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

(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 expression densitometric in mouse gastrocnemius ( n = 5 ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ 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

(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 densitometric ( n = 5 ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ 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

(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 chains in mouse gastrocnemius densitometric ( n = 4 ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ 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

(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 of total ( I ) - bound ( J ) iron in mouse quadriceps ( n = 4 - 5 ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ 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 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

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

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 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

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

(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 in transversal sections of mouse gastrocnemius corresponding ( n = 3 - 4 ) . Scale bar = 50µm . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ 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

(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 ferric citrate . ( n = 3 )

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

(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 diameter of C2C12 myotubes stained for myosin heavy chain ( G ) or human myoblast - derived myotubes ( H ) after 48h treatment with C26 CM ferric citrate ( n = 3 per condition ) . Scale bars = 50 100 µm , respectively . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by one - way Anova with Bonferroni ' s correction Data are mean ± . ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 compared to control # # 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 O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 survival of C26 - 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 data , n represents number of biological replicates . Statistical significance was calculated by chi - square for survival curves Data are mean ± . ∗ p < 0 . 05 ∗ ∗ ∗ p < 0 . 001 compared to control # 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

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

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

(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 content ( n = 3 - 7 ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 compared to control # 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

(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 densitometric of phospho - AMPK total AMPK ( stripped re - blotted ) in gastrocnemius of C26 - bearing mice after iron carboxymaltose supplementation ( n = 6 ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 compared to control # 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of ACOT 1 ( F ) , MCD ( G ) , CPT1B ( H ) normalized to GAPDH in gastrocnemius ( n = 5 - 10 ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 compared to control # 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

(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 , expressed in of baseline force ( A ) absolute values normalized to height ( B ) before / after single dose of iron carboxymaltose ( 15mg / kg , 7 subjects ) . Data information : For data , n represents In graph ( A ) , boxes represent range of values with median value being central band whiskers .

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 interface between RBD ACE2 . density , shown as pink meshes , is contoured at 12 σ .

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

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 ) , M . smegmatis merodiploid mutant S152Y ribosomes ( n = 9 ) . Mistranslation was determined by assessing misreading at His245 ( CAC codon ) of F - luc gene that was replaced by near - cognate codon CGC or non - cognate codon AGA , both encoding for Arg . Results are derived by calculating mutant hFluc / hRluc activity related to wild - type hFluc / hRluc activity .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 ) , M . smegmatis merodiploid mutant S152Y ribosomes ( n = 9 ) . To determine read - through Asp357 ( GAC codon ) of F - luc gene was replaced by nonsense TGA stop codon . Results are derived by calculating mutant hFluc / hRluc activity related to wild - type hFluc / hRluc activity .

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

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

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

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

O O O O B-MEASURED_VAR 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

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

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

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 MT - CO1 - AGA mRNA using mutant A1555G mitohybrid ribosomes in presence of tobramycin ( 0 - 20 µM ) . Autoradiography of immunoprecipitated MT - CO1 ; 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 MT - CO1 TGA - polyA constructs used for in - vitro translation are schematized .

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

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 of 14C - Lys 35S - Met labelled immunoprecipitated MT - CO1 . In - vitro translation using MT - CO1 - TGA mRNA , MT - CO1 - AGA mRNA , mutant A1555G mitohybrid ribosomes in presence of tobramycin ( 0 - 20 µM ) . 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 in presence of tobramycin , incorporation of 35S - Met 35S - Cys in MT - CO1 . Following in - organello translation were immunoprecipitated analyzed by autoradiography . Lane 1 , 3 : controls ; lanes 2 , 4 : in - organello translation in presence of 1000 µM tobramycin . MT - CO1 bands were quantified of 35S - Cys / 35S - Met calculated , 35S - Cys / 35S - Met in absence of tobramycin was set as 1 ( n = 3 ) , error bars indicate .

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

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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).


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

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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 in presence of tobramycin ( 0 - 750 µM ) . were 35S - Met labeled loaded either directly on SDS - PAGE ( total , top ) or following poly - lysine immunoprecipitation ( bottom ) . * MT - CO1 * * 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 of MT - CO1 in HEK293 MRPS5 wild - type HEK293 MRPS5 mutant V336Y . In - organello mitochondrial translation in presence of 35S - Met 750 µM tobramycin . were loaded either directly on SDS - PAGE ( total ) or following poly - lysine immunoprecipitation analyzed by autoradiography . For total samples MT - CO1 band was quantified , for poly - lysine immunoprecipitated samples extended MT - CO1 band was quantified of extended MT - CO1 / total MT - CO1 calculated . 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 - ) .

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

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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 mutant mouse cortex . A OCR in brain mitochondria from 19 - months mutant mice ( black circle ) compared with OCR from wild - type mice ( circle ) . sequential injection of mitochondrial substrates inhibitors is indicated by dotted lines Values corresponding to 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 mutant mouse cortex . Baseline OCR , effect of ( p < 0 . 0001 ) 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 mutant mouse cortex . C State 3 OCR ( after addition of ADP ) , effect of ( p < 0 . 001 ) 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 mutant mouse cortex . D State 3 uncoupled OCR ( state 3u after addition of FCCP ) , effect of genotype ( p < 0 . 0001 ) - 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.


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

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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 open field ( ANOVA : genotype F1 , 24 = 39 . 40 p < 0 . 0001 , x genotype F2 , 26 = 8 . 204 p = 0 . 0017 ) . Mutants showed little exploratory activity at 9 - 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 - transition , % time spent in bright compartment ( ANOVA : genotype F1 , 16 = 7 . 393 p = 0 . 0152 ) . Reduced scores at 9 - months old indicate increased anxiety .

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

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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 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 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 .

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 reversal ( ANOVA : day F4 , 104 = 21 . 14 p < 0 . 0001 , genotype F1 , 26 = 13 . 11 p = 0 . 0013 , x genotype F2 , 26 = 8 . 204 p = 0 . 0017 ) . groups learned : controls showed reduced performance with increasing , mutants showed poor performance already at 9 - 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 non - spatial strategy choice ( ANOVA : genotype F1 , 26 = 18 . 31 p = 0 . 0002 , F2 , 26 = 4 . 486 p = 0 . 0212 , x genotype F2 , 26 = 7 . 068 p = 0 . 0035 ) . Number of wall approaches increased with in controls was prematurely increased in mutants at 9 - 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 10 % of 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 in controls was prematurely reduced in mutants at 9 - 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.


Noise exposure 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 ( open bars ) , ( p < 0 . 0001 , two - way ANOVA for combined 108 110 dB data ) . Frequencies of 12 , 24 32 kHz were selected for measurement to cover mid - frequency to high - frequency regions of cochlea . n = 6 for baseline ; n = 6 for baseline mutants ; n = 3 for 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 UVR8 HY5 VP - peptides versus COP1 WD40 domain . top panel represents heats detected during each injection . bottom panel represents integrated heats of each injection , fitted to a one - site binding model ( solid ) . following concentrations were typically used ( titrant into ) : UVR8 - COP1 ( 2500 μM in 175 μM ) ; HY5 - COP1 ( 1500 μM in 175 μM ) . insets show dissociation constants ( Kd ) stoichiometries of binding ( N ) ( ± standard deviation ) .

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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 HY5 UVR8 VP - peptides versus different COP1 WD40 domain mutants following concentrations were typically used ( titrant into ) : 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 ) . insets show dissociation constants ( Kd ) . stoichiometries of binding ( N ) for UVR8 - COP1Lys422Ala = 0 . 87 ± 0 . 2 ; HY5 - COP1Lys422Ala = 0 . 83 ± 0 . 2 ; HY5 - COP1Tyr441Ala = 0 . 92 ± 0 . 3 ( measurements ± standard deviation ; n . d . : no detectable binding ) .

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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).


of representative individuals ( F ) of 4 - day - old seedlings grown with or without supplemental UV - B . scale bar for lines represent 5 mm except for cop1 - 5 where 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 cop1 - 5 . # 1 # 2 : independent transgenic lines .

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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.


of hypocotyl lengths ( G ) of 4 - day - old seedlings grown with or without supplemental UV - B . scale bar for lines represent 5 mm except for cop1 - 5 where scale bars represent 1 mm . Violin box plots are shown for n > 60 seedlings ; upper lower hinges correspond to first third quartiles , horizontal in interior of box indicates 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 cop1 - 5 . # 1 # 2 : independent transgenic lines .

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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 of ( H ) HY5 expression . Four - day - old seedlings grown in were exposed to narrowband UV - B for 2 hours ( + UV - B ) , or not ( - UV - B ) . Error bars represent 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 cop1 - 5 . # 1 # 2 : independent transgenic lines .

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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 of ( I ) RUP2 expression . Four - day - old seedlings grown in were exposed to narrowband UV - B for 2 hours ( + UV - B ) , or not ( - UV - B ) . Error bars represent 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 cop1 - 5 . # 1 # 2 : independent transgenic lines .

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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 between COP1 WD40 domain full - UVR8 pre - monomerized by UV - B . Integrated heats are shown in solid , purple squares . For comparison , an ITC experiment between UVR8 VP peptide COP1 WD40 domain ( from Fig 1B ) is also shown in open , purple squares . following concentrations were typically used ( titrant into ) : COP1 - UVR8 + UV - B ( 130 μM in 20 μM ) . inset shows 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

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 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 . following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - UVR8 + UV - B ( highest 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

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 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 . following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - UVR8ValPro / AlaAla + UV - B ( highest 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

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 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 . following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - HY5 ( highest 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

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 of COP1 - HY5 interaction in presence of UVR8 . ( Top ) Normalized Miller Units were calculated as a 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 for 3 biological repetitions are shown . ( Bottom ) representative filter lift assays . AD , activation domain ; BD , 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 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 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 phenotype of wild - type ( Ws ) , uvr8 - 7 uvr8 - 7 / Pro35S : UVR8HY5C44 , uvr8 - 7 / Pro35S : UVR8HY5VP uvr8 - 7 / Pro35S : UVR8TRIB1 seedlings grown for 4 days under ( - UV - B ) or supplemented with UV - B ( + UV - B ) . scale bar represents 5 mm .

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

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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 are shown ( n = 3 ) .

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


E . Quantitative real - time PCR of RUP2 , CHS , HY5 ELIP2 expression in wild - type ( Ws ) , uvr8 - 7 uvr8 - 7 / Pro35S : UVR8HY5C44 , uvr8 - 7 / Pro35S : UVR8HY5VP uvr8 - 7 / Pro35S : UVR8TRIB1 4 - day old seedlings grown under in response to 2h of UV - B ( + UV - B vs . - UV - B ) . Error bars represent of 3 biological replicates . Note : primers used to detect 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

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 of COP1 - HY5 interaction in presence of UVR8HY5C44 chimera . ( Left pane ) Normalized Miller Units were calculated as a 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 for 3 biological repetitions are shown . ( Right panel ) Representative filter lift assays of yeast analyzed in left panel . AD , activation domain ; BD , 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 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 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 UVR8HY5C44 chimera pre - monomerized by UV - B versus 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 . following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - UVR8HY5C44 + UV - B ( highest 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

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 . - exclusion chromatography of purified , recombinant chimeric expressed in Sf9 insect in presence absence of UV - B .

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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 COP1 WD40 domain ( WT ) COP1Lys422Ala as determined by ITC . 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 COL3 VP - peptide ( left ) CRY1 VP - peptide ( right ) versus COP1 WD40 COP1Lys422Ala , with a table summarizing their corresponding affinities . following concentrations were typically used ( titrant into ) : 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 ) . inset shows dissociation constant ( Kd ) . 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 ( 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 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 wild - type ( Ws ) , co - 11 , cop1 - 5 / Pro35S : YFP - COP1 cop1 - 5 / Pro35S : YFP - COP1Lys422Ala 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 . of flowering time . Means SD are shown ( n = 14 ) . D . Number of rosette cauline leaves at flowering . Means 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 of CRY2 actin ( loading control ) in wild - type ( Col ) , cry2 - 1 , wild - type ( Ws ) , cop1 - 5 / Pro35S : YFP - COP1 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 CO VP - peptide ( left ) CRY2 VP - peptide ( right ) versus COP1 WD40 COP1Lys422Ala WD40 domains , with a table summarizing their corresponding affinities . following concentrations were typically used ( titrant into ) : 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 ) . 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 ) ( 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

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 ( G ) full - activated CRY2 ( H ) CRY2NC80 versus COP1 WD40 domain obtained by GCI experiments . Sensorgrams of injected are shown in red , with their respective heterogenous ligand binding model fits in black . following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - CRY2 ( highest 14 μM ) , CRY2NC80 ( highest 450 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

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 of Mpl in healthy donors ' ( HD ) platelets shows two distinct bands representing mature glycoprotein ( top band ) ER ‐ glycosylated form ( bottom band ) . is ~ 1 .

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 of HEL whole 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 ratios of mature : immature Mpl for at least three independent ( 2 for JAK2V617F transfection ) blotting experiments under protocol shown in ( B ) . on two − Student t − statistical , there is a significant difference ( p = 0 . 0001 ) in of Mpl isoforms in transfected with JAK2WT by comparison with control . Differences were also statistically significant in HEL transfected with either JAK2V617F or JAK2L611V / V617F by comparison to controls ( p = 0 . 0159 p = 0 . 0009 , respectively ) . mature : immature is also significantly higher in transfected with JAK2WT than in 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 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 ) ( D ) , showing relative band of mature immature Mpl normalized for of β ‐ actin in each lane .

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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 Mpl ‐ Jak2 in different lines analyzed by immunoblotting with anti ‐ Mpl anti ‐ JAK2 antibodies .

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


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

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 image of K562 ( JAK2WT + / + ) transiently transfected with cDNA coding for MplmOrange2 . Red arrow in this image points to plasma 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 E ) Confocal obtained after transient expression of MplmOrange2 immuno ‐ labeling of endogenous JAK2 in K562 ( D ) or HEL ( E ) . Profiles of ( across indicated by a arrow ) are showing a good colocalization of Mpl JAK2WT in K562 at plasma ( black arrow heads ) but no colocalization have been observed between MplmOrange2 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 G ) Confocal obtained after transient coexpression of MplmOrange2 either JAK2WTmCitrine ( F ) or JAK2V617FmCitrine ( G ) in K562 . Profiles of ( arrows ) are showing a very good colocalization of MplmOrange2 with JAK2WTmCitrine , especially at plasma ( black arrow heads ) , but a very poor colocalization with JAK2V617FmCitrine ( grey arrow heads ) . Nuclei are labeled with Hoechst 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 labeled live for 30 min with an anti ‐ CD235a ‐ Atto655 ( glycophorin A ) antibody before treatment with 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 non ‐ treated with Duolink reagents .

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


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

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


E F ) K562Mpl ‐ mKO2 submitted to Duolink reaction . arrow heads are pointing at example of Duolink positive reactions at plasma , while green arrow heads are pointing at positive Duolink reactions within cytoplasm . Nuclei are labeled with Hoechst 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 ) K562 ( B ) treated with non ‐ targeting ( NT ) or anti ‐ JAK2 siRNA analyzed by western blot .

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


C ) Confocal live ( upper panel ) , or fixed ( lower left panel ) , imaging of K562 expressing MpleGFP or MplmKO2 treated with NT or anti ‐ JAK2 siRNA showing a difference of distribution of intracellular Mpl in siRNA treated compare to control . After treatment with anti ‐ JAK2 siRNA , Mpl strongly colocalizes with ER marker , calnexin . This colocalization in treated vs non ‐ treated is quantified shown in Figure C lower right panel . Nuclei are labeled with Hoechst 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 colocalize with autophagic lysosomal markers in K562 HEL . A - M ) Confocal obtained after transient expression of MplmOrange2 or MplmKO2 indicated labeling in K562 ( A - F ) or HEL ( G - M ) . Plots at right of each image show intensities for each emission spectrum at points along drawn across each image . Black arrows in plots point to overlap between MplmKO2 either ER ‐ tracker ( C , I ) , or Sec31a ( B , J ) that marks ER exit sites . MplmOrange2 did not colocalize with markers for Golgi ( Golgin ‐ 97 ) or early endosomes ( EEA1 ) in either . However , MplmKO2 colocalized greatly with autophagic ( LC3 ) ( E , L ) lysosomal ( LAMP1 ) ( F , M ) markers . Nuclei are labeled with Hoechst 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 obtained after transient expression of Mpl tagged with mKO2 miniSOG in K562 ( A ) HEL ( B ) . Electron of K562 ( C ) HEL ( D ) transiently expressing MplmKO2 ‐ miniSOG . precipitate indicates presence of miniSOG ‐ tagged Mpl after photo ‐ precipitation of DAB . boxes indicate original location of higher magnification 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 treated in same conditions as Figure C , showing MplmKO2 ‐ miniSOG 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 treated in same conditions as Figure D , 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 ) .

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 of streptavidin ‐ immunoprecipitated surface ‐ biotinylated , probed with an anti ‐ Mpl

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


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

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

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 of phosphorylation in HEL , K562Mpl ‐ mKO2 K562Mpl ‐ mOrange2 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 B ) Surface biotinylation total lysates western blot of HEL or K562Mpl ‐ mKO2 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 exosomal fraction western blot of HEL K562Mpl ‐ mKO2 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 of HEL treated , or not , with Bafilomycin A1 at 100 nm for 3 h then stimulated or not with Tpo at 25 ng / mL for 30 min . of 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 total lysates western blot of HEL 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 surface biotinylation western blot of HEL 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 surface biotinylation western blot of HEL overexpressing either GRASP55 - V5 or HA - STX5 .

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


B , HeLa were treated with DMSO or 2µM Mito - C for 15 min immunostained with anti - TOM20 ( green ) antibody DAPI ; cropped areas show mitochondria 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 , of mitochondria ( on TOM20 immunostaining as shown in B ) from HeLa treated with DMSO or 2µM Mito - C for 15 min or 2h , expressed as of fragmented mitochondria ( of total ) . Errors bars show standard error of mean ( ) ( 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 transfected with NAF - 1 - mRFP ( red ) treated with fluoMito - C ( green ) . / graph illustrates codistribution of fluoMito - C 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 of MitoNEET expression in HeLa transfected with CISD1 siRNA ; Bar Chart ( right panel ) shows replicates of MitoNEET expression . Errors bars show 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 of NAF - 1 expression in HeLa transfected with CISD2 siRNA ; graph ( right panel ) shows replicates of NAF - 1 expression . Errors bars show 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 transfected with siRNA to reduce expression of MitoNEET ( CISD1 ) , NAF - 1 ( CISD2 ) or MiNT ( CISD3 ) immunostained with anti TOM20 antibody . Arrowheads indicate fragmented mitochondria .

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

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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 transfected as described in G .

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


G , Western blot of NAF - 1 expression in stably transfected with control shRNA , shRNA targeting reduction of NAF - 1 expression ( shCISD2 ) , or shRNA targeting reduction of NAF - 1 expression complemented with plasmid derived expression of NAF - 1 ( shCISD2 + CISD2 ) ; western blot of NAF - 1 is shown on accompanying bar chart . Errors bars show 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 , of mitochondrial in EM from transfected as described in G . Errors bars show standard error of mean ( ) ( 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 treated or not , with 2µM Mito - C for 15 min were immunostained with anti - TOM20 ( red ) anti - DRP1 ( green ) ; arrowheads in far - right panel indicate recruitment of DRP1 onto mitochondrial surface ( TOM20 ) . B , of DRP1 signal on TOM20 positive structures . Errors bars show standard error of mean ( ) ( 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 of DRP1 , GAPDH ( marker ) TOM20 ( mitochondrial marker ) in mitochondrial fractions treated or not , with 2µM Mito - C as indicated . D , of western blots showed in C expressed as a distribution of DRP1 in mitochondrial fractions . Errors bars show 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 were transfected with DRP1K38A mutant mCherry treated or not , with Mito - C ( T for transfected , NT for not transfected ) . F , of mitochondrial observed in E . Errors bars show standard error of mean ( ) . ( 30 - 35 each with an average of 15 - 20 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 ( EM ) from HeLa treated with 2µM Mito - C or DMSO for 120 minutes . Empty arrowheads indicate ER - mitochondria contact sites . H , of ER - mitochondria contact sites events ( reported in 50µm² section ; 50 from an average of 30 from triplicate independent experiments were analyzed ) . Errors bars show standard error of mean ( ) . I , of ER - mitochondria contact sites density ( in µm ; 30 from an average of 30 from triplicate independent experiments were analyzed ) . Errors bars show standard error of mean ( ) .

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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 were treated with or without ( DMSO ) Mito - C , infected with dengue fixed after 48h of infection . were then immunostained for endogenous TOM20 ( green channel ) , viral non - structural NS5 ( red ) DAPI ( Blue ) . Cropped areas illustrate mitochondrial 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 , of mitochondrial observed in b . Errors bars show standard error of mean ( ) . ( 15 - 20 each with an average of 20 - 25 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 - 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 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 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 IP from HEK293 expressing myc - UBQLN1 Rv1926 - V5 ( D ) or Rv1566 - V5 ( E ) . IP was performed using antibodies directed against myc , V5 , or isotype control ( IgG ) .

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(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 transfected with vector control , myc - UBQLN1 , GFP , or V5 - parkin . WB of input ( I ) , 5th wash ( W5 ) , elution ( E ) , probed with indicated antibodies . Actin is a loading control for lysate Ag85b for pellet .

(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 transfected with myc - UBQLN1 - ΔUBA , myc - UBQLN1 - ΔUBL , myc - UBQLN1 - UBA . WB containing input , 5th wash , elution were incubated with anti - myc antibody . predicted sizes of myc - UBQLN1 - ΔUBL , myc - UBQLN1 - ΔUBA , myc - UBQLN1 - UBA are 55 kD , 50 kD , 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 input 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 immunostained with UBQLN1 antibody . IFN - γ was added 1d prior to infection as indicated . ( B ) of results from A from 3 independent experiments with at least 100 analyzed per experiment ; * * * P < 0 . 001 , Fisher ' s exact , comparing proportion of UBQLN1 positive versus negative Mtb in indicated samples .

(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.