Mean body weight of male wild type ( WT , n = 15 ) , NYKO ( n = 12 ) and heterozygous NYKO ( het NYKO , n = 7 ) mice from 4 - 32 weeks . Unpaired t - test was used for the comparison of two groups , ordinary one - way ANOVA for the comparison of three groups . *

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

Mean body weight of male wild type (WT, n = 15), NYKO (n = 12) and heterozygous NYKO (het NYKO, n = 7) mice from 4-32 weeks. Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups. *


B Body composition analysis by nuclear magnetic resonance ( NMR ) of 31 - 32 week old male WT ( n = 12 ) and NYKO ( n = 7 ) mice . Unpaired t - test was used for the comparison of two groups , ordinary one - way ANOVA for the comparison of three groups . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . Data are means ± SEM .

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

B Body composition analysis by nuclear magnetic resonance (NMR) of 31-32 week old male WT (n = 12) and NYKO (n = 7) mice. Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups. *P ≤ 0.05, *** P ≤ 0.001, ****P ≤ 0.0001. Data are means ± SEM.


C Whole - body energy expenditure per kg lean mass of 26 week old male WT ( n = 5 ) and NYKO ( n = 5 ) mice after disease onset . Unpaired t - test was used for the comparison of two groups , ordinary one - way ANOVA for the comparison of three groups .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Whole-body energy expenditure per kg lean mass of 26 week old male WT (n = 5) and NYKO (n = 5) mice after disease onset. Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups.


Walking beam test of 6 - 7 week old ( WT , n = 10 ; NYKO , n = 10 ) , 17 - 18 week old ( WT , n = 8 ; NYKO , n = 9 ) and 31 - 32 week old mice ( WT , n = 12 ; NYKO , n = 19 ; het NYKO , n = 9 ) . Unpaired t - test was used for the comparison of two groups , ordinary one - way ANOVA for the comparison of three groups . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . Data are means ± SEM .

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

Walking beam test of 6-7 week old (WT, n = 10; NYKO, n = 10), 17-18 week old (WT, n = 8; NYKO, n = 9) and 31-32 week old mice (WT, n = 12; NYKO, n = 19; het NYKO, n = 9). Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups. *P ≤ 0.05, *** P ≤ 0.001, ****P ≤ 0.0001. Data are means ± SEM.


Walking beam test of 6 - 7 week old ( WT , n = 10 ; NYKO , n = 10 ) , 17 - 18 week old ( WT , n = 8 ; NYKO , n = 9 ) and 31 - 32 week old mice ( WT , n = 12 ; NYKO , n = 19 ; het NYKO , n = 9 ) . Hind limb slips per run are shown in E . Unpaired t - test was used for the comparison of two groups , ordinary one - way ANOVA for the comparison of three groups . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . Data are means ± SEM .

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

Walking beam test of 6-7 week old (WT, n = 10; NYKO, n = 10), 17-18 week old (WT, n = 8; NYKO, n = 9) and 31-32 week old mice (WT, n = 12; NYKO, n = 19; het NYKO, n = 9). Hind limb slips per run are shown in E. Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups. *P ≤ 0.05, *** P ≤ 0.001, ****P ≤ 0.0001. Data are means ± SEM.


F Representative images of 31 - 32 week old WT and NYKO mice showing aberrant positioning of hind limbs .

F Representative images of 31-32 week old WT and NYKO mice showing aberrant positioning of hind limbs.


Brain weights were monitored at 31 - 32 weeks of age ( WT , n = 15 ; NYKO , n = 14 ) . Unpaired t - test , ns = not significant . Data are means ± SEM .

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

Brain weights were monitored at 31-32 weeks of age (WT, n = 15; NYKO, n = 14). Unpaired t-test, ns = not significant. Data are means ± SEM.


B Nissl stainings of sagittal sections across brain regions from 31 - 32 week old WT and NYKO mice . 4x and 40x enlargements are shown in the left and right panels , respectively . Scale bars 4x = 500 µm , 40x = 50 µm .

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

B Nissl stainings of sagittal sections across brain regions from 31-32 week old WT and NYKO mice. 4x and 40x enlargements are shown in the left and right panels, respectively. Scale bars 4x = 500 µm, 40x = 50 µm.


C Morphometric analysis of different brain regions from 31 - 32 week old WT ( n = 6 ) and NYKO ( n = 6 ) mice . GCL = Granule cell layer , ML = Molecular layer . Unpaired t - test , ns = not significant . Data are means ± SEM .

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

C Morphometric analysis of different brain regions from 31-32 week old WT (n = 6) and NYKO (n = 6) mice. GCL = Granule cell layer, ML = Molecular layer. Unpaired t-test, ns = not significant. Data are means ± SEM.


Immunoblot analysis of tissue lysates ( FB = forebrain ; CB = cerebellum ) from 31 - 32 week old WT and NYKO mice , using GFAP - specific and , as a loading control , GAPDH - specific antibodies . Unpaired t - test , ns = not significant . Data are means ± SEM .

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

Immunoblot analysis of tissue lysates (FB = forebrain; CB = cerebellum) from 31-32 week old WT and NYKO mice, using GFAP-specific and, as a loading control, GAPDH-specific antibodies. Unpaired t-test, ns = not significant. Data are means ± SEM.


B Transverse semithin sections of spinal cords of WT and NYKO mice of the indicated age . Sections were stained with toluidine blue . Orange arrows indicate degenerating neurons ( Myelin dense bodies , MDBs ) . Scale bars 25 µm .

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

B Transverse semithin sections of spinal cords of WT and NYKO mice of the indicated age. Sections were stained with toluidine blue. Orange arrows indicate degenerating neurons (Myelin dense bodies, MDBs). Scale bars 25 µm.


C Myelin dense bodies ( MDBs ) in dorso - lateral tracts of 6 - 7 week old WT ( n = 4 ) and NYKO ( n = 4 ) , 17 - 18 week old WT ( n = 4 ) and NYKO ( n = 4 ) and 31 - 32 week old WT ( n = 6 - 7 ) and NYKO ( n = 5 - 6 ) mice spinal cords . Unpaired t - test , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Myelin dense bodies (MDBs) in dorso-lateral tracts of 6-7 week old WT (n = 4) and NYKO (n = 4), 17-18 week old WT (n = 4) and NYKO (n = 4) and 31-32 week old WT (n = 6-7) and NYKO (n = 5-6) mice spinal cords. Unpaired t-test, *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


D Immunoblots of 31 - 32 week old spinal cord lysates . GAPDH was used to control for gel loading .

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

D Immunoblots of 31-32 week old spinal cord lysates. GAPDH was used to control for gel loading.


E mRNA levels of proinflammatory cytokines from spinal cords of 31 - 32 week old mice ( WT , n = 5 ; NYKO , n = 5 ) . Transcript levels were normalized to Hprt mRNA levels . Unpaired t - test , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± SEM .

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

E mRNA levels of proinflammatory cytokines from spinal cords of 31-32 week old mice (WT, n = 5; NYKO, n = 5). Transcript levels were normalized to Hprt mRNA levels. Unpaired t-test, *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


Fluorescence images of isolated cortical neurons from Yme1lfl / fl animals expressing CAG - GFP ( WT ) or CAG - Cre - IRES - GFP ( Yme1l - / - ) at DIV12 and stained with TOMM20 to visualize mitochondria . Scale bars 30 µm .

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

Fluorescence images of isolated cortical neurons from Yme1lfl/fl animals expressing CAG-GFP (WT) or CAG-Cre-IRES-GFP (Yme1l-/-) at DIV12 and stained with TOMM20 to visualize mitochondria. Scale bars 30 µm.


B - E Quantification of total axon length ( WT , n = 13 neurons ; Yme1l - / - , n = 13 neurons ) , total dendritic length ( WT , n = 12 neurons ; Yme1l - / - , n = 13 neurons ) , axonal branch points ( WT , n = 12 neurons ; Yme1l - / - , n = 13 neurons ) and number of dendrites ( WT , n = 12 neurons ; Yme1l - / - , n = 13 neurons ) . Individual neurons originate from two independent preparations . Unpaired t - test , ns = not significant , * P ≤ 0 . 05 .

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

B - E Quantification of total axon length (WT, n = 13 neurons; Yme1l-/-, n = 13 neurons), total dendritic length (WT, n = 12 neurons; Yme1l-/-, n = 13 neurons), axonal branch points (WT, n = 12 neurons; Yme1l-/-, n = 13 neurons) and number of dendrites (WT, n = 12 neurons; Yme1l-/-, n = 13 neurons). Individual neurons originate from two independent preparations. Unpaired t-test, ns = not significant, *P ≤ 0.05.


F , G Kymographs showing mitochondrial movements in representative axons . Vertical lines correspond to stationary mitochondria and diagonal lines to moving mitochondria . Mitochondria were stained using Mitotracker . In total 39 axonal segments were analyzed for WT control neurons and 37 axonal segments for Yme1l - / - neurons at DIV7 . Data are means of three independent experiments ± SEM . Unpaired t - test , ns = not significant , * P ≤ 0 . 05 .

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

F, G Kymographs showing mitochondrial movements in representative axons. Vertical lines correspond to stationary mitochondria and diagonal lines to moving mitochondria. Mitochondria were stained using Mitotracker. In total 39 axonal segments were analyzed for WT control neurons and 37 axonal segments for Yme1l-/- neurons at DIV7. Data are means of three independent experiments ± SEM. Unpaired t-test, ns = not significant, *P ≤ 0.05.


H , I Kymographs showing mitochondrial movements in representative axons . Vertical lines correspond to stationary mitochondria and diagonal lines to moving mitochondria . Mitochondria were stained using Mitotracker . In total 120 axonal segments were analyzed for WT control neurons and 107 axonal segments for Yme1l - / - neurons at DIV10 - 14 . Data are means of three independent experiments ± SEM . Unpaired t - test , ns = not significant , * P ≤ 0 . 05 .

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

H, I Kymographs showing mitochondrial movements in representative axons. Vertical lines correspond to stationary mitochondria and diagonal lines to moving mitochondria. Mitochondria were stained using Mitotracker. In total 120 axonal segments were analyzed for WT control neurons and 107 axonal segments for Yme1l-/- neurons at DIV10-14. Data are means of three independent experiments ± SEM. Unpaired t-test, ns = not significant, *P ≤ 0.05.


J Axonal area occupied by mitochondria in Yme1l - / - neurons relative to WT controls . In total 120 axonal segments were analyzed for WT control neurons and 107 axonal segments for Yme1l - / - neurons at DIV10 - 14 . Data are means of three independent experiments ± SEM . Unpaired t - test , ns = not significant , * P ≤ 0 . 05 .

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 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

J Axonal area occupied by mitochondria in Yme1l-/- neurons relative to WT controls. In total 120 axonal segments were analyzed for WT control neurons and 107 axonal segments for Yme1l-/- neurons at DIV10-14. Data are means of three independent experiments ± SEM. Unpaired t-test, ns = not significant, *P ≤ 0.05.


A , B Oxygen consumption rates of spinal cord mitochondria isolated from WT ( n = 4 ) and NYKO ( n = 4 ) mice in the presence of pyruvate - glutamate - malate ( C I ) and pyruvate - glutamate - malate + succinate ( C I + C II ) as substrates . State 3 ( substrates + ADP ) , state 4 ( + oligomycin ) , uncoupled ( + FCCP ) . Unpaired t - test , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , ns = not significant . Data are means ± SEM .

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

A, B Oxygen consumption rates of spinal cord mitochondria isolated from WT (n = 4) and NYKO (n = 4) mice in the presence of pyruvate-glutamate-malate (C I) and pyruvate-glutamate-malate + succinate (C I + C II) as substrates. State 3 (substrates + ADP), state 4 (+ oligomycin), uncoupled (+ FCCP). Unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ns=not significant. Data are means ± SEM.


C , D TEM of dorso - lateral tracts of spinal cords of 6 - 7 week old WT ( n = 4 , 1209 mitochondria ) and NYKO ( n = 4 , 1792 mitochondria ) mice , 17 - 18 week old WT ( n = 4 , 442 mitochondria ) and NYKO ( n = 4 , 724 mitochondria ) mice and 31 - 32 week old WT ( n = 3 , 764 mitochondria ) and NYKO ( n = 3 , 817 mitochondria ) mice . Scale bars , 200 nm . Unpaired t - test , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , ns = not significant . Data are means ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 TEM of dorso-lateral tracts of spinal cords of 6-7 week old WT (n = 4, 1209 mitochondria) and NYKO (n = 4, 1792 mitochondria) mice, 17-18 week old WT (n = 4, 442 mitochondria) and NYKO (n = 4, 724 mitochondria) mice and 31-32 week old WT (n = 3, 764 mitochondria) and NYKO (n = 3, 817 mitochondria) mice. Scale bars, 200 nm. Unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ns=not significant. Data are means ± SEM.


A TEM analysis of sagittal spinal cord sections of 6 - 7 week old WT and NYKO mice . Scale bars 500 nm .

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

A TEM analysis of sagittal spinal cord sections of 6-7 week old WT and NYKO mice. Scale bars 500 nm.


B Frequency distribution of mitochondrial lengths in spinal cords of 6 - 7 week old WT ( 182 mitochondria ) and NYKO mice ( 192 mitochondria ) . Kruskal - Wallis test , * P < 0 .

O O O O O O O O O O O O O O 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 Frequency distribution of mitochondrial lengths in spinal cords of 6-7 week old WT (182 mitochondria) and NYKO mice (192 mitochondria). Kruskal-Wallis test, *P < 0.05


C TEM analysis of cerebellar sections of 28 - 32 week old WT and NYKO mice . Scale bars 1 µm . D Frequency distribution of mitochondrial aspect ratios were calculated from WT ( n = 210 ) and NYKO ( n = 230 ) mitochondria . Mann - Whitney test , * * * * P < 0 . 0001 . Data are presented as median values .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 TEM analysis of cerebellar sections of 28-32 week old WT and NYKO mice. Scale bars 1 µm. D Frequency distribution of mitochondrial aspect ratios were calculated from WT (n = 210) and NYKO (n = 230) mitochondria. Mann-Whitney test, ****P < 0.0001. Data are presented as median values.


Immunoblot analysis of forebrain , cerebellar and spinal cord lysates from 31 - 32 week old WT and NYKO mice ( n = 3 ) using the indicated antibodies . SDHA and LONP1 were used to control for gel loading . * indicates unspecific antibody binding .

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

Immunoblot analysis of forebrain, cerebellar and spinal cord lysates from 31-32 week old WT and NYKO mice (n = 3) using the indicated antibodies. SDHA and LONP1 were used to control for gel loading. * indicates unspecific antibody binding.


Immunoblot analysis of retinal and spinal cord lysates from 6 - 7 week old WT , NYKO , NOKO and NYOKO mice ( n = 3 ) using the indicated antibodies . SDHA was used as a loading control . * was not reproducibly detectable in all experiments .

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

Immunoblot analysis of retinal and spinal cord lysates from 6-7 week old WT, NYKO, NOKO and NYOKO mice (n = 3) using the indicated antibodies. SDHA was used as a loading control. * was not reproducibly detectable in all experiments.


B Frequency distribution of mitochondrial lengths in spinal cords of 6 - 7 week old NYOKO mice ( 176 mitochondria ) compared to WT Kruskal - Wallis test , ns = not significant . Data were analyzed using one - way ANOVA .

O O O O O O O O O O O O O O O O O O O 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 Frequency distribution of mitochondrial lengths in spinal cords of 6-7 week old NYOKO mice (176 mitochondria) compared to WT Kruskal-Wallis test, ns = not significant. Data were analyzed using one-way ANOVA.


Representative images of eyes and lenses from 6 - 7 week old WT and mice lacking OMA1 ( NOKO ) or both YME1L and OMA1 ( NYOKO ) in the nervous system . Orange dashed lines mark eye morphology . Scale bars 2 . 5 mm .

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

Representative images of eyes and lenses from 6-7 week old WT and mice lacking OMA1 (NOKO) or both YME1L and OMA1 (NYOKO) in the nervous system. Orange dashed lines mark eye morphology. Scale bars 2.5 mm.


D Immunoblot analysis of retina lysates of 6 - 7 week old WT , NYKO , NOKO and NYOKO mice ( n = 3 ) . SDHA was used to control for gel loading .

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

D Immunoblot analysis of retina lysates of 6-7 week old WT, NYKO, NOKO and NYOKO mice (n = 3). SDHA was used to control for gel loading.


E Transverse semithin sections of spinal cords of 6 - 7 week old WT , NOKO and NYOKO mice . Sections were stained with toluidine blue . Orange arrows point to MDBs indicating degenerating neurons . Scale bars 25 µm .

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

E Transverse semithin sections of spinal cords of 6-7 week old WT, NOKO and NYOKO mice. Sections were stained with toluidine blue. Orange arrows point to MDBs indicating degenerating neurons. Scale bars 25 µm.


F MDBs in dorso - lateral tracts of 6 - 7 week old WT ( n = 3 ) , NOKO ( n = 3 ) , and NYOKO ( n = 3 ) mice . Data were analyzed using one - way ANOVA . * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± SEM .

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

F MDBs in dorso-lateral tracts of 6-7 week old WT (n = 3), NOKO (n = 3), and NYOKO (n = 3) mice. Data were analyzed using one-way ANOVA. ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


G Walking beam test of 6 - 7 week old WT ( n = 6 ) , NOKO ( n = 5 ) and NYOKO ( n = 7 ) mice . Data were analyzed using one - way ANOVA . * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± SEM .

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

G Walking beam test of 6-7 week old WT (n = 6), NOKO (n = 5) and NYOKO (n = 7) mice. Data were analyzed using one-way ANOVA. ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


B In vitro substrate ubiquitylation assay for Hrd1 with Ubc7 ( left ) and Ubc6 ( right ) . Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub ( K48R ) . Top : representative immunoblot using a poly - clonal α - RNase A antibody ; " no E3 " reactions do not contain S - Hrd1 , " no ATP " reactions do not contain ATP , but S - Hrd1 . The RNase - Ub2 band co - migrates with a nonspecific band ( # ) common to all samples . Bottom : Quantification of RNase - Ub signals is shown . Values are reported as means ± standard deviation ( n = 3 ) . Significances for pairwise comparisons were determined by One - way ANOVA Test ; * p < 0 . 05 . For clarity , only significances related to the " no E3 " control of a given E2 are shown .

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 B-CONTROLLED_VAR 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 I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 In vitro substrate ubiquitylation assay for Hrd1 with Ubc7 (left) and Ubc6 (right). Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub(K48R). Top: representative immunoblot using a poly-clonal α-RNase A antibody; "no E3" reactions do not contain S-Hrd1, "no ATP" reactions do not contain ATP, but S-Hrd1. The RNase-Ub2 band co-migrates with a nonspecific band (#) common to all samples. Bottom: Quantification of RNase-Ub signals is shown. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity, only significances related to the "no E3" control of a given E2 are shown.


C In vitro Ub nucleophile discharge assays for Hrd1 with U7BR / Ubc7 ( top - yellow ) and Ubc6 ( bottom - green ) with ethanolamine as nucleophile . Representative Coomassie gels are shown .

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

C In vitro Ub nucleophile discharge assays for Hrd1 with U7BR/Ubc7 (top - yellow) and Ubc6 (bottom - green) with ethanolamine as nucleophile. Representative Coomassie gels are shown.


D Quantification of Ub nucleophile discharge assays for Hrd1 with U7BR / Ubc7 ( left ) and Ubc6 ( right ) . Plots of E2 ~ Ub discharge ( dots ) as a function of time with first - order reaction models fitted to the discharge data ( lines ) are shown . Values for each time point are reported as means ± standard deviation ( n = 3 ) . Insets show reaction rates derived from these fits . Significances were determined by student T - test ; * p < 0 . 05 . ( E ) Stimulation of U7BR / Ubc7 and Ubc6 discharge activities by Hrd1 . E2 stimulation is reported as the ratio of rates derived from RING - catalyzed reactions and " no E3 " controls in D . Values are reported as means ± standard deviation ( n = 3 ) . Significance was determined by student T - test ; * p < 0 . 05 .

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

D Quantification of Ub nucleophile discharge assays for Hrd1 with U7BR/Ubc7 (left) and Ubc6 (right). Plots of E2~Ub discharge (dots) as a function of time with first-order reaction models fitted to the discharge data (lines) are shown. Values for each time point are reported as means ± standard deviation (n = 3). Insets show reaction rates derived from these fits. Significances were determined by student T-test; * p < 0.05. (E) Stimulation of U7BR/Ubc7 and Ubc6 discharge activities by Hrd1. E2 stimulation is reported as the ratio of rates derived from RING-catalyzed reactions and "no E3" controls in D. Values are reported as means ± standard deviation (n = 3). Significance was determined by student T-test; * p < 0.05.


D Quantification of Ub nucleophile discharge assays for Hrd1 with U7BR / Ubc7 ( left ) and Ubc6 ( right ) . Plots of E2 ~ Ub discharge ( dots ) as a function of time with first - order reaction models fitted to the discharge data ( lines ) are shown . Values for each time point are reported as means ± standard deviation ( n = 3 ) . Insets show reaction rates derived from these fits . Significances were determined by student T - test ; * p < 0 . 05 .

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

D Quantification of Ub nucleophile discharge assays for Hrd1 with U7BR/Ubc7 (left) and Ubc6 (right). Plots of E2~Ub discharge (dots) as a function of time with first-order reaction models fitted to the discharge data (lines) are shown. Values for each time point are reported as means ± standard deviation (n = 3). Insets show reaction rates derived from these fits. Significances were determined by student T-test; * p < 0.05.


A In vitro Ub nucleophile discharge assays for U7BR / Ubc7 with indicated Hrd1 ( left ) and Doa10 ( right ) variants . E2 stimulation is shown as the ratio of rates derived from RING - catalyzed reactions and the " no E3 " control ( see Fig . EV2C - D ) . Values are reported as means ± standard deviation ( n = 3 ) . Significances for pairwise comparisons were determined by One - way ANOVA Test ; * p < 0 . 05 . For clarity only significances related to the " no E3 " control are shown . In addition , reactions for Hrd1 ( 400R ) and Doa10 ( 94R ) are significantly faster than all other reactions and the reaction for Doa10 ( 94H ) is significantly faster than that for Doa10 ( 94E ) . The wild - type E3s are identified by black frames for each set .

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

A In vitro Ub nucleophile discharge assays for U7BR/Ubc7 with indicated Hrd1 (left) and Doa10 (right) variants. E2 stimulation is shown as the ratio of rates derived from RING-catalyzed reactions and the "no E3" control (see Fig. EV2C-D). Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity only significances related to the "no E3" control are shown. In addition, reactions for Hrd1(400R) and Doa10(94R) are significantly faster than all other reactions and the reaction for Doa10(94H) is significantly faster than that for Doa10(94E). The wild-type E3s are identified by black frames for each set.


B In vitro Ub chain formation assay by Ubc7 with indicated Hrd1 ( left ) and Doa10 ( right ) variants in the presence of indicated Cue1 variants . Rates for reactions of mono - Ub to di - Ub and di - Ub to tri - Ub with fluorescently - labeled Ub are shown on a logarithmic scale . Values are reported as means ± standard deviation ( n = 3 ) . Significances for pairwise comparisons were determined by One - way ANOVA Test ; * p < 0 . 05 . For clarity only significances related to the " no E3 " control of given reaction set are shown .

O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 In vitro Ub chain formation assay by Ubc7 with indicated Hrd1 (left) and Doa10 (right) variants in the presence of indicated Cue1 variants. Rates for reactions of mono-Ub to di-Ub and di-Ub to tri-Ub with fluorescently-labeled Ub are shown on a logarithmic scale. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity only significances related to the "no E3" control of given reaction set are shown.


D Protein degradation in indicated yeast strains monitored by pulse - chase experiments for the Hrd1 model substrate PrA * - 3xHA ( left ) and by CHX decay assays for the Doa10 model substrate Deg1 - eGFP2 ( right ) . Values for each time point are reported as means ± standard deviation ( n = 4 for PrA * - 3xHA and n = 3 for Deg1 - eGFP2 ) .

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

D Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the Hrd1 model substrate PrA*-3xHA (left) and by CHX decay assays for the Doa10 model substrate Deg1-eGFP2 (right). Values for each time point are reported as means ± standard deviation (n = 4 for PrA*-3xHA and n = 3 for Deg1-eGFP2).


A Protein degradation in indicated yeast strains monitored by pulse - chase experiments for the Hrd1 model substrate PrA * - 3xHA ( left ) and by CHX decay assays for the Doa10 model substrate Deg1 - eGFP2 ( right ) . Values for each time point are reported as means ± standard deviation ( n = 4 for PrA * - 3xHA and n = 3 for Deg1 - eGFP2 ) . Data for control strains ( Hrd1 ( 400R ) wt , Δhrd1 , Doa10 ( 94H ) wt and Δdoa10 ) is the same as in Fig . 3D .

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

A Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the Hrd1 model substrate PrA*-3xHA (left) and by CHX decay assays for the Doa10 model substrate Deg1-eGFP2 (right). Values for each time point are reported as means ± standard deviation (n = 4 for PrA*-3xHA and n = 3 for Deg1-eGFP2). Data for control strains (Hrd1(400R) wt, Δhrd1, Doa10(94H) wt and Δdoa10) is the same as in Fig. 3D.


B In vitro substrate ubiquitylation assay for Hrd1 variants with Ubc7 . Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub ( K48R ) . Top : representative immunoblot using a poly - clonal α - RNase A antibody ; " no E3 " reaction does not contain any S - Hrd1 , " no ATP " reaction does not contain ATP , but wild - type S - Hrd1 ( 400R ) . The RNase - Ub2 band co - migrates with a nonspecific band ( # ) common to all samples . Bottom : Quantification of RNase - Ub signals is shown . Values are reported as means ± standard deviation ( n = 3 ) . Significances for pairwise comparisons were determined by One - way ANOVA Test ; * p < 0 . 05 . For clarity , only significances related to the " no E3 " control are shown . In addition , the reaction containing S - Hrd1 ( 400R ) shows significantly more ubiquitylation than those with other E3 variants .

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

B In vitro substrate ubiquitylation assay for Hrd1 variants with Ubc7. Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub(K48R). Top: representative immunoblot using a poly-clonal α-RNase A antibody; "no E3" reaction does not contain any S-Hrd1, "no ATP" reaction does not contain ATP, but wild-type S-Hrd1(400R). The RNase-Ub2 band co-migrates with a nonspecific band (#) common to all samples. Bottom: Quantification of RNase-Ub signals is shown. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity, only significances related to the "no E3" control are shown. In addition, the reaction containing S-Hrd1(400R) shows significantly more ubiquitylation than those with other E3 variants.


C In vitro Ub nucleophile discharge assays for Ubc6 with indicated Doa10 variants . E2 stimulation is shown as the ratio of rates derived from RING - catalyzed reactions and the " no E3 " control ( see Fig . EV2E ) . Values are reported as means ± standard deviation ( n = 3 ) . Significances for pairwise comparisons were determined by One - way ANOVA Test ; * p < 0 . 05 . For clarity only significances related to the " no E3 " control are shown . In addition , the reaction for Doa10 ( 94R ) is significantly faster than that for Doa10 ( 94H ) and ( 94E ) . The wild - type E3 is identified by a black frame . The scale of the y - axis was set identical to that of plots from other discharge assays

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

C In vitro Ub nucleophile discharge assays for Ubc6 with indicated Doa10 variants. E2 stimulation is shown as the ratio of rates derived from RING-catalyzed reactions and the "no E3" control (see Fig. EV2E). Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity only significances related to the "no E3" control are shown. In addition, the reaction for Doa10(94R) is significantly faster than that for Doa10(94H) and (94E). The wild-type E3 is identified by a black frame. The scale of the y-axis was set identical to that of plots from other discharge assays


C In vitro Ub nucleophile discharge assays for Ubc6 with indicated Doa10 variants . E2 stimulation is shown as the ratio of rates derived from RING - catalyzed reactions and the " no E3 " control ( see Fig . EV2E ) . Values are reported as means ± standard deviation ( n = 3 ) . Significances for pairwise comparisons were determined by One - way ANOVA Test ; * p < 0 . 05 . For clarity only significances related to the " no E3 " control are shown . In addition , the reaction for Doa10 ( 94R ) is significantly faster than that for Doa10 ( 94H ) and ( 94E ) . The wild - type E3 is identified by a black frame . The scale of the y - axis was set identical to that of plots from other discharge

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


D In vitro substrate ubiquitylation assay for Doa10 variants with Ubc7 ( left ) and Ubc6 ( right ) . Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub ( K48R ) . Top : representative immunoblot using a poly - clonal α - RNase A antibody ; " no E3 " reaction does not contain any S - Doa10 , " no ATP " reaction does no contain ATP , but wild - type S - Doa10 ( 94H ) . The RNase - Ub2 band co - migrates with a nonspecific band ( # ) common to all samples . Bottom : Quantification of RNase - Ub signals is shown . Values are reported as means ± standard deviation ( n = 3 ) . Significances for pairwise comparisons were determined by One - way ANOVA Test ; * p < 0 . 05 . For clarity , only significances related to the " no E3 " control of a given E2 are shown . In addition , the reaction containing Ubc7 and S - Hrd1 ( 400R ) shows significantly more ubiquitylation than those with Ubc7 and other E3 variants .

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

D In vitro substrate ubiquitylation assay for Doa10 variants with Ubc7 (left) and Ubc6 (right). Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub(K48R). Top: representative immunoblot using a poly-clonal α-RNase A antibody; "no E3" reaction does not contain any S-Doa10, "no ATP" reaction does no contain ATP, but wild-type S-Doa10(94H). The RNase-Ub2 band co-migrates with a nonspecific band (#) common to all samples. Bottom: Quantification of RNase-Ub signals is shown. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity, only significances related to the "no E3" control of a given E2 are shown. In addition, the reaction containing Ubc7 and S-Hrd1(400R) shows significantly more ubiquitylation than those with Ubc7 and other E3 variants.


A E2 ~ Ub hydrolysis assays for U7BR / Ubc7 and Ubc6 . E3 - independent Ub discharge assays for both E2s in PBS ( absence of nucleophile = hydrolysis ) were performed at 32 ° C . Left : representative Coomassie gels . Right : Plots of E2 ~ Ub hydrolysis ( dots ) as a function of time with first - order reaction models fitted to the discharge data ( lines ) are shown . Values for each time point are reported as mean ± standard deviation ( n = 3 ) .

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

A E2~Ub hydrolysis assays for U7BR/Ubc7 and Ubc6. E3-independent Ub discharge assays for both E2s in PBS (absence of nucleophile = hydrolysis) were performed at 32°C. Left: representative Coomassie gels. Right: Plots of E2~Ub hydrolysis (dots) as a function of time with first-order reaction models fitted to the discharge data (lines) are shown. Values for each time point are reported as mean ± standard deviation (n = 3).


B HSQC - TROSY experiments comparing free and E2 - conjugated ( 15N ) Ub . Top : CSPs of ( 15N ) Ub ( G76C ) conjugated to indicated E2s via disulfide bond ( 775 µM each ) compared to free ( 15N ) Ub ( G76C ) are shown ; Ub resides known to be involved in the closed conformation interface are underlined in grey ; * = number of resonances with perturbations too large to be assigned confidently ( shown as off - scale in diagram ) . Bottom : Spectra of resonances for selected residues in Ub ' s hydrophobic patch for free Ub ( G76C ) ( gray ) and Ub conjugated to Ubc6 ( green ) and U7BR / Ubc7 ( yellow ) .

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

B HSQC-TROSY experiments comparing free and E2-conjugated (15N)Ub. Top: CSPs of (15N)Ub(G76C) conjugated to indicated E2s via disulfide bond (775 µM each) compared to free (15N)Ub(G76C) are shown; Ub resides known to be involved in the closed conformation interface are underlined in grey; * = number of resonances with perturbations too large to be assigned confidently (shown as off-scale in diagram). Bottom: Spectra of resonances for selected residues in Ub's hydrophobic patch for free Ub(G76C) (gray) and Ub conjugated to Ubc6 (green) and U7BR/Ubc7 (yellow).


Protein degradation in indicated yeast strains monitored by pulse - chase experiments for the Hrd1 model substrates PrA * - 3xHA Values for each time point are reported as means ± standard deviation ( n = 4 for PrA * - 3xHA For A , data for control strains ( Hrd1 ( 400R ) wt , Hrd1 ( 400H ) and Δhrd1 ) are the same as in Fig . 3D and 4A .

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

Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the Hrd1 model substrates PrA*-3xHA Values for each time point are reported as means ± standard deviation (n = 4 for PrA*-3xHA For A, data for control strains (Hrd1(400R) wt, Hrd1(400H) and Δhrd1) are the same as in Fig. 3D and 4A.


Protein degradation in indicated yeast strains monitored by CHX decay assays for the Doa10 model substrates Deg1 - eGFP2 Values for each time point are reported as means ± standard deviation n = 3 for Deg1 - eGFP2

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

Protein degradation in indicated yeast strains monitored by CHX decay assays for the Doa10 model substrates Deg1-eGFP2 Values for each time point are reported as means ± standard deviation n = 3 for Deg1-eGFP2


Protein degradation in indicated yeast strains monitored by CHX decay assays for the Doa10 model substrates Deg1 - eGFP2 Values for each time point are reported as means ± standard deviation n = 3 for Deg1 -

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


Protein degradation in indicated yeast strains monitored by CHX decay assays for the Doa10 model substrates FLAG - Sbh2 Values for each time point are reported as means ± standard deviation n = 3 for FLAG - Sbh2

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

Protein degradation in indicated yeast strains monitored by CHX decay assays for the Doa10 model substrates FLAG-Sbh2 Values for each time point are reported as means ± standard deviation n = 3 for FLAG-Sbh2


Protein degradation in indicated yeast strains monitored by CHX decay assays for the Doa10 model substrates FLAG - Sbh2 Values for each time point are reported as means ± standard deviation n = 3 for FLAG -

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


Protein degradation in indicated yeast strains monitored by pulse - chase experiments for the Hrd1 model substrates PrA * - 3xHA Values for each time point are reported as means ± standard deviation ( n = 4 for PrA * - 3xHA For D , data for control strains ( Doa10 ( 94H ) wt and Δdoa10 ) are the same as in Fig 3D and 4A .

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

Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the Hrd1 model substrates PrA*-3xHA Values for each time point are reported as means ± standard deviation (n = 4 for PrA*-3xHA For D, data for control strains (Doa10(94H) wt and Δdoa10) are the same as in Fig 3D and 4A.


Protein degradation in indicated yeast strains monitored by pulse - chase experiments for the lysine - less CPY * - K0 - HA Values for each time point are reported as means ± standard deviation

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Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the lysine-less CPY*-K0-HA Values for each time point are reported as means ± standard deviation


Protein degradation in indicated yeast strains monitored by pulse - chase experiments for the lysine - less CPY * - K0 - HA Values for each time point are reported as means ± standard

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


Protein degradation in indicated yeast strains monitored by pulse - chase experiments for FLAG - Sbh2 Values for each time point are reported as means ± standard deviation n = 3 for FLAG - Sbh2

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 B-MEASURED_VAR

Protein degradation in indicated yeast strains monitored by pulse-chase experiments for FLAG-Sbh2 Values for each time point are reported as means ± standard deviation n = 3 for FLAG-Sbh2


Protein degradation in indicated yeast strains monitored by pulse - chase experiments for FLAG - Sbh2 Values for each time point are reported as means ± standard deviation n = 3 for FLAG -

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C . Confocal images of U2OS cells transiently expressing lifeAct - GFP wild type and mutants of lifeAct residues interacting with F - actin , cells were additionally stained with SiR - actin to confirm the actin filaments . The line scan as indicated with yellow line on the cells shows the extent of lifeAct ( green ) and SiR - actin ( magenta ) co - staining of actin structures . Scale bar = 5µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal images of U2OS cells transiently expressing lifeAct-GFP wild type and mutants of lifeAct residues interacting with F-actin, cells were additionally stained with SiR-actin to confirm the actin filaments. The line scan as indicated with yellow line on the cells shows the extent of lifeAct (green) and SiR-actin (magenta) co-staining of actin structures. Scale bar = 5µm.


D . Binding affinities calculated from titration data of co - sedimentation assays of lifeAct - 14 ( 1 . 2µM ) and lifeAct - 17 ( 2 . 2µM ) as indicated . Data points for each concentration were averaged from 3 independent experiments , error bars represent S . D between independent experiments .

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D. Binding affinities calculated from titration data of co-sedimentation assays of lifeAct-14 (1.2µM) and lifeAct-17 (2.2µM) as indicated. Data points for each concentration were averaged from 3 independent experiments, error bars represent S.D between independent experiments.


C . Representative TIRF images of lifeAct binding experiments as indicated . The remaining lifeAct concentration images are shown in Supplement Figure 4 . Scale bar = 5µm .

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C. Representative TIRF images of lifeAct binding experiments as indicated. The remaining lifeAct concentration images are shown in Supplement Figure 4. Scale bar = 5µm.


D . Mean ratio of lifeAct fluorescent intensity bound to phalloidin and jasplakinolide F - actin ( mean and s . e . m ; n = 2 or 3 independent experiments with > 50 actin filament for each set ) .

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D. Mean ratio of lifeAct fluorescent intensity bound to phalloidin and jasplakinolide F-actin (mean and s.e.m; n = 2 or 3 independent experiments with >50 actin filament for each set).


A . Representative Coomassie stained SDS - PAGE gels of UTRN - ABD and UTRN - V87D co - sedimentation with F - actin . Pellet ( top ) and supernatant ( bottom ) fractions of individual co - sedimentation reactions of increasing utrophin concentrations , uncropped gel images of all the co - sedimentation reactions are presented in Supplement Figure 6 .

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A. Representative Coomassie stained SDS-PAGE gels of UTRN-ABD and UTRN-V87D co-sedimentation with F-actin. Pellet (top) and supernatant (bottom) fractions of individual co-sedimentation reactions of increasing utrophin concentrations, uncropped gel images of all the co-sedimentation reactions are presented in Supplement Figure 6.


B . Apparent Kd indicated was calculated from the titration data of co - sedimentation assays of utrophin wildtype ( 1 . 8µM ) and mutants ; H29A ( 2 . 8µM ) , I22D ( 38µM ) , V87D ( > 100µM ) and N109A ( 55µM ) as indicated . Data points for each concentration were averaged from 3 independent experiments , error bars represent S . D between independent experiments .

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B. Apparent Kd indicated was calculated from the titration data of co-sedimentation assays of utrophin wildtype (1.8µM) and mutants; H29A (2.8µM), I22D (38µM), V87D (>100µM) and N109A (55µM) as indicated. Data points for each concentration were averaged from 3 independent experiments, error bars represent S.D between independent experiments.


C . Confocal images of U20S cells transfected with GFP tagged UTRN - mini and mCherry UTRN - ABD , stained with SIR - Actin shows F - actin structures , mainly stress fibers . Scale bar = 5µm .

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C. Confocal images of U20S cells transfected with GFP tagged UTRN-mini and mCherry UTRN-ABD, stained with SIR-Actin shows F-actin structures, mainly stress fibers. Scale bar = 5µm.


Electron microscopy revealed a number of demyelinated or degenerated axons in PLP - 150Q mice at the age of 5 months . LAQ ( 5 mg / kg ) treatment at 3 months of age for 2 months improved myelination in PLP - 150Q mice . Scale bars : 2μm ( Low magnification ) and 0 . 5 μm ( High magnification ) .

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Electron microscopy revealed a number of demyelinated or degenerated axons in PLP-150Q mice at the age of 5 months. LAQ (5 mg/kg) treatment at 3 months of age for 2 months improved myelination in PLP-150Q mice. Scale bars: 2μm (Low magnification) and 0.5 μm (High magnification).


G - ratios , which were calculated and plotted against axon diameter with linear regression , were shown beneath the micrographs and were significantly decreased in PLP - 150Q mice after LAQ treatment ( G = 0 . 6418 ± 0 . 0191 ) , compared with age - matched vehicle group ( G = 0 . 7727 ± 0 . 0203 ) . One - way ANOVA with Tukey ' s test . * P = 0 . 0166 . At least 182 axons per genotype were examined from 3 mice in each group .

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G-ratios, which were calculated and plotted against axon diameter with linear regression, were shown beneath the micrographs and were significantly decreased in PLP-150Q mice after LAQ treatment (G=0.6418±0.0191), compared with age-matched vehicle group (G=0.7727±0.0203). One-way ANOVA with Tukey's test. *P=0.0166. At least 182 axons per genotype were examined from 3 mice in each group.


Western blotting showing that LAQ ( 5 or 25 mg / kg ) treatment for 2 months up - regulates multiple myelin proteins ( MBP , MOBP and MOG ) in the corpus callosum in PLP - 150Q mice , which were treated from 3 months of age . Ratios of MBP , MOBP or MOG to vinculin obtained from 3 independent experiments were presented on the right . One - way ANNOVA followed with Tukey ' s test . MBP : * * * P = 0 . 0009 ; MOG : * * * P = 0 . 0003 ; MOBP : * * P = 0 . 0067 . Data are mean ±

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Western blotting showing that LAQ (5 or 25 mg/kg) treatment for 2 months up-regulates multiple myelin proteins (MBP, MOBP and MOG) in the corpus callosum in PLP-150Q mice, which were treated from 3 months of age. Ratios of MBP, MOBP or MOG to vinculin obtained from 3 independent experiments were presented on the right. One-way ANNOVA followed with Tukey's test. MBP: ***P=0.0009; MOG: ***P=0.0003; MOBP: **P=0.0067. Data are mean ±SEM


Quantitative PCR of the transcript expression of myelin associated genes ( MBP and MOG ) in PLP - 150Q mouse corpus callosum at 5 months of age after LAQ ( 5 or 25 mg / kg ) treatment for 2 months . Student ' s t - test . MBP : * * * P = 0 . 0007 ; MOG : * * * P = 0 . 0009 . Data are mean ± SEM ( n = 3 ) .

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Quantitative PCR of the transcript expression of myelin associated genes (MBP and MOG) in PLP-150Q mouse corpus callosum at 5 months of age after LAQ (5 or 25 mg/kg) treatment for 2 months. Student's t-test. MBP: ***P=0.0007; MOG: ***P=0.0009. Data are mean ±SEM (n=3).


The MBP DNA promoter was inserted into the pGL4 . 1 luciferase report vector and was co - expressed with N - terminal MYRF ( nMYRF ) and mHTT to assess its transcription activity via the luciferase assay . MYRF markedly enhanced the MBP promoter activity . N - terminal mutant HTT significantly inhibited the reporter activity , * * * P < 0 . 001 ; which was reversed by LAQ ( 5 μM ) treatment , * * P < 0 . 01 . The ratios were obtained from 3 independent experiments . One - way ANNOVA followed with Tukey ' s test . Data are mean ± SEM .

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The MBP DNA promoter was inserted into the pGL4.1 luciferase report vector and was co-expressed with N-terminal MYRF (nMYRF) and mHTT to assess its transcription activity via the luciferase assay. MYRF markedly enhanced the MBP promoter activity. N-terminal mutant HTT significantly inhibited the reporter activity, ***P< 0.001; which was reversed by LAQ (5 μM) treatment, **P< 0.01. The ratios were obtained from 3 independent experiments. One-way ANNOVA followed with Tukey's test. Data are mean ±SEM.


Immunoprecipitation of transgenic mHTT from 3 - month - old PLP - 150Q mouse brains revealed that 5 or 25 mg / kg LAQ treatments for 2 months reduced the interaction between transgenic mutant HTT and MYRF . fMYRF : full - length MYRF ; nMYRF : N - terminal MYRF . The ratio of immunoprecipitated MYRF to input obtained from 3 independent experiments was shown on the right . One - way ANNOVA followed with Tukey ' s test . * * P = 0 . 009 . Data are mean ± SEM .

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Immunoprecipitation of transgenic mHTT from 3-month-old PLP-150Q mouse brains revealed that 5 or 25 mg/kg LAQ treatments for 2 months reduced the interaction between transgenic mutant HTT and MYRF. fMYRF: full-length MYRF; nMYRF: N-terminal MYRF. The ratio of immunoprecipitated MYRF to input obtained from 3 independent experiments was shown on the right. One-way ANNOVA followed with Tukey's test. **P=0.009. Data are mean ±SEM.


Western blotting of the phosphorylated - NF - kB , - Akt , - JNK in PLP - 150Q mouse corpus callosum at 3 months of age showing that LAQ ( 5 or 25 mg / kg ) treatment for 2 months could dephosphorylate these proteins . Ratios of the phosphorylated proteins to their total proteins obtained from 3 independent experiments were presented on the right . One - way ANNOVA with Tukey ' s test . NF - kB : * * * P = 0 . 00067 ( left ) and 0 . 00086 ( right ) ; JNK : * * * P = 0 . 00013 ( left ) and 0 . 00047 ( right ) ; Akt : * * P = 0 . 0014 and * * * P = 0 . 00012 . Data are mean ± SEM .

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Western blotting of the phosphorylated- NF-kB, -Akt, -JNK in PLP-150Q mouse corpus callosum at 3 months of age showing that LAQ (5 or 25 mg/kg) treatment for 2 months could dephosphorylate these proteins. Ratios of the phosphorylated proteins to their total proteins obtained from 3 independent experiments were presented on the right. One-way ANNOVA with Tukey's test. NF-kB: ***P=0.00067(left) and 0.00086(right); JNK: ***P=0.00013(left) and 0.00047(right); Akt: **P=0.0014 and ***P=0.00012. Data are mean ±SEM.


In vitro phosphorylation assay of N - terminal MYRF ( nMYRF ) . GST fusion proteins containing nMYRF were incubated overnight with brain lysates from PLP - 23Q or PLP - 150Q mice that were treated with vehicle or 5 mg / kg LAQ ( left panel ) . GST fusion proteins containing wild type N - terminal MYRF , S259A , or S261A were incubated with PLP - 150Q mouse tissue lysates ( right panel ) . The beads were then centrifuged and analyzed by western blotting with anti - GST ( lower panels ) and anti - phosphor - serine ( upper panels ) . Note that LAQ treatment could eliminate mutant HTT ( PLP - 150Q ) - mediated MYRF phosphorylation and that S259A substitution prevents MYRF phosphorylation .

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In vitro phosphorylation assay of N-terminal MYRF (nMYRF). GST fusion proteins containing nMYRF were incubated overnight with brain lysates from PLP-23Q or PLP-150Q mice that were treated with vehicle or 5 mg/kg LAQ (left panel). GST fusion proteins containing wild type N-terminal MYRF, S259A, or S261A were incubated with PLP-150Q mouse tissue lysates (right panel). The beads were then centrifuged and analyzed by western blotting with anti-GST (lower panels) and anti-phosphor-serine (upper panels). Note that LAQ treatment could eliminate mutant HTT (PLP-150Q)-mediated MYRF phosphorylation and that S259A substitution prevents MYRF phosphorylation.


Co - immunoprecipitation of nMYRF and N - terminal HTT ( 1 - 212 aa ) containing 150Q in HEK293 cells . Five μM LAQ treatment decreased the phosphorylation of immunoprecipitated MYRF and the amount of co - immunoprecipitated HTT . Λ - PPase served as the positive control for the dephosphorylation of MYRF . The ratios of the pho - serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under the blots , which were obtained from 3 independent western blotting experiments . One - way ANNOVA with Tukey ' s test . * * * P = 6 . 51x10 - 5 . Data are mean ± SEM .

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Co-immunoprecipitation of nMYRF and N-terminal HTT (1-212 aa) containing 150Q in HEK293 cells. Five μM LAQ treatment decreased the phosphorylation of immunoprecipitated MYRF and the amount of co-immunoprecipitated HTT. Λ-PPase served as the positive control for the dephosphorylation of MYRF. The ratios of the pho-serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under the blots, which were obtained from 3 independent western blotting experiments. One-way ANNOVA with Tukey's test. ***P=6.51x10-5. Data are mean ±SEM.


Co - transfection of nMYRF with N - terminal mutant HTT in HEK293 cells and immunoprecipitation of nMYRF with or without LAQ ( 5 μM ) treatment . Coomassie blue staining confirms the presence of immunoprecipitated N - terminal MYRF bands ( arrow ) .

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Co-transfection of nMYRF with N-terminal mutant HTT in HEK293 cells and immunoprecipitation of nMYRF with or without LAQ (5 μM) treatment. Coomassie blue staining confirms the presence of immunoprecipitated N-terminal MYRF bands (arrow).


Transcriptional activity of wide type N - terminal MYRF , S259A , or S261A with the MBP promoter reporter in HEK293 cells was detected using a luciferase assay . Compared to wide type MYRF or S261A , S259A ( non - phosphorylated ) remained transcriptional activity that was not inhibited by N - terminal mutant HTT . The ratios were obtained from 3 independent experiments . One - way ANNOVA with Tukey ' s test . * * * P < 0 . 001 . Data are mean ± SEM .

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Transcriptional activity of wide type N-terminal MYRF, S259A, or S261A with the MBP promoter reporter in HEK293 cells was detected using a luciferase assay. Compared to wide type MYRF or S261A, S259A (non-phosphorylated) remained transcriptional activity that was not inhibited by N-terminal mutant HTT. The ratios were obtained from 3 independent experiments. One-way ANNOVA with Tukey's test. ***P< 0.001. Data are mean ±SEM.


Co - transfection of wide type N - terminal MYRF , S259A , or S261A with N - terminal mutant HTT in HEK293 cells and immunoprecipitation of MYRF . Compared to wide type MYRF and S261A , less S259A ( non - phosphorylated ) was precipitated with mutant HTT . The ratios of the pho - serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under the blots and were obtained from 3 independent experiments . One - way ANNOVA with Tukey ' s test . * * * P = 2 . 30x10 - 5 . Data are mean ± SEM .

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Co-transfection of wide type N-terminal MYRF, S259A, or S261A with N-terminal mutant HTT in HEK293 cells and immunoprecipitation of MYRF. Compared to wide type MYRF and S261A, less S259A (non-phosphorylated) was precipitated with mutant HTT. The ratios of the pho-serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under the blots and were obtained from 3 independent experiments. One-way ANNOVA with Tukey's test. ***P=2.30x10-5 . Data are mean ±SEM.


Western blotting analysis of the corpus callosum in PLP - 23Q or PLP - 150Q mice with anti - pS259 and an antibody to total MYRF . Note that LAQ ( 5 mg / kg ) treatment for 2 months decreased the phosphorylation in both full length ( fMYRF ) and N - terminal MYRF ( nMYRF ) in PLP - 150Q mice .

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Western blotting analysis of the corpus callosum in PLP-23Q or PLP-150Q mice with anti-pS259 and an antibody to total MYRF. Note that LAQ (5 mg/kg) treatment for 2 months decreased the phosphorylation in both full length (fMYRF) and N-terminal MYRF (nMYRF) in PLP-150Q mice.


Ratios of phosphor - S259 - MYRF ( pMYRF ) and total MYRF to GAPDH . The ratios were obtained from 3 independent western blotting experiments . Student ' s t - test ; * * * P = 0 . 0007 . Data are mean ± SEM .

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Ratios of phosphor-S259-MYRF (pMYRF) and total MYRF to GAPDH. The ratios were obtained from 3 independent western blotting experiments. Student's t-test; ***P=0.0007. Data are mean ±SEM.


Anti - Ser259 - MYRF immunohistochemical staining of the corpus callosum ( CC , indicated between two dotted lines ) of PLP - 23Q and PLP - 150Q mice showing decreased expression of Ser259 - MYRF by LAQ ( 5 mg / kg ) treatment for 2 months . Micrographs at 10X ( upper ) and 40X ( lower ) are shown . Scale bars : 40 μm .

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Anti-Ser259-MYRF immunohistochemical staining of the corpus callosum (CC, indicated between two dotted lines) of PLP-23Q and PLP-150Q mice showing decreased expression of Ser259-MYRF by LAQ (5 mg/kg) treatment for 2 months. Micrographs at 10X (upper) and 40X (lower) are shown. Scale bars: 40 μm.


Quantitative statistics of the cells stained by anti - Ser259 MYRF in each filed was presented . Twenty random fields ( 40X ) of each section were examined to count anti - Ser259 positive cells , n = 3 mice in each group . One - way ANNOVA with Tukey ' s test . * * * P = 0 . 0003 ; * * P = 0 . 0039 . Data are mean ± SEM .

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Quantitative statistics of the cells stained by anti-Ser259 MYRF in each filed was presented. Twenty random fields (40X) of each section were examined to count anti-Ser259 positive cells, n=3 mice in each group. One-way ANNOVA with Tukey's test. ***P=0.0003; **P=0.0039. Data are mean ±SEM.


Western blotting analysis of MBP and Ser259 - MYRF expression in the postmortem brain cortex tissues in 4 HD patients and 4 non - HD control individuals . Ratios of MBP to GAPDH , or Ser259 ( pMYRF ) to total MYRF are presented on the right . One - way ANNOVA with Tukey ' s test . MBP : * * * P = 0 . 0004 ; pMYRF : * * P = 0 . 0051 . Data are mean ± SEM .

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Western blotting analysis of MBP and Ser259-MYRF expression in the postmortem brain cortex tissues in 4 HD patients and 4 non-HD control individuals. Ratios of MBP to GAPDH, or Ser259 (pMYRF) to total MYRF are presented on the right. One-way ANNOVA with Tukey's test. MBP: ***P=0.0004; pMYRF: **P=0.0051. Data are mean ±SEM.


Western blotting analysis of the expression of Ser259 ( pMYRF ) and total MYRF ( full - length : fMYRF and N - terminal : nMYRF ) in 13 - months - old HD - KI and WT mouse cortex . Ratios of pMYRF to total MYRF obtained from 3 independent experiments were presented beneath the blots . One - way ANNOVA with Tukey ' s test . * * * P = 0 . 00083 . Data are mean ± SEM .

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Western blotting analysis of the expression of Ser259 (pMYRF) and total MYRF (full-length: fMYRF and N-terminal: nMYRF) in 13-months-old HD-KI and WT mouse cortex. Ratios of pMYRF to total MYRF obtained from 3 independent experiments were presented beneath the blots. One-way ANNOVA with Tukey's test. ***P=0.00083. Data are mean ±SEM.


Heatmaps of gene expression in PLP - 150Q mouse corpus callosum at 5 months after LAQ ( 5 mg / kg ) treatment for 2 months revealed 27 significantly upregulated and 103 significantly downregulated genes .

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Heatmaps of gene expression in PLP-150Q mouse corpus callosum at 5 months after LAQ (5 mg/kg) treatment for 2 months revealed 27 significantly upregulated and 103 significantly downregulated genes.


The individual networks of up - regulated gene were heat mapped typically , on the clusters of " REGULATE _ OF _ PHOSPHORUS _ METABOLIC _ PROCESS " , and " POSITIVE _ REGULATE _ OF _ PHOSPHORUS _ METABOLIC _ PROCESS "

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The individual networks of up-regulated gene were heat mapped typically, on the clusters of "REGULATE_OF_PHOSPHORUS_METABOLIC_PROCESS", and "POSITIVE_REGULATE_OF_PHOSPHORUS_METABOLIC _PROCESS"


Western blotting showing down - regulation of PRKG2 in PLP - 150Q mouse corpus callosum at 3 months of age by 5 mg / kg LAQ . Ratios of PRKG2 or phosphorylated VASP ( p - VASP ) to GAPDH obtained from 3 independent experiments were presented on the right . One - way ANNOVA with Tukey ' s test . PRKG2 : * * P = 0 . 0076 ( left ) and * * * P = 0 . 0015 ( right ) ; p - VASP : * * * P = 3 . 86x10 - 5 ( left ) and 1 . 01x10 - 5 ( right ) . Data are mean ± SEM .

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Western blotting showing down-regulation of PRKG2 in PLP-150Q mouse corpus callosum at 3 months of age by 5 mg/kg LAQ. Ratios of PRKG2 or phosphorylated VASP (p-VASP) to GAPDH obtained from 3 independent experiments were presented on the right. One-way ANNOVA with Tukey's test. PRKG2: **P=0.0076(left) and ***P=0.0015(right); p-VASP: ***P=3.86x10-5(left) and 1.01x10-5(right). Data are mean ±SEM.


Quantitative PCR of PRKG2 or PRKG1 gene expression in PLP - 150Q mouse corpus callosum at 5 months of age after LAQ ( 5 mg / kg ) treatment for 2 months . Only PRKG2 was decreased by LAQ . n = 3 mice in each group . Student ' s t - test ; * * P = 0 . 0073 ; * * * P = 0 . 00041 . Data are mean ± SEM .

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Quantitative PCR of PRKG2 or PRKG1 gene expression in PLP-150Q mouse corpus callosum at 5 months of age after LAQ (5 mg/kg) treatment for 2 months. Only PRKG2 was decreased by LAQ. n=3 mice in each group. Student's t-test; **P=0.0073; ***P=0.00041. Data are mean ±SEM.


The different PRKG2 promoter regions were inserted into the pGL4 . 1 luciferase report vector . The core promoter reporter vector of PRKG2 ( - 472 / + 131 ) was determined for transcription activity in cultured N2a cells , obtained from 3 independent experiments . One - way ANNOVA with Tukey ' s test ; * * * P < 0 . 001 . Data are mean ± SEM .

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The different PRKG2 promoter regions were inserted into the pGL4.1 luciferase report vector. The core promoter reporter vector of PRKG2 (-472/+131) was determined for transcription activity in cultured N2a cells, obtained from 3 independent experiments. One-way ANNOVA with Tukey's test; ***P<0.001. Data are mean ±SEM.


The treatment of LAQ ( 5 or 10 μM ) caused a marked inhibition on the transcription activity of the PRKG2 promoter detected by the luciferase assay from 3 independent experiments . One - way ANNOVA with Tukey ' s test . * * * P < 0 . 001 . Data are mean ± SEM .

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The treatment of LAQ (5 or 10 μM) caused a marked inhibition on the transcription activity of the PRKG2 promoter detected by the luciferase assay from 3 independent experiments. One-way ANNOVA with Tukey's test. ***P<0.001. Data are mean ±SEM.


The qPCR analysis of the transcripts of Cyp1a1 and Ugt1a6a , which were mediated by AhR , in the PLP - 150Q mouse brain ( corpus callosum ) . Note that Cyp1a1 and Ugt1a6a were significantly increased by LAQ ( 5 mg / kg ) . n = 3 mice in each group . Student ' s t - test ; Cyp1a1 : * * * P = 0 . 00042 ; Ugt1a6a ; * * * P = 0 . 00057 . Data are mean ± SEM .

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

The qPCR analysis of the transcripts of Cyp1a1 and Ugt1a6a, which were mediated by AhR, in the PLP-150Q mouse brain (corpus callosum). Note that Cyp1a1 and Ugt1a6a were significantly increased by LAQ (5 mg/kg). n=3 mice in each group. Student's t-test; Cyp1a1: ***P=0.00042; Ugt1a6a; ***P=0.00057. Data are mean ±SEM.


The PRKG2 core promoter activity in N2a cells transfected with AhR siRNA or its scrambled siRNA control and then treated with 5 μM LAQ . The values of promoter activity via luciferase report assay were obtained from three independent experiments . One - way ANNOVA with Tukey ' s test . * * * P < 0 . 001 . Data are mean ± SEM .

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The PRKG2 core promoter activity in N2a cells transfected with AhR siRNA or its scrambled siRNA control and then treated with 5 μM LAQ. The values of promoter activity via luciferase report assay were obtained from three independent experiments. One-way ANNOVA with Tukey's test. ***P< 0.001. Data are mean ±SEM.


The semi - PCR detection of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti - AhR in ChIP assay . The N2a cells were treated with 10 μM LAQ or 10 nM 2 , 3 , 7 , 8 - TCDD or DMSO for 12 hr . The quantification of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti - AhR in ChIP assay . The N2a cells were treated with 10 μM LAQ or 10 nM 2 , 3 , 7 , 8 - TCDD or DMSO for 12 hr . The results were obtained from three independent experiments . One - way ANNOVA with Tukey ' s test . LAQ : * * * P = 0 . 00065 ; 2 , 3 , 7 , 8 - TCDD : * * * P = 0 . 00014 . Data are mean ±

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

The semi-PCR detection of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti-AhR in ChIP assay. The N2a cells were treated with 10 μM LAQ or 10 nM 2,3,7,8-TCDD or DMSO for 12 hr. The quantification of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti-AhR in ChIP assay. The N2a cells were treated with 10 μM LAQ or 10 nM 2,3,7,8-TCDD or DMSO for 12 hr. The results were obtained from three independent experiments. One-way ANNOVA with Tukey's test. LAQ: ***P=0.00065; 2,3,7,8-TCDD: ***P=0.00014. Data are mean ±SEM


Western blotting showing that the phosphorylation of transfected N - terminal MYRF ( pMYRF ) was promoted by treatment with the PKG activator of 8 - Br - cGMP ( 10 or 100 μM ) for 1 hr or co - transfected 150Q - HTT for 24 hr , which was blocked by treatment of the PKG inhibitors of RKRARKE ( 50 μM ) or KT5823 ( 5 μM ) for 12 hr . trans - nMYRF : transfected N - terminal MYRF .

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Western blotting showing that the phosphorylation of transfected N-terminal MYRF (pMYRF) was promoted by treatment with the PKG activator of 8-Br-cGMP (10 or 100 μM) for 1 hr or co-transfected 150Q-HTT for 24 hr, which was blocked by treatment of the PKG inhibitors of RKRARKE (50 μM) or KT5823 (5 μM) for 12 hr. trans-nMYRF: transfected N-terminal MYRF.


Western blotting of N2a cell line that was transfected with AAV - PRKG2 for 48 hr . The blots were probed with anti - Ser259 ( pMYRF ) antibody . Note that overexpression of PRKG2 increased phosphorylation of N - terminal MYRF ( nMYRF ) in a dose - dependent manner .

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Western blotting of N2a cell line that was transfected with AAV-PRKG2 for 48 hr. The blots were probed with anti-Ser259 (pMYRF) antibody. Note that overexpression of PRKG2 increased phosphorylation of N-terminal MYRF (nMYRF) in a dose-dependent manner.


Stereotaxic injection of AAV - GFP virus into the corpus callosum ( CC , indicated between two dotted lines ) in the mouse brain . Ctx : cortex , Str : striatum . Scale bar : 100 μm . . Immunohistochemical staining with anti - Ser259 ( pMYRF ) showing that AAV - PRKG2 , but not AAV - GFP , promotes MYRF phosphorylation in the corpus callosum . Scale bar : 40 μm .

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Stereotaxic injection of AAV-GFP virus into the corpus callosum (CC, indicated between two dotted lines) in the mouse brain. Ctx: cortex, Str: striatum. Scale bar: 100 μm..   Immunohistochemical staining with anti-Ser259 (pMYRF) showing that AAV-PRKG2, but not AAV-GFP, promotes MYRF phosphorylation in the corpus callosum. Scale bar: 40 μm.


Quantitative analysis of the pMYRF - or total MYRF - positive cells in each filed ( 40X ) . Twenty random fields in each section were examined , n = 3 mice in each group . One - way ANNOVA with Tukey ' s test ; * * * P = 8 . 79x10 - 5 . Data are mean ± SEM .

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Quantitative analysis of the pMYRF- or total MYRF-positive cells in each filed (40X). Twenty random fields in each section were examined, n=3 mice in each group. One-way ANNOVA with Tukey's test; ***P=8.79x10-5. Data are mean ±SEM.


Double immunofluorescent staining with antibodies to PRKG2 and Ser259 showing that AAV - PRKG2 , but not AAV - GFP ( green ) , promotes MYRF phosphorylation ( red ) in the injected corpus callosum . Scale bar : 40 μm .

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Double immunofluorescent staining with antibodies to PRKG2 and Ser259 showing that AAV-PRKG2, but not AAV-GFP (green), promotes MYRF phosphorylation (red) in the injected corpus callosum. Scale bar: 40 μm.


Western blotting analysis of the mouse corpus callosum tissues using anti - Ser259 ( pMYRF ) , phosphor - PRKG2 ( pPRKG2 ) or phosphor - VASP ( pVASP ) . Note that overexpression of mouse PRKG2 leads to the phosphorylation of MYRF and VASP . fMYRF : full - length MYRF , nMYRF : N - terminal MYRF .

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Western blotting analysis of the mouse corpus callosum tissues using anti-Ser259 (pMYRF), phosphor-PRKG2 (pPRKG2) or phosphor-VASP (pVASP). Note that overexpression of mouse PRKG2 leads to the phosphorylation of MYRF and VASP. fMYRF: full-length MYRF, nMYRF: N-terminal MYRF.


Construction of AAV - U6 - gRNA - CMV - RFP vector expressing PRKG2 gRNA under the U6 promoter and RFP protein under the CMV promoter , respectively . AAV - gRNA - PRKG2 was injected into the corpus callosum ( CC , indicated between two dotted lines ) in PLP - 150Q / Cas9 mouse at 2 months of age and the injected tissues were isolated 4 weeks after injection ( lower panel ) . Ctx : cortex , Str : striatum , LV : lateral ventricle . Scale bar : 100

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Construction of AAV-U6-gRNA-CMV-RFP vector expressing PRKG2 gRNA under the U6 promoter and RFP protein under the CMV promoter, respectively. AAV-gRNA-PRKG2 was injected into the corpus callosum (CC, indicated between two dotted lines) in PLP-150Q/Cas9 mouse at 2 months of age and the injected tissues were isolated 4 weeks after injection (lower panel). Ctx: cortex, Str: striatum, LV: lateral ventricle. Scale bar: 100 μm


Western blotting analysis of the PLP - 150Q / Cas9 mouse corpus callosum tissues using the antibody to Ser259 ( pMYRF ) , phosphor - PRKG2 or MBP . Note that reduction of mouse PRKG2 by CRISPR / Cas9 leads to the decreased MYRF phosphorylation and the increased expression of MBP . The ratios of pMYRF to MYRF or MBP to GAPDH obtained from 3 independent experiments were presented under the blots . Student ' s t - test ; pMYRF : * * * P = 0 . 00026 ; MBP ; * * P = 0 . 0036 . Data are presented as mean ± SEM . fMYRF : full - length MYRF , nMYRF : N - terminal MYRF .

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Western blotting analysis of the PLP-150Q/Cas9 mouse corpus callosum tissues using the antibody to Ser259 (pMYRF), phosphor-PRKG2 or MBP. Note that reduction of mouse PRKG2 by CRISPR/Cas9 leads to the decreased MYRF phosphorylation and the increased expression of MBP. The ratios of pMYRF to MYRF or MBP to GAPDH obtained from 3 independent experiments were presented under the blots. Student's t-test; pMYRF: ***P=0.00026; MBP; **P=0.0036. Data are presented as mean ±SEM. fMYRF: full-length MYRF, nMYRF: N-terminal MYRF.


Immunohistochemical staining with antibody to Ser259 showed that knocking down PRKG2 reduced MYRF phosphorylation in the corpus callosum of PLP - 150Q mouse . Scale bar : 40 μm .

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Immunohistochemical staining with antibody to Ser259 showed that knocking down PRKG2 reduced MYRF phosphorylation in the corpus callosum of PLP-150Q mouse. Scale bar: 40 μm.


Quantitative analysis of the pMYRF - positive cells in each filed ( 40X ) . Twenty random fields in each section were examined , n = 3 mice in each group . One - way ANNOVA with Tukey ' s test ; * * * P = 0 . 00022 . Data are mean ± SEM .

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Quantitative analysis of the pMYRF-positive cells in each filed (40X). Twenty random fields in each section were examined, n=3 mice in each group. One-way ANNOVA with Tukey's test; ***P=0.00022. Data are mean ±SEM.


Immunofluorescent staining with antibodies to Ser259 or MBP showed the AAV - PRKG2 - gRNA ( red ) injection increased MBP expression ( green ) as compared with AAV - gRNA - control . Scale bar : 40 μm .

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Immunofluorescent staining with antibodies to Ser259 or MBP showed the AAV- PRKG2-gRNA (red) injection increased MBP expression (green) as compared with AAV-gRNA-control. Scale bar: 40 μm.


Quantitative analysis of the MBP - positive cells in the corpus callosum injected with control - gRNA or PRKG2 - gRNA . Twenty random fields ( 40X ) in each section were examined . n = 3 mice in each group . One - way ANNOVA with Tukey ' s test ; * * * P = 4 . 43x10 - 5 . Data are mean ± SEM .

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Quantitative analysis of the MBP-positive cells in the corpus callosum injected with control-gRNA or PRKG2-gRNA. Twenty random fields (40X) in each section were examined. n=3 mice in each group. One-way ANNOVA with Tukey's test; ***P=4.43x10-5. Data are mean ±SEM.


A Proliferation of melanoma cell lines treated with LDHA inhibitor GSK - 2837808A ( 10 μM ) for the indicated duration under hypoxic ( 1 % O2 ) and normoxic conditions . Data information : Statistical analysis was performed by two - way Anova for time - dependent proliferation changes and by one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . In ( A ) statistical comparison for the treatments that showed a significant change .

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A Proliferation of melanoma cell lines treated with LDHA inhibitor GSK-2837808A (10 μM) for the indicated duration under hypoxic (1% O2) and normoxic conditions. Data information: Statistical analysis was performed by two-way Anova for time-dependent proliferation changes and by one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. In (A) statistical comparison for the treatments that showed a significant change.


B ( left ) Immunoblotting of LDHA in melanoma cell lines transfected with non - targeting siRNA ( NT - siRNA ) or LDHA - specific siRNA for 72 hr . ( right ) Proliferation of melanoma cell lines transfected with NT - siRNA or LDHA - specific siRNA for the indicated timepoints . Data information : Statistical analysis was performed by two - way Anova for time - dependent proliferation changes and by one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . In ( B ) , statistical comparison for the treatments that showed a significant change .

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B (left) Immunoblotting of LDHA in melanoma cell lines transfected with non-targeting siRNA (NT-siRNA) or LDHA-specific siRNA for 72 hr. (right) Proliferation of melanoma cell lines transfected with NT-siRNA or LDHA-specific siRNA for the indicated timepoints. Data information: Statistical analysis was performed by two-way Anova for time-dependent proliferation changes and by one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. In (B), statistical comparison for the treatments that showed a significant change.


Immunoblotting of LDHA of MeWo and A375 cells stably transduced with empty vector ( EV ) or LDHA - specific shRNAs .

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Immunoblotting of LDHA of MeWo and A375 cells stably transduced with empty vector (EV) or LDHA-specific shRNAs.


proliferation under normoxic ( D ) conditions of MeWo and A375 cells stably transduced with empty vector ( EV ) or LDHA - specific shRNAs . Data information : Statistical analysis was performed by two - way Anova for time - dependent proliferation changes and by one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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proliferation under normoxic (D) conditions of MeWo and A375 cells stably transduced with empty vector (EV) or LDHA-specific shRNAs. Data information: Statistical analysis was performed by two-way Anova for time-dependent proliferation changes and by one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


proliferation under hypoxic conditions of MeWo and A375 cells stably transduced with empty vector ( EV ) or LDHA - specific shRNAs . Data information : Statistical analysis was performed by two - way Anova for time - dependent proliferation changes and by one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . statistical significance is denoted by blue ( for si / sh - LDHA # 1 , hypoxia ) and black ( for sish - LDHA # 2 , hypoxia ) stars respectively .

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proliferation under hypoxic conditions of MeWo and A375 cells stably transduced with empty vector (EV) or LDHA-specific shRNAs. Data information: Statistical analysis was performed by two-way Anova for time-dependent proliferation changes and by one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. statistical significance is denoted by blue (for si/sh-LDHA#1, hypoxia) and black (for sish-LDHA#2, hypoxia) stars respectively.


G Anchorage - independent growth of MeWo and A375 cells stably transduced with empty vector ( EV ) or LDHA - specific shRNAs . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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G Anchorage-independent growth of MeWo and A375 cells stably transduced with empty vector (EV) or LDHA-specific shRNAs. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


B , C Glucose ( B ) and glutamine ( Gln , C ) levels in the medium after treatment of melanoma cell lines with LDHAi for 72 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups , N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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B, C Glucose (B) and glutamine (Gln, C) levels in the medium after treatment of melanoma cell lines with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


D Medium Gln levels after transfection of melanoma cells with non - targeting ( NT ) or LDHA - specific siRNA for 96 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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D Medium Gln levels after transfection of melanoma cells with non-targeting (NT) or LDHA-specific siRNA for 96 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


E GC - MS - based quantification of intracellular Gln levels in cells Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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

E GC-MS-based quantification of intracellular Gln levels in cells Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.