C distribution of wild type K262R FOXP3 . Hela were transfected with expression vectors encoding either wild type FOXP3 or K262R mutant . relative overlap of FOXP3 signal DAPI - stained nuclei was observed by microscopy . ( blue : DAPI , green : FOXP3 , Scale bars : 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 O O O O O O

C Cellular distribution of wild type and K262R FOXP3. Hela cells were transfected with expression vectors encoding either wild type FOXP3 or the K262R mutant. The relative overlap of FOXP3 protein signal and DAPI-stained nuclei was observed by fluorescence microscopy. (blue: DAPI, green: FOXP3, Scale bars: 50 μm).


D Immunostaining of FOXP3 in Traf6fl / flCD4Cre + wild type ( Traf6fl / flCD4Cre - ) derived suspensions of lymph node spleen . ( blue : DAPI , green : FOXP3 , Scale bars : 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

D Immunostaining of FOXP3 in Traf6fl/flCD4Cre+ and wild type (Traf6fl/flCD4Cre-) derived suspensions of lymph node and spleen cells. (blue: DAPI, green: FOXP3, Scale bars: 50 μm).


E of FOXP3 distribution in murine Tregs . degree of FOXP3 - coIocalization in CD4 + / FOXP3 + isolated from lymph nodes spleens of Traf6fl / flCD4Cre + wild type ( Traf6fl / flCD4Cre - ) mice was determined by Imagestream , frequencies of with either a low or high probability of FOXP3 distribution are shown ( red : stain , green : FOXP3 , Scale bars : 10 um ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of FOXP3 distribution in murine Tregs. The degree of FOXP3-nuclear coIocalization in CD4+/FOXP3+ cells isolated from the lymph nodes and spleens of Traf6fl/flCD4Cre+ and wild type (Traf6fl/flCD4Cre-) mice was determined by Imagestream analysis, and the frequencies of cells with either a low or high probability of nuclear FOXP3 distribution are shown (red: nuclear stain, green:FOXP3, Scale bars: 10 um).


in vitro suppressive potency of wild type - K262R Foxp3 - expressing T . Naïve CD4 + T were purified from Thy1 . 1 + BALB / c mice subjected to retroviral transduction to express either wild type FOXP3 or a K262R mutant resistant to ubiquitination that lysine residue 262 . These engineered " Tregs " were then co - cultured with naïve responder CD4 + T stained with CFSE at indicated ratios activated with anti - CD3 / CD28 antibodies . Dilution of CFSE signal by responder populations ( i . e . proliferation ) was assessed by flow cytometry .

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

The in vitro suppressive potency of wild type- and K262R Foxp3-expressing T cells. Naïve CD4+ T cells were purified from Thy1.1+ BALB/c mice and subjected to retroviral transduction to express either wild type FOXP3 or a K262R mutant resistant to ubiquitination that lysine residue 262. These engineered "Tregs" were then co-cultured with naïve responder CD4+ T cells stained with CFSE at the indicated ratios and activated with anti-CD3/CD28 antibodies. Dilution of CFSE signal by the responder populations (i.e. proliferation) was assessed by flow cytometry.


C In vivo suppression of colitis by wild type - K262R mutant - Foxp3 expressing T . 2x105 naïve CD4 + transductants from A were mixed with 1x106 colitogenic naïve CD4 + T from wild type mice transferred i . v . into Rag2 - / - mice . Rag2 - / - mice receiving no transfers , naïve CD4 + T alone , or purified wild type Tregs served as controls ( n = 6 / group ) . Recipient mouse weights were monitored weekly for each 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

C In vivo suppression of colitis by wild type- and K262R mutant-Foxp3 expressing T cells. 2x105 naïve CD4+ transductants from A were mixed with 1x106 colitogenic naïve CD4+ T cells from wild type mice and transferred i.v. into Rag2-/- mice. Rag2-/- mice receiving no cell transfers, naïve CD4+ T cells alone, or purified wild type Tregs served as controls (n= 6/group). Recipient mouse body weights were monitored weekly for each group.


D , E After 8 , colons were excised , fixed in buffered formalin , processed for H E ( D ) histopathology scoring ( E ) . Scale bars shown in 1 mm ) .

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

D, E After 8 weeks, colons were excised, fixed in buffered formalin, and processed for H and E staining (D) and histopathology scoring (E). Scale bars shown in 1 mm).


B Western blot of liver lysates ( 100 µg ) shows that intein - mediated trans - splicing efficiently reconstitutes full - eGFP . Single AAV : n = 5 ; AAV - intein : n = 5 . Arrows indicate both full - eGFP excised inteins . C of eGFP bands . Values are reported as mean ± . Each dot represents eGFP band from injected with either single AAV n = 5 or AAV intein n = 5 .

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

B Western blot analysis of liver lysates (100 µg) shows that intein-mediated protein trans-splicing efficiently reconstitutes full-length eGFP. Single AAV: n=5; AAV-intein: n=5. Arrows indicate both full-length eGFP and excised inteins. C Quantification of eGFP protein bands. Values are reported as mean±SEM. Each dot represents the eGFP protein band quantification from animals injected with either single AAV n=5 or AAV intein n=5.


B Western blot of lysates of HEK293 72 hours post - transfection with various F8 variants . Neg , non - transfected .

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

B Western blot analysis of lysates of HEK293 cells 72 hours post-transfection with the various F8 variants. Neg, non-transfected cells.


C Chromogenic of F8 activity in medium of transfected are reported as International Units / deciliter ( IU / dl ) . Data are presented as mean ± . Each dot within same group corresponds to a biological replicate : Neg n = 5 ; Wild - type n = 8 ; N6 n = 8 ; SQ n = 7 ; V3 n = 6 . Significant differences between groups were assessed using Kruskal - Wallis followed by posthoc : Nemenyi ' s - Pairs Rank Comparison . Kruskal - Wallis p value = 2 . 88e - 05 . * * indicates significant difference between N6 Wild - type groups : p value ≤ 0 . 01 . * indicates significant difference between V3 Wild - type groups : p value ≤ 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Chromogenic assay of F8 activity in the medium of transfected cells are reported as International Units/deciliter (IU/dl). Data are presented as mean±SEM. Each dot within the same group corresponds to a biological replicate: Neg n=5; Wild-type n=8; N6 n=8; SQ n=7; V3 n=6. Significant differences between groups were assessed using The Kruskal-Wallis test followed by the posthoc analysis: Nemenyi's All-Pairs Rank Comparison Test. The Kruskal-Wallis test p value = 2.88e-05. **indicates the significant difference between the N6 and the Wild-type groups: p value ≤ 0.01.*indicates the significant difference between the V3 and the Wild-type groups: p value ≤ 0.05.


A Western blot ( WB ) of lysates of HEK293 72 hours post - transfection ( n = 3 ; biological replicates ) with either Npu inteins or Heterologous ( N - intein DnaB + C - intein DnaE ) split - inteins . I + II , N6 split - intein ; I + II ( Het . ) , heterologous split - intein ; I , 5 ' N6 coding ( CDS ) - N - DnaE ; I ( N - int B ) , 5 ' N6 CDS - N - DnaB . II , C - DnaE - 3 ' N6 CDS . Excised inteins ( ∼ 12kDa ) are present only in down part of blot when I + II ( Npu inteins ) are provided . Arrows indicate full - N6 , single halves excised inteins .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot (WB) of protein lysates of HEK293 cells 72 hours post-transfection (n=3; biological replicates) with either Npu inteins or Heterologous (N-intein DnaB + C-intein DnaE) split-inteins. I + II, N6 split-intein proteins; I+II (Het.), heterologous split-intein proteins; I, 5'N6 coding sequence (CDS)-N-DnaE protein; I (N-int B), 5'N6 CDS-N-DnaB. II, C-DnaE-3'N6 CDS protein. Excised inteins (∼12kDa) are present only in the down part of the blot when I + II (Npu inteins) are provided. Arrows indicate the full-length N6 protein, single halves and excised inteins.


B WB of medium of transfected showing secreted ( n = 3 ; biological replicates ) . Arrows indicate full - N6 as well as single halves excised inteins .

O O O O O O 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 WB of medium of the transfected cells showing the secreted proteins (n=3; biological replicates). Arrows indicate the full-length N6 protein as well as single halves and excised inteins.


C Chromogenic performed on medium of transfected to detect F8 activity reported as International Units / deciliter ( IU / dl ) . Data are presented as mean ± . Each dot within each group represent a different biological replicate ( n ) : I + II n = 3 ; I + II ( Het . ) n = 3 ; I ( N - int E ) n = 3 ; I ( N - int B ) n = 3 ; II ( C - int E ) n = 3 ; Neg n = 4 . Significant differences between groups were assessed using Kruskal - Wallis rank sum Kruskal − Wallis , p value = 0 . 013 . * indicates significant difference between I + II I ( N - int E ) groups : p value ≤ 0 . 05 . * * * indicates significant difference between I + II Neg groups : p value ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Chromogenic assay performed on the medium of transfected cells to detect F8 activity levels reported as International Units/deciliter (IU/dl). Data are presented as mean ±SEM. Each dot within each group represent a different biological replicate (n): I + II n=3; I+II (Het.) n=3; I (N-int E) n=3; I (N-int B) n=3; II (C-int E) n=3; Neg n=4. Significant differences between groups were assessed using Kruskal-Wallis rank sum test Kruskal−Wallis, p value = 0.013. *indicates the significant difference between the I+II and the I (N-int E) groups: p value ≤ 0.05. ***indicates the significant difference between the I+II and the Neg groups: p value ≤0.001.


A Western blot ( WB ) of lysates of HEK293 72 hpt with AAV - N6 split - intein plasmids with codon - optimised set . I + II , N6 split - intein ; I , 5 ' N6 CDS - N - DnaE ; II , C - DnaE - 3 ' N6 CDS . ( n = 3 ; biological replicates ) . Arrows indicate full - N6 , excised inteins both single halves . Codop : codon - optimised .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot (WB) of protein lysates of HEK293 cells 72 hpt with the AAV-N6 split- intein plasmids and with the codon-optimised set. I + II, N6 split-intein proteins; I, 5' N6 CDS-N-DnaE protein; II, C-DnaE-3'N6 CDS protein. (n=3; biological replicates). Arrows indicate the full-length N6 protein, excised inteins and both single halves. Codop: codon-optimised.


B WB of medium from transfected showing increased secretion of Codop - N6 full - compared to non - codon - optimised . ( n = 3 ; biological replicates ) . Arrows indicate full - N6 , excised inteins both single halves .

O O O O O O O O O O O O O O O O O O O O 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 WB of medium from the transfected cells showing increased secretion of the Codop-N6 full-length protein compared to the non-codon-optimised. (n=3; biological replicates). Arrows indicate the full-length N6 protein, excised inteins and both single halves.


C Chromogenic performed on medium from transfected to measure F8 activity reported as International Units / deciliter ( IU / dl ) ( n = 3 ; biological replicates ) . Data are presented as mean ± . Significant differences between groups were assessed using Kruskal - Wallis p value = 0 . 027 . * indicates significant difference between I + II ( N6 split - intein ) II ( C - DnaE - 3 ' N6 CDS ) groups : p value ≤ 0 . 05 . * * indicates significant difference between Codop I + II I ( 5 ' N6 CDS - N - DnaE ) codop groups : p value ≤ 0 . 01 . * * indicates significant difference between Codop I + II II ( C - DnaE - 3 ' N6 CDS ) codop groups : p value ≤ 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Chromogenic assay performed on the medium from transfected cells to measure F8 activity levels reported as International Units/deciliter (IU/dl) (n=3; biological replicates).Data are presented as mean ±SEM. Significant differences between groups were assessed using Kruskal-Wallis test p value = 0.027. *indicates the significant difference between the I+II (N6 split-intein) and the II (C-DnaE-3'N6 CDS) groups: p value ≤ 0.05. **indicates the significant difference between the Codop I+II and the I (5' N6 CDS-N-DnaE) codop groups: p value ≤ 0.01. **indicates the significant difference between the Codop I+II and the II (C-DnaE-3'N6 CDS) codop groups: p value ≤ 0.01.


A Alkaline gel Southern blot of AAV . AAV was hybridized to a probe specific for HLP promoter . Neg , AAV treated with Dnase I ; CodopV3 , AAV - CodopV3 ; I , AAV - 5 ' N6 - N - intein ; II , AAV - C - intein - 3 ' N6 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Alkaline gel Southern blot analysis of AAV DNA. AAV DNA was hybridized to a probe specific for the HLP promoter. Neg, AAV DNA treated with Dnase I; CodopV3, AAV-CodopV3; I, AAV-5'N6-N-intein; II, AAV-C-intein-3' N6.


B Chromogenic performed on plasma samples to detect F8 activity in AAV - treated mice compared to controls groups . Data are presented as mean ± . same were analysed at various time - points . Each dot within same group within different groups of treatment corresponds to a single . baseline includes ( n = 20 ) before treatment : CodopV3 : n = 8 ; N6 intein : n = 5 ; CodopN6 intein : n = 5 . statistical difference between groups has been assessed at 4weeks post - injection ( wpi ) with One - way Anova p value = 0 . 056 ; at 8 wpi with Kruskal - Wallis p value = 0 . 68 ; at 12wpi with Kruskal - Wallis p value = 0 . 45 ; at 16wpi with Kruskal - Wallis p value = 0 . 58 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Chromogenic assay performed on plasma samples to detect F8 activity in AAV-treated mice compared to controls groups. Data are presented as mean ±SEM. The same animals were analysed at the various time-points. Each dot within the same group and within different groups of treatment corresponds to a single animal. The baseline includes all animals (n=20) before the treatment: CodopV3: n=8; N6 intein: n=5; CodopN6 intein: n=5. The statistical difference between groups has been assessed at 4weeks post-injection (wpi) with the One-way Anova p value = 0.056; at 8 wpi with the Kruskal-Wallis test p value = 0.68; at 12wpi with the Kruskal-Wallis test p value = 0.45; at 16wpi with the Kruskal-Wallis test p value = 0.58.


C Activated partial thromboplastin time ( aPTT ) performed on plasma samples both at baseline at last time - point of ( 16 wpi ) . Data are presented as mean ± . Each dot within each group of treatment corresponds to different : baseline includes before treatment n = 21 ; CodopV3 : n = 8 ; N6 intein : n = 5 ; CodopN6 intein : n = 5 . Significant differences between groups were assessed using Kruskal - Wallis . * * indicates significant difference between baseline N6 intein groups : p value ≤ 0 . 01 . * indicates significant difference between baseline CodopN6 intein groups : p value ≤ 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Activated partial thromboplastin time (aPTT) assay performed on plasma samples both at the baseline and at the last time-point of the analysis (16 wpi). Data are presented as mean ±SEM. Each dot within each group of treatment corresponds to different animals: The baseline includes all animals before the treatment n=21; CodopV3: n=8; N6 intein: n=5; CodopN6 intein: n=5. Significant differences between groups were assessed using the Kruskal-Wallis test. **indicates the significant difference between the baseline and the N6 intein groups: p value ≤ 0.01.*indicates the significant difference between the baseline and the CodopN6 intein groups: p value ≤ 0.05.


D Tail - clip reported as bleeding time in seconds . Significant differences between groups were assessed using One - way Anova p value is = 0 . 11 . Data are presented as mean ± . Each dot corresponds to a different . Knockout ( K . O . ) group : n = 4 ; CodopV3 : n = 4 ; N6 intein : n = 4 ; CodopN6 intein : n =

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Tail-clip assay reported as bleeding time in seconds. Significant differences between groups were assessed using One-way Anova test p value is = 0.11.Data are presented as mean±SEM. Each dot corresponds to a different animal. Knockout (K.O.) group: n=4; CodopV3: n=4; N6 intein: n=4; CodopN6 intein: n=


E Haemoglobin content measured as Optical Density ( OD ) at 416 nm after tail - clip . Significant differences between groups were assessed using One - way Anova . Data are reported as mean ± . Each dot corresponds to a single . Knockout ( K . O . ) group : n = 4 ; CodopV3 : n = 4 ; N6 intein : n = 4 ; CodopN6 intein : n = 6 .

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

E Haemoglobin content measured as Optical Density (OD) at 416 nm after the tail-clip assay. Significant differences between groups were assessed using One-way Anova test. Data are reported as mean±SEM. Each dot corresponds to a single animal. Knockout (K.O.) group: n=4; CodopV3: n=4; N6 intein: n=4; CodopN6 intein: n=6.


F Western blot of liver lysates ( 100 µg ) from either CodopN6 intein - treated mice ( n = 6 ) or untreated haemophilic mice ( n = 2 ) . A lysate from HEK293 transfected with N6 full - plasmid was used as positive control ( 50 µg ) . Arrows point at N6 full - ( N6 ) , 5 ' half of N6 ( 5 ' N6 ) 3 ' half of N6 ( 3 ' N6 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot analysis of liver lysates (100 µg) from either CodopN6 intein-treated mice (n=6) or untreated haemophilic mice (n=2). A lysate from HEK293 cells transfected with the N6 full-length plasmid was used as positive control (50 µg). Arrows point at N6 full-length protein (N6), the 5' half of N6 (5'N6) and 3' half of N6 (3'N6).


A amount of anti - F8 antibodies analysed by indirect ELISA is reported in Arbitrary Units / milliliter ( AU / ml ) . Each numbered bar represents a single mouse . CodopV3 , AAV - CodopV3 injected group n = 8 ; N6 intein , AAV - N6 intein injected group n = 5 ; CodopN6 intein , AAV - CodopN6 intein injected group n = 5 . Significant differences were assessed as follow : Paired T - has been used for CodopV3 n = 5 : p value ≤ 0 . 001 ; Wilcoxon for CodopV3 n = 3 : p value ≤ 0 . 05 ; Paired T - has been used for N6 intein n = 5 : p value ≤ 0 . 05 ; Paired T - has been used for CodopN6 intein n = 5 p value ≤ 0 . 01 .

A The amount of anti-F8 antibodies analysed by indirect ELISA is reported in Arbitrary Units/milliliter (AU/ml). Each numbered bar represents a single mouse. CodopV3, AAV-CodopV3 injected group n=8; N6 intein, AAV-N6 intein injected group n=5; CodopN6 intein, AAV-CodopN6 intein injected group n=5. Significant differences were assessed as follow: Paired T-test has been used for CodopV3 n=5: p value ≤ 0.001; Wilcoxon test for CodopV3 n=3: p value ≤ 0.05; Paired T-test has been used for N6 intein n=5: p value ≤ 0.05; Paired T- test has been used for CodopN6 intein n=5 p value ≤ 0.01.


B Bethesda performed on plasma samples mice injected with AAV - CodopV3 ; on plasma of injected with AAV - N6 intein n = 3 plasma of injected with AAV - CodopN6 intein n = 2 ; plasma of a wild - type was used as a control . dotted indicates threshold above which anti F8 - antibodies are considered inhibitors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Bethesda assay performed on plasma samples mice injected with AAV-CodopV3; on the plasma of animals injected with AAV-N6 intein n=3 and the plasma of animals injected with AAV-CodopN6 intein n=2; plasma of a wild-type animal was used as a control. The dotted line indicates the threshold above which anti F8-antibodies are considered inhibitors.


C Pro - inflammatory cytokines followed over - time reported as of baseline ( time point before injection ) . Data are represented as mean ± . Five belonging to CodopV3 group were analysed . Significant differences were assessed using Kruskal − Wallis ; for IP - 10 : * indicates p value ≤ 0 . 05 between 0 4 wpi ; * indicates p value ≤ 0 . 05 between 0 8 wpi ; * indicates p value ≤ 0 . 05 between 0 16 wpi . ( n = 20 ) ; for eotaxin : * indicates p value ≤ 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Pro-inflammatory cytokines levels followed over-time and reported as percentage of the baseline levels (time point before the injection). Data are represented as mean±SEM. Five animals belonging to the CodopV3 group were analysed. Significant differences were assessed using the Kruskal−Wallis test; for IP-10: * indicates the p value ≤ 0.05 between 0 and 4 wpi; * indicates the p value ≤ 0.05 between 0 and 8 wpi; * indicates the p value ≤ 0.05 between 0 and 16 wpi. (n=20); for eotaxin: * indicates the p value ≤ 0.05.


A Representative of eyes lenses from 6 - 7 week old wildtype ( WT ) nervous system - specific YME1L knockout ( NYKO ) mice . Orange dashed lines mark eye . Scale bars 5 mm .

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

A Representative images of eyes and lenses from 6-7 week old wildtype (WT) and nervous system-specific YME1L knockout (NYKO) mice. Orange dashed lines mark eye morphology. Scale bars 5 mm.


Retinal sagittal cross - sections from 6 - 7 week old mice stained with hematoxylin eosin . NFL = Nerve fiber layer , IPL = inner plexiform layer , INL = inner layer , OPL = outer plexiform layer , ONL = outer layer , R & C = Rods cones . Scale bars 30

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

Retinal sagittal cross-sections from 6-7 week old mice stained with hematoxylin and eosin. NFL = Nerve fiber layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer, R&C = Rods and cones. Scale bars 30 µ


of nuclei in OPL ( = 1000 µm2 ) from retina cross - sections of 6 - 7 week old WT ( n = 4 ) NYKO ( n = 4 ) mice . Data were analyzed using unpaired t - , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± .

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

Quantification of nuclei in OPL (area = 1000 µm2) from retina cross-sections of 6-7 week old WT (n = 4) and NYKO (n = 4) mice. Data were analyzed using unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


D Immunoblot of retinal lysates from 6 - 7 week old WT NYKO mice . GFAP was used as a marker for reactive astrogliosis SDHA as a loading control . Data were analyzed using unpaired t - , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 retinal lysates from 6-7 week old WT and NYKO mice. GFAP was used as a marker for reactive astrogliosis and SDHA as a loading control. Data were analyzed using unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


E mRNA of proinflammatory cytokines NF - κB target genes from 6 - 7 week old retinas ( WT , n = 5 ; NYKO , n = 5 ) . Transcript were normalized to Hprt mRNA . Data were analyzed using unpaired t - , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and NF-κB target genes from 6-7 week old retinas (WT, n = 5; NYKO, n = 5). Transcript levels were normalized to Hprt mRNA levels. Data were analyzed using unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


mRNA of Fgf21 from 6 - 7 week old retinas ( WT , n = 5 ; NYKO , n = 5 ) . Transcript were normalized to Hprt mRNA . Data were analyzed using unpaired t - , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± .

mRNA levels of Fgf21 from 6-7 week old retinas (WT, n = 5; NYKO, n = 5). Transcript levels were normalized to Hprt mRNA levels. Data were analyzed using unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


G Transmission electron of optic nerves from 6 - 7 week old WT NYKO mice . Scale bars 2 µm .

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

G Transmission electron micrographs of optic nerves from 6-7 week old WT and NYKO mice. Scale bars 2 µm.


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

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of 6 - 7 week old ( WT , n = 10 ; NYKO , n = 10 ) , 17 - 18 week old ( WT , n = 8 ; NYKO , n = 9 ) 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 - was used for comparison of two groups , ordinary one - way ANOVA for comparison of three groups . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . Data are means ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of 31 - 32 week old WT 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 of ( WT , n = 15 ; NYKO , n = 14 ) . Unpaired t - , ns = not significant . Data are means ± .

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 NYKO mice . 4x 40x enlargements are shown in left 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

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

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

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 NYKO mice of indicated . 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

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 ) NYKO ( n = 4 ) , 17 - 18 week old WT ( n = 4 ) NYKO ( n = 4 ) 31 - 32 week old WT ( n = 6 - 7 ) NYKO ( n = 5 - 6 ) mice spinal cords . Unpaired t - , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± .

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

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


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

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


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

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 of total axon ( WT , n = 13 neurons ; Yme1l - / - , n = 13 neurons ) , total dendritic ( WT , n = 12 neurons ; Yme1l - / - , n = 13 neurons ) , axonal branch points ( WT , n = 12 neurons ; Yme1l - / - , n = 13 neurons ) number of dendrites ( WT , n = 12 neurons ; Yme1l - / - , n = 13 neurons ) . neurons originate from two independent preparations . Unpaired t - , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 diagonal lines to moving mitochondria . Mitochondria were stained using Mitotracker . In total 39 axonal segments were analyzed for WT control neurons 37 axonal segments for Yme1l - / - neurons at DIV7 . Data are means of three independent experiments ± . Unpaired t - , 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 O O O O O O O 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 diagonal lines to moving mitochondria . Mitochondria were stained using Mitotracker . In total 120 axonal segments were analyzed for WT control neurons 107 axonal segments for Yme1l - / - neurons at DIV10 - 14 . Data are means of three independent experiments ± . Unpaired t - , 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 O O O O O O O O O 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 occupied by mitochondria in Yme1l - / - neurons relative to WT controls . In total 120 axonal segments were analyzed for WT control neurons 107 axonal segments for Yme1l - / - neurons at DIV10 - 14 . Data are means of three independent experiments ± . Unpaired t - , 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 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 ) NYKO ( n = 4 ) mice in presence of pyruvate - glutamate - malate ( C I ) pyruvate - glutamate - malate + succinate ( C I + C II ) as substrates . State 3 ( substrates + ADP ) , state 4 ( + oligomycin ) , uncoupled ( + FCCP ) . Unpaired t - , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , ns = not significant . Data are means ± .

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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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

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

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

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 ) NYKO mice ( 192 mitochondria ) . Kruskal - Wallis , * 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

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 of cerebellar sections of 28 - 32 week old WT NYKO mice . Scale bars 1 µm . D Frequency distribution of mitochondrial aspect ratios were calculated from WT ( n = 210 ) NYKO ( n = 230 ) mitochondria . Mann - Whitney , * * * * 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

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 of forebrain , cerebellar spinal cord lysates from 31 - 32 week old WT NYKO mice ( n = 3 ) using indicated antibodies . SDHA 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

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

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 , ns = not significant . Data were analyzed using one - way ANOVA .

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

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

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 of retina lysates of 6 - 7 week old WT , NYKO , NOKO 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

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 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

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 ) , NYOKO ( n = 3 ) mice . Data were analyzed using one - way ANOVA . * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 for Hrd1 with Ubc7 ( left ) Ubc6 ( right ) . Ubc7 reactions contained equimolar amounts of Cue1 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 . RNase - Ub2 band co - migrates with a nonspecific band ( # ) common to samples . Bottom : 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 ; * p < 0 . 05 . For clarity , only significances related to " no E3 " control of a given E2 are shown .

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

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 ) Ubc6 ( bottom - green ) with ethanolamine as nucleophile . Representative Coomassie gels are shown .

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

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

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of Ub nucleophile discharge assays for Hrd1 with U7BR / Ubc7 ( left ) Ubc6 ( right ) . Plots of E2 ~ Ub discharge ( dots ) as a function of time with first - order reaction models fitted to 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 - ; * p < 0 . 05 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) Doa10 ( right ) variants . E2 stimulation is shown as of rates derived from RING - catalyzed reactions " 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 ; * p < 0 . 05 . For clarity only significances related to " no E3 " control are shown . In addition , reactions for Hrd1 ( 400R ) Doa10 ( 94R ) are significantly faster than other reactions reaction for Doa10 ( 94H ) is significantly faster than that for Doa10 ( 94E ) . wild - type E3s are identified by black frames for each set .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 by Ubc7 with indicated Hrd1 ( left ) Doa10 ( right ) variants in presence of indicated Cue1 variants . Rates for reactions of mono - Ub to di - Ub 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 ; * p < 0 . 05 . For clarity only significances related to " no E3 " control of given reaction set are shown .

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 for Hrd1 variants with Ubc7 . Ubc7 reactions contained equimolar amounts of Cue1 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 ) . RNase - Ub2 band co - migrates with a nonspecific band ( # ) common to samples . Bottom : 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 ; * p < 0 . 05 . For clarity , only significances related to " no E3 " control are shown . In addition , reaction containing S - Hrd1 ( 400R ) shows significantly more ubiquitylation than those with other E3 variants .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 of rates derived from RING - catalyzed reactions " 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 ; * p < 0 . 05 . For clarity only significances related to " no E3 " control are shown . In addition , reaction for Doa10 ( 94R ) is significantly faster than that for Doa10 ( 94H ) ( 94E ) . wild - type E3 is identified by a black frame . scale of y - axis was set identical to that of plots from other discharge assays

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of rates derived from RING - catalyzed reactions " 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 ; * p < 0 . 05 . For clarity only significances related to " no E3 " control are shown . In addition , reaction for Doa10 ( 94R ) is significantly faster than that for Doa10 ( 94H ) ( 94E ) . wild - type E3 is identified by a black frame . scale of y - axis was set identical to that of plots from other discharge

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 for Doa10 variants with Ubc7 ( left ) Ubc6 ( right ) . Ubc7 reactions contained equimolar amounts of Cue1 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 ) . RNase - Ub2 band co - migrates with a nonspecific band ( # ) common to samples . Bottom : 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 ; * p < 0 . 05 . For clarity , only significances related to " no E3 " control of a given E2 are shown . In addition , reaction containing Ubc7 S - Hrd1 ( 400R ) shows significantly more ubiquitylation than those with Ubc7 other E3 variants .

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


A E2 ~ Ub hydrolysis assays for U7BR / Ubc7 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 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 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 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

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 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 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 ) Ub conjugated to Ubc6 ( green ) U7BR / Ubc7 ( yellow ) .

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 B-MEASURED_VAR I-MEASURED_VAR I-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 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).


degradation in indicated yeast strains monitored by - chase experiments for 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 ) Δhrd1 ) are same as in Fig . 3D 4A .

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.


degradation in indicated yeast strains monitored by CHX decay assays for 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 O O O O O 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 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


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

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degradation in indicated yeast strains monitored by CHX decay assays for 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-Sbh2


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


degradation in indicated yeast strains monitored by - chase experiments for 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 Δdoa10 ) are same as in Fig 3D 4A .

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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.


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

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


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

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


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


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

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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.


D . Binding affinities calculated from titration data of co - sedimentation assays of lifeAct - 14 ( 1 . 2µM ) lifeAct - 17 ( 2 . 2µM ) as indicated . Data points for each 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 of lifeAct binding experiments as indicated . remaining lifeAct 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 of lifeAct bound to phalloidin jasplakinolide F - actin ( mean 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 UTRN - V87D co - sedimentation with F - actin . Pellet ( top ) supernatant ( bottom ) fractions of co - sedimentation reactions of increasing utrophin concentrations , uncropped gel of 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 titration data of co - sedimentation assays of utrophin wildtype ( 1 . 8µM ) mutants ; H29A ( 2 . 8µM ) , I22D ( 38µM ) , V87D ( > 100µM ) N109A ( 55µM ) as indicated . Data points for each were averaged from 3 independent experiments , error bars represent S . D between independent 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 O O O O O O O O O O O O O O O O O O O O O

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

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 of 5 months . LAQ ( 5 mg / kg ) treatment at 3 months of for 2 months improved myelination in PLP - 150Q mice . Scale bars : 2μm ( Low magnification ) 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 plotted against axon diameter with linear regression , were shown beneath were significantly decreased in PLP - 150Q mice after LAQ treatment ( G = 0 . 6418 ± 0 . 0191 ) , compared with - matched vehicle group ( G = 0 . 7727 ± 0 . 0203 ) . One - way ANOVA with Tukey ' s . * 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 ( MBP , MOBP MOG ) in corpus callosum in PLP - 150Q mice , which were treated from 3 months of . Ratios of MBP , MOBP or MOG to vinculin obtained from 3 independent experiments were presented on right . One - way ANNOVA followed with Tukey ' s . 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 transcript expression of myelin associated genes ( MBP MOG ) in PLP - 150Q mouse corpus callosum at 5 months of after LAQ ( 5 or 25 mg / kg ) treatment for 2 months . Student ' s t - . MBP : * * * P = 0 . 0007 ; MOG : * * * P = 0 . 0009 . Data are mean ± ( 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).


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

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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 interaction between transgenic mutant HTT MYRF . fMYRF : full - MYRF ; nMYRF : N - terminal MYRF . of immunoprecipitated MYRF to input obtained from 3 independent experiments was shown on right . One - way ANNOVA followed with Tukey ' s . * * P = 0 . 009 . Data are mean ± .

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

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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 of N - terminal MYRF ( nMYRF ) . GST fusion 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 containing wild type N - terminal MYRF , S259A , or S261A were incubated with PLP - 150Q mouse tissue lysates ( right panel ) . beads were then centrifuged analyzed by western blotting with anti - GST ( lower panels ) anti - phosphor - serine ( upper panels ) . Note that LAQ treatment could eliminate mutant HTT ( PLP - 150Q ) - mediated MYRF phosphorylation 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 N - terminal HTT ( 1 - 212 aa ) containing 150Q in HEK293 . Five μM LAQ treatment decreased phosphorylation of immunoprecipitated MYRF amount of co - immunoprecipitated HTT . Λ - PPase served as positive control for dephosphorylation of MYRF . ratios of pho - serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under blots , which were obtained from 3 independent western blotting experiments . One - way ANNOVA with Tukey ' s . * * * P = 6 . 51x10 - 5 . Data are mean ± .

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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 immunoprecipitation of nMYRF with or without LAQ ( 5 μM ) treatment . Coomassie blue confirms 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 MBP promoter reporter in HEK293 was detected using a luciferase . Compared to wide type MYRF or S261A , S259A ( non - phosphorylated ) remained transcriptional activity that was not inhibited by N - terminal mutant HTT . ratios were obtained from 3 independent experiments . One - way ANNOVA with Tukey ' s . * * * P < 0 . 001 . Data are mean ± .

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

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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 of corpus callosum in PLP - 23Q or PLP - 150Q mice with anti - pS259 an antibody to total MYRF . Note that LAQ ( 5 mg / kg ) treatment for 2 months decreased phosphorylation in both full ( fMYRF ) 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 ) total MYRF to GAPDH . ratios were obtained from 3 independent western blotting experiments . Student ' s t - ; * * * P = 0 . 0007 . Data are mean ± .

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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 of corpus callosum ( CC , indicated between two dotted lines ) of PLP - 23Q PLP - 150Q mice showing decreased expression of Ser259 - MYRF by LAQ ( 5 mg / kg ) treatment for 2 months . at 10X ( upper ) 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 stained by anti - Ser259 MYRF in each filed was presented . Twenty random fields ( 40X ) of each section were examined to anti - Ser259 positive , n = 3 mice in each group . One - way ANNOVA with Tukey ' s . * * * P = 0 . 0003 ; * * P = 0 . 0039 . Data are mean ± .

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

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

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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 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.


networks of up - regulated gene were heat mapped typically , on clusters of " REGULATE _ OF _ PHOSPHORUS _ METABOLIC _ PROCESS " , " 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 by 5 mg / kg LAQ . Ratios of PRKG2 or phosphorylated VASP ( p - VASP ) to GAPDH obtained from 3 independent experiments were presented on right . One - way ANNOVA with Tukey ' s . PRKG2 : * * P = 0 . 0076 ( left ) * * * P = 0 . 0015 ( right ) ; p - VASP : * * * P = 3 . 86x10 - 5 ( left ) 1 . 01x10 - 5 ( right ) . Data are mean ± .

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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 after LAQ ( 5 mg / kg ) treatment for 2 months . Only PRKG2 was decreased by LAQ . n = 3 mice in each group . Student ' s t - ; * * P = 0 . 0073 ; * * * P = 0 . 00041 . Data are mean ± .

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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.


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

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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.


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

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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.


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

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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.


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

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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.