown for 39 days in long days conditions.


C . Quantification of flowering time . Means and SD are shown ( n = 14 ) . D . Number of rosette and cauline leaves at flowering . Means and SD are shown ( n = 14 ) .

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C. Quantification of flowering time. Means and SD are shown (n = 14). D. Number of rosette and cauline leaves at flowering. Means and SD are shown (n = 14).


E . Immunoblot analysis of CRY2 and actin ( loading control ) protein levels in wild - type ( Col ) , cry2 - 1 , wild - type ( Ws ) , cop1 - 5 / Pro35S : YFP - COP1 and cop1 - 5 / Pro35S : YFP - COP1Lys422Ala seedlings grown for 4 days in darkness .

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E. Immunoblot analysis of CRY2 and actin (loading control) protein levels in wild-type (Col), cry2-1, wild-type (Ws), cop1-5/Pro35S:YFP-COP1 and cop1-5/Pro35S:YFP-COP1Lys422Ala seedlings grown for 4 days in darkness.


F . ITC assays between the CO VP - peptide ( left ) and CRY2 VP - peptide ( right ) versus the COP1 WD40 and the COP1Lys422Ala WD40 domains , with a table summarizing their corresponding affinities . The following concentrations were typically used ( titrant into cell ) : CO - COP1 ( 2240 μM in 195 μM ) ; CO - COP1Lys422Ala ( 1200 μM in 120 μM ) ; CRY2 - COP1 ( 750 μM in 70 μM ) ; CRY2 - COP1Lys422Ala ( 3000 μM in 175 μM ) . The stoichiometries of binding ( N ) for CO - COP1 = 1 . 03 ± 0 . 00 ; CO - COP1Lys422Ala = 0 . 89 ± 0 . 03 ; CRY2 - COP1 = 1 . 04 ± 0 . 20 ; CRY2 - COP1Lys422Ala = 1 ( fixed ) ( All measurements ± standard deviation ) .

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F. ITC assays between the CO VP-peptide (left) and CRY2 VP-peptide (right) versus the COP1 WD40 and the COP1Lys422Ala WD40 domains, with a table summarizing their corresponding affinities. The following concentrations were typically used (titrant into cell): CO - COP1 (2240 μM in 195 μM); CO - COP1Lys422Ala (1200 μM in 120 μM); CRY2 - COP1 (750 μM in 70 μM); CRY2 - COP1Lys422Ala (3000 μM in 175 μM). The stoichiometries of binding (N) for CO - COP1 = 1.03 ± 0.00; CO - COP1Lys422Ala = 0.89 ± 0.03; CRY2 - COP1 = 1.04 ± 0.20; CRY2 - COP1Lys422Ala = 1 (fixed) (All measurements ± standard deviation).


G , H . Binding kinetics of the ( G ) full - length activated CRY2 and ( H ) CRY2NC80 versus the COP1 WD40 domain obtained by GCI experiments . Sensorgrams of protein injected are shown in red , with their respective heterogenous ligand binding model fits in black . The following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - CRY2 ( highest concentration 14 μM ) , CRY2NC80 ( highest concentration 450 μM ) . ka = association rate constant , kd = dissociation rate constant , Kd = dissociation constant .

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G, H. Binding kinetics of the (G) full-length activated CRY2 and (H) CRY2NC80 versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of protein injected are shown in red, with their respective heterogenous ligand binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - CRY2 (highest concentration 14 μM), CRY2NC80 (highest concentration 450 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant.


A ) Western blot analysis of Mpl in healthy donors ' ( HD ) platelets shows two distinct bands representing the mature glycoprotein ( top band ) and the ER ‐ core glycosylated form ( bottom band ) . The ratio is ~ 1 .

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A) Western blot analysis of Mpl in healthy donors' (HD) platelets shows two distinct bands representing the mature glycoprotein (top band) and the ER‐core glycosylated form (bottom band). The ratio is ~ 1.


B ) Western blot analysis of HEL whole cell lysate after transfection with cDNA coding for JAK2WT or JAK2V617F , alone or cotransfected with cDNA coding for Mpl .

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B) Western blot analysis of HEL whole cell lysate after transfection with cDNA coding for JAK2WT or JAK2V617F, alone or cotransfected with cDNA coding for Mpl.


C ) Summary of the ratios of mature : immature Mpl for at least three independent ( 2 for the JAK2V617F transfection ) blotting experiments under the protocol shown in ( B ) . Based on two − tailed Student t − test statistical analysis , there is a significant difference ( p = 0 . 0001 ) in the ratio of Mpl isoforms in cells transfected with JAK2WT by comparison with control cells . Differences were also statistically significant in HEL cells transfected with either JAK2V617F or JAK2L611V / V617F by comparison to controls ( p = 0 . 0159 and p = 0 . 0009 , respectively ) . The mature : immature ratio is also significantly higher in cells transfected with JAK2WT than in cells transfected with JAK2V617F or JAK2L611V / V617F ( p < 0 . 01 ) .

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C) Summary of the ratios of mature:immature Mpl for at least three independent (2 for the JAK2V617F transfection) blotting experiments under the protocol shown in (B). Based on two−tailed Student t−test statistical analysis, there is a significant difference (p = 0.0001) in the ratio of Mpl isoforms in cells transfected with JAK2WT by comparison with control cells. Differences were also statistically significant in HEL cells transfected with either JAK2V617F or JAK2L611V/V617F by comparison to controls (p = 0.0159 and p = 0.0009, respectively). The mature:immature ratio is also significantly higher in cells transfected with JAK2WT than in cells transfected with JAK2V617F or JAK2L611V/V617F (p < 0.01).


D ) Western blot analysis of K562 lysates after transfection with cDNA coding for Mpl , alone or cotransfected with cDNA coding for JAK2WT or JAK2V617F .

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D) Western blot analysis of K562 lysates after transfection with cDNA coding for Mpl, alone or cotransfected with cDNA coding for JAK2WT or JAK2V617F.


E ) Summary of data in ( A ) , ( B ) and ( D ) , showing the relative band intensity of mature and immature Mpl and normalized for levels of β ‐ actin in each lane .

E) Summary of data in (A), (B) and (D), showing the relative band intensity of mature and immature Mpl and normalized for levels of β‐actin in each lane.


F ) Immunoprecipitation of the Mpl ‐ Jak2 complex in different cell lines analyzed by immunoblotting with anti ‐ Mpl and anti ‐ JAK2 antibodies .

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F) Immunoprecipitation of the Mpl‐Jak2 complex in different cell lines analyzed by immunoblotting with anti‐Mpl and anti‐JAK2 antibodies.


Confocal fluorescent images of HEL cells ( JAK2V617F + / + ) after immuno ‐ fluorescence labeling of endogenous Mpl ( A ) , or after transient transfection with cDNA coding for a chimeric Mpl protein fused to mOrange2 ( B ) .

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Confocal fluorescent images of HEL cells (JAK2V617F+/+) after immuno‐fluorescence labeling of endogenous Mpl (A), or after transient transfection with cDNA coding for a chimeric Mpl protein fused to mOrange2 (B).


C ) Confocal fluorescence image of K562 cells ( JAK2WT + / + ) transiently transfected with cDNA coding for MplmOrange2 . Red arrow in this image points to plasma membrane localization .

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C) Confocal fluorescence image of K562 cells (JAK2WT+/+) transiently transfected with cDNA coding for MplmOrange2. Red arrow in this image points to plasma membrane localization.


D and E ) Confocal fluorescent images obtained after transient expression of MplmOrange2 and immuno ‐ fluorescent labeling of endogenous JAK2 in K562 ( D ) or HEL ( E ) cells . Profiles of fluorescence ( across individual cells and indicated by a white arrow ) are showing a good colocalization of Mpl and JAK2WT in K562 cells at the plasma membrane ( black arrow heads ) but no colocalization have been observed between MplmOrange2 and JAK2V617F in HEL .

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D and E) Confocal fluorescent images obtained after transient expression of MplmOrange2 and immuno‐fluorescent labeling of endogenous JAK2 in K562 (D) or HEL (E) cells. Profiles of fluorescence (across individual cells and indicated by a white arrow) are showing a good colocalization of Mpl and JAK2WT in K562 cells at the plasma membrane (black arrow heads) but no colocalization have been observed between MplmOrange2 and JAK2V617F in HEL.


F and G ) Confocal fluorescent images obtained after transient coexpression of MplmOrange2 and either JAK2WTmCitrine ( F ) or JAK2V617FmCitrine ( G ) in K562 cells . Profiles of fluorescence ( white arrows ) are showing a very good colocalization of MplmOrange2 with JAK2WTmCitrine , especially at the plasma membrane ( black arrow heads ) , but a very poor colocalization with JAK2V617FmCitrine ( grey arrow heads ) . Nuclei are labeled with Hoechst and scale bars = 5 µm . R : Pearson ' s correlation coefficient .

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F and G) Confocal fluorescent images obtained after transient coexpression of MplmOrange2 and either JAK2WTmCitrine (F) or JAK2V617FmCitrine (G) in K562 cells. Profiles of fluorescence (white arrows) are showing a very good colocalization of MplmOrange2 with JAK2WTmCitrine, especially at the plasma membrane (black arrow heads), but a very poor colocalization with JAK2V617FmCitrine (grey arrow heads). Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.


A ) K562 cells labeled live for 30 min with an anti ‐ CD235a ‐ Atto655 ( glycophorin A ) antibody before treatment with the Duolink reagents .

A) K562 cells labeled live for 30 min with an anti‐CD235a‐Atto655 (glycophorin A) antibody before treatment with the Duolink reagents.


B ) K562Mpl ‐ mKO2 cells non ‐ treated with the Duolink reagents .

B) K562Mpl‐mKO2 cells non‐treated with the Duolink reagents.


C and D ) HEL cells primed live for 30 min with an anti ‐ CD235a ‐ Atto655 antibody before treatment with the Duolink reagents .

C and D) HEL cells primed live for 30 min with an anti‐CD235a‐Atto655 antibody before treatment with the Duolink reagents.


E and F ) K562Mpl ‐ mKO2 cells submitted to the Duolink reaction . White arrow heads are pointing at example of Duolink positive reactions at the plasma membrane , while green arrow heads are pointing at positive Duolink reactions within the cytoplasm . Nuclei are labeled with Hoechst and scale bars = 5 µm . R : Pearson ' s correlation coefficient .

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E and F) K562Mpl‐mKO2 cells submitted to the Duolink reaction. White arrow heads are pointing at example of Duolink positive reactions at the plasma membrane, while green arrow heads are pointing at positive Duolink reactions within the cytoplasm. Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.


HEL ( A ) and K562 ( B ) cells treated with non ‐ targeting ( NT ) or anti ‐ JAK2 siRNA analyzed by western blot .

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HEL (A) and K562 (B) cells treated with non‐targeting (NT) or anti‐JAK2 siRNA analyzed by western blot.


C ) Confocal live cell ( upper panel ) , or fixed cells ( lower left panel ) , imaging of K562 cells expressing MpleGFP or MplmKO2 treated with NT or anti ‐ JAK2 siRNA showing a difference of distribution of intracellular Mpl in the siRNA treated cells compare to the control cells . After treatment with anti ‐ JAK2 siRNA , Mpl strongly colocalizes with the ER marker , calnexin . This colocalization in treated vs non ‐ treated cells is quantified and shown in Figure C lower right panel . Nuclei are labeled with Hoechst and scale bars = 5 µm .

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C) Confocal live cell (upper panel), or fixed cells (lower left panel), imaging of K562 cells expressing MpleGFP or MplmKO2 treated with NT or anti‐JAK2 siRNA showing a difference of distribution of intracellular Mpl in the siRNA treated cells compare to the control cells. After treatment with anti‐JAK2 siRNA, Mpl strongly colocalizes with the ER marker, calnexin. This colocalization in treated vs non‐treated cells is quantified and shown in Figure C lower right panel. Nuclei are labeled with Hoechst and scale bars = 5 µm.


Intracellular Mpl pools partially colocalize with ER markers and colocalize with autophagic and lysosomal markers in K562 and HEL cells . A - M ) Confocal fluorescent images obtained after transient expression of MplmOrange2 or MplmKO2 and indicated labeling in K562 ( A - F ) or HEL ( G - M ) cells . Plots at right of each image show fluorescence intensities for each emission spectrum at points along the line drawn across each image . Black arrows in the plots point to overlap between MplmKO2 and either ER ‐ tracker ( C , I ) , or Sec31a ( B , J ) that marks ER exit sites . MplmOrange2 did not colocalize with markers for the Golgi ( Golgin ‐ 97 ) or early endosomes ( EEA1 ) in either cell line . However , MplmKO2 colocalized greatly with autophagic ( LC3 ) ( E , L ) and lysosomal ( LAMP1 ) ( F , M ) markers . Nuclei are labeled with Hoechst and scale bars = 5 µm . R : Pearson ' s correlation coefficient .

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Intracellular Mpl pools partially colocalize with ER markers and colocalize with autophagic and lysosomal markers in K562 and HEL cells. A-M) Confocal fluorescent images obtained after transient expression of MplmOrange2 or MplmKO2 and indicated labeling in K562 (A-F) or HEL (G-M) cells. Plots at right of each image show fluorescence intensities for each emission spectrum at points along the line drawn across each image. Black arrows in the plots point to overlap between MplmKO2 and either ER‐tracker (C,I), or Sec31a (B,J) that marks ER exit sites. MplmOrange2 did not colocalize with markers for the Golgi (Golgin‐97) or early endosomes (EEA1) in either cell line. However, MplmKO2 colocalized greatly with autophagic (LC3) (E,L) and lysosomal (LAMP1) (F,M) markers. Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.


Confocal fluorescent images obtained after transient expression of Mpl tagged with mKO2 and miniSOG in K562 ( A ) and HEL ( B ) cells . Electron micrographs of K562 ( C ) and HEL ( D ) cells transiently expressing MplmKO2 ‐ miniSOG . Dark precipitate indicates the presence of the miniSOG ‐ tagged Mpl protein after photo ‐ precipitation of DAB . White boxes indicate original location of higher magnification images with corresponding letters .

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Confocal fluorescent images obtained after transient expression of Mpl tagged with mKO2 and miniSOG in K562 (A) and HEL (B) cells. Electron micrographs of K562 (C) and HEL (D) cells transiently expressing MplmKO2‐miniSOG. Dark precipitate indicates the presence of the miniSOG‐tagged Mpl protein after photo‐precipitation of DAB. White boxes indicate original location of higher magnification images with corresponding letters.


E - G ) High magnification electron micrograph of K562 cells treated in the same conditions as Figure C , showing MplmKO2 ‐ miniSOG dark precipitate into autophagic carrier vesicles ( black arrows , Figure E ) . Osmiophilic reaction products also heavily stain mature AP in these preparations ( black arrows , Figure G ) .

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E-G) High magnification electron micrograph of K562 cells treated in the same conditions as Figure C, showing MplmKO2‐miniSOG dark precipitate into autophagic carrier vesicles (black arrows, Figure E). Osmiophilic reaction products also heavily stain mature AP in these preparations (black arrows, Figure G).


H ) High magnification electron micrograph of HEL cells treated in the same conditions as Figure D , cells transiently expressing MplmKO2 - miniSOG , showing different stage of formation of APs ( black arrows numbered from stages 1 - 4 ) . Scale bars = 5 µm ( A - D ) or 100 nm ( E - H ) .

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H) High magnification electron micrograph of HEL cells treated in the same conditions as Figure D, cells transiently expressing MplmKO2-miniSOG , showing different stage of formation of APs (black arrows numbered from stages 1-4). Scale bars = 5 µm (A-D) or 100 nm (E-H).


A ) Western blot analysis and quantification of streptavidin ‐ immunoprecipitated surface ‐ biotinylated proteins , probed with an anti ‐ Mpl

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A) Western blot analysis and quantification of streptavidin‐immunoprecipitated surface‐biotinylated proteins, probed with an anti‐Mpl antibody


B and C ) Confocal images of K562 cells transiently expressing MplmOrange2 . Cells in ( B ) are untreated . Cells in ( C ) were stimulated 60 ' with Tpo , washed and chased for 15 min before imaging .

B and C) Confocal images of K562 cells transiently expressing MplmOrange2. Cells in (B) are untreated. Cells in (C) were stimulated 60' with Tpo, washed and chased for 15 min before imaging.


D ) Quantification of plasma membrane fluorescence in resting and Tpo ‐ stimulated cells . Mean fluorescence at the membrane was estimated by averaging the total fluorescence after values equal to or below background . A two ‐ tailed Student t ‐ test shows a significant difference in the membrane fluorescence after stimulation with Tpo followed by Tpo removal . Scale bars = 5 µm .

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D) Quantification of plasma membrane fluorescence in resting and Tpo‐stimulated cells. Mean fluorescence at the membrane was estimated by averaging the total fluorescence after values equal to or below background. A two‐tailed Student t‐test shows a significant difference in the membrane fluorescence after stimulation with Tpo followed by Tpo removal. Scale bars = 5 µm.


E ) Western blot analysis of protein phosphorylation in HEL , K562Mpl ‐ mKO2 and K562Mpl ‐ mOrange2 cells after Tpo stimulation ( 50 ng / mL Tpo , 10 min ) .

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E) Western blot analysis of protein phosphorylation in HEL, K562Mpl‐mKO2 and K562Mpl‐mOrange2 cells after Tpo stimulation (50 ng/mL Tpo, 10 min).


A and B ) Surface biotinylation and total lysates western blot analysis of HEL or K562Mpl ‐ mKO2 cells after treatments for 60 min with Tpo , ionomycin ( I ) , PMA , Mon or A23187 .

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A and B) Surface biotinylation and total lysates western blot analysis of HEL or K562Mpl‐mKO2 cells after treatments for 60 min with Tpo, ionomycin (I), PMA, Mon or A23187.


C ) Total lysate , surface biotinylation and exosomal fraction western blot analysis of HEL and K562Mpl ‐ mKO2 cells treated with Mon or A23187 for 15 h .

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C) Total lysate, surface biotinylation and exosomal fraction western blot analysis of HEL and K562Mpl‐mKO2 cells treated with Mon or A23187 for 15 h.


D ) Western blot analysis of HEL cells treated , or not , with Bafilomycin A1 at 100 nm for 3 h and then stimulated or not with Tpo at 25 ng / mL for 30 min . The quantification of the blot is shown .

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D) Western blot analysis of HEL cells treated, or not, with Bafilomycin A1 at 100 nm for 3 h and then stimulated or not with Tpo at 25 ng/mL for 30 min. The quantification of the blot is shown.


E ) Surface biotinylation and total lysates western blot analysis of HEL cells after treatments for 30 min with Tpo or for 3 h with 3 − MA ( 3 − Methyladenine ) or Rp .

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E) Surface biotinylation and total lysates western blot analysis of HEL cells after treatments for 30 min with Tpo or for 3 h with 3−MA (3−Methyladenine) or Rp.


F ) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing ATG5 K130R ‐ V5 6 h post ‐ transfection .

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F) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing ATG5 K130R‐V5 6 h post‐transfection.


G ) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing either GRASP55 - V5 or HA - STX5 .

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G) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing either GRASP55-V5 or HA-STX5.


B , HeLa cells were treated with DMSO or 2µM Mito - C for 15 min and immunostained with anti - TOM20 ( green ) antibody and DAPI ; cropped areas show the mitochondria morphology changes .

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B, HeLa cells were treated with DMSO or 2µM Mito-C for 15 min and immunostained with anti-TOM20 (green) antibody and DAPI; cropped areas show the mitochondria morphology changes.


C , Quantification of mitochondria morphology ( based on TOM20 immunostaining as shown in B ) from HeLa cells treated with DMSO or 2µM Mito - C for 15 min or 2h , expressed as ratio of fragmented mitochondria ( percentage of total ) . Errors bars show the standard error of the mean ( SEM ) ( n = 100 , technical replicates ) .

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C, Quantification of mitochondria morphology (based on TOM20 immunostaining as shown in B) from HeLa cells treated with DMSO or 2µM Mito-C for 15 min or 2h, expressed as ratio of fragmented mitochondria (percentage of total). Errors bars show the standard error of the mean (SEM) (n=100, technical replicates).


E , Time lapse video - microscopy snapshot on HeLa cells transfected with NAF - 1 - mRFP ( red ) and treated with fluoMito - C ( green ) . The distance / intensity fluorescence quantification graph illustrates the codistribution of fluoMito - C and NAF - 1 - mRFP .

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E, Time lapse video-microscopy snapshot on HeLa cells transfected with NAF-1-mRFP (red) and treated with fluoMito-C (green). The distance/intensity fluorescence quantification graph illustrates the codistribution of fluoMito-C and NAF-1-mRFP.


A , Western blot analysis of MitoNEET expression in HeLa cells transfected with CISD1 siRNA ; Bar Chart ( right panel ) shows replicates quantification of MitoNEET expression . Errors bars show the standard deviation ( SD ) of 3 independent experiments .

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A, Western blot analysis of MitoNEET expression in HeLa cells transfected with CISD1 siRNA; Bar Chart (right panel) shows replicates quantification of MitoNEET expression. Errors bars show the standard deviation (SD) of 3 independent experiments.


B , Western blot analysis of NAF - 1 expression in HeLa cells transfected with CISD2 siRNA ; graph ( right panel ) shows the replicates quantification of NAF - 1 expression . Errors bars show the standard deviation ( SD ) of 3 independent experiments .

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B, Western blot analysis of NAF-1 expression in HeLa cells transfected with CISD2 siRNA; graph (right panel) shows the replicates quantification of NAF-1 expression. Errors bars show the standard deviation (SD) of 3 independent experiments.


D , HeLa cells transfected with siRNA to reduce expression of MitoNEET ( CISD1 ) , NAF - 1 ( CISD2 ) or MiNT ( CISD3 ) and immunostained with anti TOM20 antibody . Arrowheads indicate fragmented mitochondria .

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D, HeLa cells transfected with siRNA to reduce expression of MitoNEET (CISD1), NAF-1 (CISD2) or MiNT (CISD3) and immunostained with anti TOM20 antibody. Arrowheads indicate fragmented mitochondria.


E , Quantification of mitochondria morphology ( based on TOM20 immunostaining as shown in D ) from HeLa cells transfected with siRNA targeting mitoNEET ( siCISD1 ) , NAF1 ( siCISD2 ) or MiNT ( siCISD3 ) . Quantification is expressed as ratio of tubular and fragmented mitochondria . Errors bars show the standard error of the mean ( SEM ) . ( n = 125 - 130 cells ) .

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E, Quantification of mitochondria morphology (based on TOM20 immunostaining as shown in D) from HeLa cells transfected with siRNA targeting mitoNEET (siCISD1), NAF1 (siCISD2) or MiNT (siCISD3). Quantification is expressed as ratio of tubular and fragmented mitochondria. Errors bars show the standard error of the mean (SEM). (n =125-130 cells).


F , Electron microscopy pictures from 150 INS - 1 cells transfected as described in G .

F, Electron microscopy pictures from 150 INS-1 cells transfected as described in G.


G , Western blot analysis of NAF - 1 expression in cells stably transfected with control shRNA , shRNA targeting reduction of NAF - 1 protein expression ( shCISD2 ) , or shRNA targeting reduction of NAF - 1 protein expression complemented with plasmid derived expression of NAF - 1 ( shCISD2 + CISD2 ) ; western blot quantification of NAF - 1 is shown on the accompanying bar chart . Errors bars show the standard deviation ( SD ) of 3 independent experiments .

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G, Western blot analysis of NAF-1 expression in cells stably transfected with control shRNA, shRNA targeting reduction of NAF-1 protein expression (shCISD2), or shRNA targeting reduction of NAF-1 protein expression complemented with plasmid derived expression of NAF-1 (shCISD2+CISD2); western blot quantification of NAF-1 is shown on the accompanying bar chart. Errors bars show the standard deviation (SD) of 3 independent experiments.


H , Quantification of mitochondrial length in EM images from cells transfected as described in G . Errors bars show the standard error of the mean ( SEM ) ( n = 260 - 270 , technical replicates ) .

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H, Quantification of mitochondrial length in EM images from cells transfected as described in G. Errors bars show the standard error of the mean (SEM) (n=260-270, technical replicates).


A , HeLa cells treated or not , with 2µM Mito - C for 15 min were immunostained with anti - TOM20 ( red ) and anti - DRP1 ( green ) ; arrowheads in the far - right panel indicate recruitment of DRP1 onto the mitochondrial surface ( TOM20 ) . B , Quantification of DRP1 signal on TOM20 positive structures . Errors bars show the standard error of the mean ( SEM ) ( n = 45 , technical replicates ) .

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A, HeLa cells treated or not, with 2µM Mito-C for 15 min were immunostained with anti-TOM20 (red) and anti-DRP1 (green); arrowheads in the far-right panel indicate recruitment of DRP1 onto the mitochondrial surface (TOM20). B, Quantification of DRP1 signal on TOM20 positive structures. Errors bars show the standard error of the mean (SEM) (n=45, technical replicates). C


C , Western blot analysis of DRP1 , GAPDH ( cytosolic marker ) and TOM20 ( mitochondrial marker ) protein in cytosolic and mitochondrial fractions treated or not , with 2µM Mito - C as indicated . D , Quantification of western blots showed in C and expressed as a distribution of DRP1 in the cytosolic and mitochondrial fractions . Errors bars show the standard deviation ( SD ) of 5 independent experiments .

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C, Western blot analysis of DRP1, GAPDH (cytosolic marker) and TOM20 (mitochondrial marker) protein in cytosolic and mitochondrial fractions treated or not, with 2µM Mito-C as indicated. D, Quantification of western blots showed in C and expressed as a distribution of DRP1 in the cytosolic and mitochondrial fractions. Errors bars show the standard deviation (SD) of 5 independent experiments. E


E , HeLa cells were transfected with DRP1K38A mutant and mCherry and treated or not , with Mito - C ( T for transfected , NT for not transfected ) . F , Quantification of the mitochondrial phenotypes observed in E . Errors bars show the standard error of the mean ( SEM ) . ( 30 - 35 images each with an average of 15 - 20 cells from triplicate independent experiments were analyzed ) .

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E, HeLa cells were transfected with DRP1K38A mutant and mCherry and treated or not, with Mito-C (T for transfected, NT for not transfected). F, Quantification of the mitochondrial phenotypes observed in E. Errors bars show the standard error of the mean (SEM). (30-35 images each with an average of 15-20 cells from triplicate independent experiments were analyzed). G


G , Electron micrographs ( EM ) from HeLa cells treated with 2µM Mito - C or DMSO for 120 minutes . Empty arrowheads indicate ER - mitochondria contact sites . H , Quantification of ER - mitochondria membrane contact sites events ( reported in 50µm² section ; 50 images from an average of 30 cells from triplicate independent experiments were analyzed ) . Errors bars show the standard error of the mean ( SEM ) . I , Quantification of ER - mitochondria membrane contact sites density ( in µm ; 30 images from an average of 30 cells from triplicate independent experiments were analyzed ) . Errors bars show the standard error of the mean ( SEM ) .

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G, Electron micrographs (EM) from HeLa cells treated with 2µM Mito-C or DMSO for 120 minutes. Empty arrowheads indicate ER-mitochondria contact sites. H, Quantification of ER-mitochondria membrane contact sites events (reported in 50µm² section; 50 images from an average of 30 cells from triplicate independent experiments were analyzed). Errors bars show the standard error of the mean (SEM). I, Quantification of ER-mitochondria membrane contact sites density (in µm; 30 images from an average of 30 cells from triplicate independent experiments were analyzed). Errors bars show the standard error of the mean (SEM). Da


B , Huh7 cells were treated with or without ( DMSO ) Mito - C , infected with dengue virus and fixed after 48h of infection . Cells were then immunostained for endogenous TOM20 ( green channel ) , viral non - structural protein NS5 ( red ) and DAPI ( Blue ) . Cropped areas illustrate the mitochondrial morphology in described conditions .

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B, Huh7 cells were treated with or without (DMSO) Mito-C, infected with dengue virus and fixed after 48h of infection. Cells were then immunostained for endogenous TOM20 (green channel), viral non-structural protein NS5 (red) and DAPI (Blue). Cropped areas illustrate the mitochondrial morphology in described conditions.


C , Quantification of the mitochondrial phenotypes observed in b . Errors bars show the standard error of the mean ( SEM ) . ( 15 - 20 images each with an average of 20 - 25 cells from triplicate independent experiments were analyzed ) .

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C, Quantification of the mitochondrial phenotypes observed in b. Errors bars show the standard error of the mean (SEM). (15-20 images each with an average of 20-25 cells from triplicate independent experiments were analyzed).


( B ) Gal4 DNA - binding domain fusions of MUPs were tested with Gal4 activation domain fusions of ubiquilins ( murine ) in Y2H .

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(B) Gal4 DNA-binding domain fusions of MUPs were tested with Gal4 activation domain fusions of ubiquilins (murine) in Y2H.


( C ) HEK293 cells transfected with indicated MUP - V5 , myc - UBQLN1 ( human ) , or empty vector were immunoprecipitated ( IP ) with anti - myc or anti - UBQLN1 antibodies . Western blot ( WB ) of IP and lysate were probed as indicated .

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(C) HEK293 cells transfected with indicated MUP-V5, myc-UBQLN1 (human), or empty vector were immunoprecipitated (IP) with anti-myc or anti-UBQLN1 antibodies. Western blot (WB) of IP and lysate were probed as indicated.


( D - E ) WB of input lysate and IP from HEK293 cells expressing myc - UBQLN1 and Rv1926 - V5 ( D ) or Rv1566 - V5 ( E ) . IP was performed using antibodies directed against myc , V5 , or isotype control ( IgG ) .

(D-E) WB of input lysate and IP from HEK293 cells expressing myc-UBQLN1 and Rv1926-V5 (D) or Rv1566-V5 (E). IP was performed using antibodies directed against myc, V5, or isotype control (IgG).


( B ) Mtb were incubated with lysate from HEK293 cells transfected with vector control , myc - UBQLN1 , GFP , or V5 - parkin . WB analysis of the input ( I ) , 5th wash ( W5 ) , and elution ( E ) , probed with indicated antibodies . Actin is a loading control for the cell lysate and Ag85b for the bacterial pellet .

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(B) Mtb were incubated with lysate from HEK293 cells transfected with vector control, myc-UBQLN1, GFP, or V5-parkin. WB analysis of the input (I), 5th wash (W5), and elution (E), probed with indicated antibodies. Actin is a loading control for the cell lysate and Ag85b for the bacterial pellet.


( C ) Mtb were incubated with lysate from HEK293 cells transfected with myc - UBQLN1 - ΔUBA , myc - UBQLN1 - ΔUBL , and myc - UBQLN1 - UBA . WB containing the input , 5th wash , and elution were incubated with anti - myc antibody . The predicted sizes of myc - UBQLN1 - ΔUBL , myc - UBQLN1 - ΔUBA , and myc - UBQLN1 - UBA are 55 kD , 50 kD , and 6 kD , respectively .

(C) Mtb were incubated with lysate from HEK293 cells transfected with myc-UBQLN1-ΔUBA, myc-UBQLN1-ΔUBL, and myc-UBQLN1-UBA. WB containing the input, 5th wash, and elution were incubated with anti-myc antibody. The predicted sizes of myc-UBQLN1-ΔUBL, myc-UBQLN1-ΔUBA, and myc-UBQLN1-UBA are 55 kD, 50 kD, and 6 kD, respectively.


( D ) Mtb were incubated with recombinant GST - UBQLN1 , GST - UBQLN1 - ΔUBL , or GST - UBQLN1 - ΔUBA . WB containing the input and elution was probed with an anti - GST antibody .

(D) Mtb were incubated with recombinant GST-UBQLN1, GST-UBQLN1-ΔUBL, or GST-UBQLN1-ΔUBA. WB containing the input and elution was probed with an anti-GST antibody.


( A ) BMDMs were infected with GFP - expressing wt Mtb or ΔesxA for 24h and immunostained with UBQLN1 antibody . IFN - γ was added 1d prior to infection as indicated . ( B ) Quantification of results from A from 3 independent experiments with at least 100 bacteria analyzed per experiment ; * * * P < 0 . 001 , Fisher ' s exact test , comparing the proportion of UBQLN1 positive versus negative Mtb in indicated samples .

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(A) BMDMs were infected with GFP-expressing wt Mtb or ΔesxA for 24h and immunostained with UBQLN1 antibody. IFN-γ was added 1d prior to infection as indicated. (B) Quantification of results from A from 3 independent experiments with at least 100 bacteria analyzed per experiment; ***P< 0.001, Fisher's exact test, comparing the proportion of UBQLN1 positive versus negative Mtb in indicated samples.


( C ) Cellular lysate from BMDMs that were treated with IFN - γ as indicated were analyzed by WB with an antibody that recognizes UBQLN1 and UBQLN2 .

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(C) Cellular lysate from BMDMs that were treated with IFN-γ as indicated were analyzed by WB with an antibody that recognizes UBQLN1 and UBQLN2.


( D ) BMDMs were transfected with siRNAs targeting UBQLN1 or a non - targeting control ( siCON ) and lysates were analyzed by WB using an antibody that recognizes UBQLN1 and UBQLN2 . ( C - D ) Actin served as a loading control .

(D) BMDMs were transfected with siRNAs targeting UBQLN1 or a non-targeting control (siCON) and lysates were analyzed by WB using an antibody that recognizes UBQLN1 and UBQLN2. (C-D) Actin served as a loading control.


( E ) BMDMs were infected with Mtb live / dead strain and treated with AnTc 24h prior to fixation .

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(E) BMDMs were infected with Mtb live/dead strain and treated with AnTc 24h prior to fixation.


( F ) BMDMs were transfected with siRNAs targeting UBQLN1 or siCON and infected with live / dead Mtb strain . IFN - γ was added 1d prior to infection if indicated . % Viable bacteria were quantified 24 and 48 hpi . Data are from 2 independent experiments . * P < 0 . 05 ; * * * P < 0 . 001 , Fisher ' s exact test , comparing the proportion of viable and non - viable bacteria in indicated samples .

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(F) BMDMs were transfected with siRNAs targeting UBQLN1 or siCON and infected with live/dead Mtb strain. IFN-γ was added 1d prior to infection if indicated. %Viable bacteria were quantified 24 and 48 hpi. Data are from 2 independent experiments. *P<0.05; ***P<0.001, Fisher's exact test, comparing the proportion of viable and non-viable bacteria in indicated samples.


( G ) BMDMs were treated with the indicated siRNAs three days prior to infection and then activated with IFN - γ one day prior to infection with H37Rv or ΔesxA . CFU were enumerated 4 and 96 hpi . * P < 0 . 05 , unpaired Student ' s t - test . Data are representative of three independent experiments .

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(G) BMDMs were treated with the indicated siRNAs three days prior to infection and then activated with IFN-γ one day prior to infection with H37Rv or ΔesxA. CFU were enumerated 4 and 96 hpi. *P< 0.05, unpaired Student's t-test. Data are representative of three independent experiments.


( H ) BMDMs transfected with siRNA targeting UBQLN1 or siCON were infected with wt Mtb ( H37Rv ) and IFN - γ was measured by

(H) BMDMs transfected with siRNA targeting UBQLN1 or siCON were infected with wt Mtb (H37Rv) and IFN-γ was measured by ELISA


( I ) UBQLN1 - silenced BMDMs and controls were infected with H37Rv or ΔesxA . ( H - I ) 24 hpi infected BMDMs were co - cultured with P25TCR - Tg CD4 + T cells , and IFN - γ was measured by ELISA . Data is representative of 3 independent experiments . * P < 0 . 05 , * * P < 0 . 005 , unpaired Student ' s t - test . ( B , F , Γ , H ) Results are mean + / - SEM ; ns , not significant .

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(I) UBQLN1-silenced BMDMs and controls were infected with H37Rv or ΔesxA. (H-I) 24 hpi infected BMDMs were co-cultured with P25TCR-Tg CD4+ T cells, and IFN-γ was measured by ELISA. Data is representative of 3 independent experiments. *P<0.05, **P<0.005, unpaired Student's t-test. (B, F, Γ, H) Results are mean +/- SEM; ns, not significant.


( A ) BMDMs treated with IFN - γ and infected for 24h with GFP - Mtb were immunostained for UBQLN1 and either ubiquitin ( using the FK2 antibody ) or p62 . GFP - LC3 BMDMs infected with DsRed - Mtb were immunostained for UBQLN1 . ( B ) Quantification of those Mtb that co - localize with at least one of the cellular markers shown in A . More than 375 total bacteria were counted in two independent experiments , and the number co - localizing with at least one marker is indicated as " total " under each bar .

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(A) BMDMs treated with IFN-γ and infected for 24h with GFP-Mtb were immunostained for UBQLN1 and either ubiquitin (using the FK2 antibody) or p62. GFP-LC3 BMDMs infected with DsRed-Mtb were immunostained for UBQLN1. (B) Quantification of those Mtb that co-localize with at least one of the cellular markers shown in A. More than 375 total bacteria were counted in two independent experiments, and the number co-localizing with at least one marker is indicated as "total" under each bar.


( C ) Wt macrophages ( Atg16L1flox / flox ; Cre - ) and autophagy - deficient macrophages ( Atg16L1flox / flox Lyz - cre ; Cre + ) were activated with IFN - γ or not , followed by infection with the live / dead Mtb reporter strain . % viable was quantified after addition of AnTc . * * P < 0 . 005 , * * * P < 0 . 0001 , Fisher ' s exact test ( in which the proportions of viable to non - viable Mtb was compared ) .

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(C) Wt macrophages (Atg16L1flox/flox; Cre-) and autophagy-deficient macrophages (Atg16L1flox/flox Lyz-cre; Cre+) were activated with IFN-γ or not, followed by infection with the live/dead Mtb reporter strain. % viable was quantified after addition of AnTc. **P<0.005, ***P<0.0001, Fisher's exact test (in which the proportions of viable to non-viable Mtb was compared).


( D ) Atg16L1flox / flox Cre + and Cre - BMDMs treated with IFN - γ and infected for 24h with GFP - Mtb were immunostained for UBQLN1 and p62 . The number of Mtb co - localizating with the cellular markers is shown . ( C - D ) At least 100 bacteria were analyzed in three independent experiments .

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(D) Atg16L1flox/flox Cre+ and Cre- BMDMs treated with IFN-γ and infected for 24h with GFP-Mtb were immunostained for UBQLN1 and p62. The number of Mtb co-localizating with the cellular markers is shown. (C-D) At least 100 bacteria were analyzed in three independent experiments.


( E ) Images from IFN - γ treated BMDMs of indicated genotype infected with GFP - Mtb and immunostained with UBQLN1 and p62 . Arrows indicates p62 + UBQLN1 + Mtb .

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(E) Images from IFN-γ treated BMDMs of indicated genotype infected with GFP-Mtb and immunostained with UBQLN1 and p62. Arrows indicates p62+ UBQLN1+ Mtb.


( A - C ) BMDMs transfected with siRNA targeting UBQLN1 or control siRNA . Images show IFN - γ treated macrophages infected with GFP - Mtb ( A - B ) or DsRed - Mtb ( C ) for 24h . Cells were immunostained for ubiquitin ( Ub ) with the FK2 antibody ( A ) or p62 ( B ) , which are shown in red . ( C ) GFP - LC3 BMDMs were infected with DsRed - Mtb . LC3 is shown in green , whereas Mtb are red . ( A - C ) Quantification shows the percentage of Mtb that colocalized with the indicated cellular marker in macrophages that were treated with IFN - γ as indicated . Results are mean + / - SEM from two independent experiments , with at least 100 bacteria analyzed per experiment . * * * P < 0 . 001 , Fisher ' s exact test ( in which the proportion of Mtb associated with the cellular marker in a given condition was compared to the proportion found in the sample treated with siCON and IFN - γ ) .

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(A-C) BMDMs transfected with siRNA targeting UBQLN1 or control siRNA. Images show IFN-γ treated macrophages infected with GFP-Mtb (A-B) or DsRed-Mtb (C) for 24h. Cells were immunostained for ubiquitin (Ub) with the FK2 antibody (A) or p62 (B), which are shown in red. (C) GFP-LC3 BMDMs were infected with DsRed-Mtb. LC3 is shown in green, whereas Mtb are red. (A-C) Quantification shows the percentage of Mtb that colocalized with the indicated cellular marker in macrophages that were treated with IFN-γ as indicated. Results are mean +/- SEM from two independent experiments, with at least 100 bacteria analyzed per experiment. ***P<0.001, Fisher's exact test (in which the proportion of Mtb associated with the cellular marker in a given condition was compared to the proportion found in the sample treated with siCON and IFN-γ).