C . Y2H interaction experiment with human AAR2 ( FL ) as bait PRP8 ( aa1755 - 2335 ) as prey . Growth on selective media is abolished when AAR2 carries a S284E phospho - mimicry acid substitution . Mutation of second candidate phospho - site , AAR2 ( T356 ) , does not influence AAR2 - PRP8 interaction .

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

C. Y2H interaction experiment with human AAR2 (FL) as bait and PRP8 (aa1755-2335) as prey. Growth on selective media is abolished when AAR2 carries a S284E phospho-mimicry amino acid substitution. Mutation of second candidate phospho-site, AAR2(T356), does not influence the AAR2-PRP8 protein interaction.


D . Interaction of AAR2 PRP8 in human . HA ‑ tagged PRP8 ( aa1755 - 2335 ) was co - expressed in HEK293 with either wildtype AAR2 , AAR2 ( S284A ) or AAR2 ( S284E ) mutant versions . Immunoprecipitation of PRP8 led to a co - precipitation of wild typeAAR2 phospho - null AAR2 ( S284A ) . In contrast , phospho - mimicry AAR2 ( S284E ) version did not bind PRP8 .

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

D. Interaction of AAR2 and PRP8 in human cells. HA‑tagged PRP8 (aa1755-2335) was co-expressed in HEK293 cells with either wildtype AAR2, AAR2(S284A) or AAR2(S284E) mutant versions. Immunoprecipitation of PRP8 led to a co-precipitation of wild typeAAR2 and phospho-null AAR2(S284A). In contrast, the phospho-mimicry AAR2(S284E) version did not bind PRP8.


E . Results of phospho - Y2H screen to identify kinases modulating interaction of AAR2 PRP8 . Growth on selective media ( SD5 ) , of strains carrying AAR2 bait PRP8 prey plasmids is reduced through co - expression of either SGK2 or CK2α1 ( CSNK2A1 ) . Growth reduction is visible in a 1 : 10 dilution series on selective agar ( SD5 ) in comparison to control media ( SD3 ) . Ctrl : non - related kinases .

O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O 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. Results of the phospho-Y2H screen to identify kinases modulating the interaction of AAR2 and PRP8. Growth on selective media (SD5), of strains carrying AAR2 bait and PRP8 prey plasmids is reduced through co-expression of either SGK2 or CK2α1(CSNK2A1). Growth reduction is visible in a 1:10 dilution series on selective agar (SD5) in comparison to the control media (SD3). Ctrl: non-related kinases.


F . Interaction of AAR2 PRP8 in human in presences of SGK CK2α1 kinases . Experiment as in D . Co - expression of wild type SGK2 CK2α1 reduces amount of AAR2 co - immunoprecipitated with RPR8 .

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

F. Interaction of AAR2 and PRP8 in human cells in the presences of SGK and CK2α1 kinases. Experiment as in D. Co-expression of wild type SGK2 and CK2α1 reduces the amount of AAR2 co-immunoprecipitated with RPR8.


B . Results of phospho - Y2H screen to identify kinases modulating interaction of ERα with 14 ‑ 3 ‑ 3β . interaction of ERα ( prey ) with 14 ‑ 3 ‑ 3β ( bait ) is promoted by co - expression of wildtype kinases of PAK5 , CDK17 , BUB1 NEK6 , visible by yeast growth on selective media in comparison to control ( three biological replicas are shown ) . coexpression of kinase ‑ dead mutants of PAK5 CDK17 also promoted interaction of ERα 14 ‑ 3 ‑ 3β . In contrast kinase ‑ dead mutant versions of BUB1 ( K821M ) NEK6 ( K74M & K75M ) did not promote Y2H interaction .

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

B. Results of the phospho-Y2H screen to identify kinases modulating the interaction of the ERα with 14‑3‑3β. The interaction of ERα (prey) with 14‑3‑3β (bait) is promoted by the co-expression of wildtype kinases of PAK5, CDK17, BUB1 and NEK6, visible by the yeast growth on selective media in comparison to the control (three biological replicas are shown). The coexpression of the kinase‑dead mutants of PAK5 and CDK17 also promoted the interaction of ERα and 14‑3‑3β. In contrast the kinase‑dead mutant versions of BUB1(K821M) and NEK6(K74M&K75M) did not promote the Y2H interaction.


C . Phosphorylation of ERα in mammalian . ERα was co - expressed with indicated wildtype kinases or kinase ‑ dead versions in HEK293 immunoprecipitated . Phosphorylation status of ERα was analyzed with an in - gel phosphostain ( Pro - Q ® Diamond phosphoprotein gel stain , Invitrogen ) , relative to whole stain ( SYPRO ® Ruby gel stain , Invitrogen ) .

O O O O B-MEASURED_VAR 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

C. Phosphorylation of ERα in mammalian cells. ERα was co-expressed with the indicated wildtype kinases or kinase‑dead versions in HEK293 cells and immunoprecipitated. Phosphorylation status of ERα was analyzed with an in-gel phosphostain (Pro-Q® Diamond phosphoprotein gel stain, Invitrogen), relative to the whole protein stain (SYPRO®Ruby protein gel stain, Invitrogen).


D . Interaction of ERα 14 ‑ 3 ‑ 3β in a GST pull - down . GST ‑ tagged 14 ‑ 3 ‑ 3β was expressed in E . coli immobilized on beads . PA ‑ tagged ERα was co - expressed with either wildtype PAK5 or NEK6 , kinase ‑ dead ( KD ) version or without a kinase in HEK293 . Immobilized 14 ‑ 3 ‑ 3β was used to pull PA ‑ tagged ERα from HEK lysates . Co - precipitation of ERα was successful in presence of wild kinases , but not in their absence or with kinase ‑ dead mutant versions .

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

D. Interaction of ERα and 14‑3‑3β in a GST pull-down assay. GST‑tagged 14‑3‑3β was expressed in E. coli and immobilized on beads. PA‑tagged ERα was co-expressed with either wildtype PAK5 or NEK6, the kinase‑dead (KD) version or without a kinase in HEK293 cells. Immobilized 14‑3‑3β was used to pull PA‑tagged ERα from the HEK lysates. Co-precipitation of ERα was successful in the presence of wild kinases, but not in their absence or with kinase‑dead mutant versions.


E . Effect of NEK6 co - expression on ERα gene activation . Relative luciferase activity of ERα in presence absence of 1 nM E2 ( 17β - estradiol ) was measured . A dose ‑ dependent reduction ( ng plasmid transfected ) in E2 - Erα ‑ dependent luciferase expression was observed in HEK293T , MCF - 7 US - OS cancer lines . This effect was not observed with NEK6 ( K74M & K75M ) kinase ‑ dead version . Graphs represent ≥ 5 biological replicates , each derived from averaging 6 technical replicates . Error bars indicate SD across biological replicates .

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

E. Effect of NEK6 co-expression on ERα gene activation. Relative luciferase activity of ERα in the presence and absence of 1 nM E2 (17β-estradiol) was measured. A dose‑dependent reduction (ng plasmid transfected) in E2-Erα‑dependent luciferase expression was observed in HEK293T, MCF-7 and US-OS cancer cell lines. This effect was not observed with the NEK6(K74M&K75M) kinase‑dead version. Graphs represent ≥ 5 biological replicates, each derived from averaging 6 technical replicates. Error bars indicate SD across the biological replicates.


( A ) Unlabeled or Cy5 - labeled dsRNA corresponding to first 200 nucleotides of either Renilla luciferase ( dsRNA - RL ) or GFP ( dsRNA - GFP ) coding sequences visualised on gel stained with ethidium bromide ( EtBr - stained , top ) or by in - gel ( bottom ) .

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

(A) Unlabeled or Cy5-labeled dsRNA corresponding to the first 200 nucleotides of either the Renilla luciferase (dsRNA-RL) or the GFP (dsRNA-GFP) coding sequences visualised on gel stained with ethidium bromide (EtBr-stained, top) or by in-gel fluorescence (bottom).


( B ) Gating strategy used in flow cytometry - . First , were selected ( side scatter ( SSC ) vs . forward scatter ( FSC ) ) gated for singlets ( SSC - Width ( W ) vs . FSC ) to hence exclude doublets or . single gate was then further analysed for DAPI , gating on live ( DAPI negative ) . DsRNA - transfected ( Cy5 - positive ) were then gated level of GFP expression analysed within that population . Flow cytometry counting beads ( gated on SSC vs . FSC plot ) were added to quantify number in each sample .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Gating strategy used in the flow cytometry-based assay. First, cells were selected (side scatter (SSC) vs. forward scatter (FSC)) and gated for singlets (SSC-Width (W) vs. FSC) to hence exclude doublets or cell aggregates. The single cell gate was then further analysed for DAPI, gating on the live cells (DAPI negative). DsRNA-transfected cells (Cy5-positive) were then gated and the level of GFP expression analysed within that population. Flow cytometry counting beads (gated on SSC vs. FSC plot) were added to quantify cell number in each sample.


( C ) mESCs stably expressing GFP ( mESCs - GFP ) were mock transfected or transfected with indicated Cy5 - labeled dsRNA level of GFP in Cy5 + was analysed 3 days later is represented as histograms ( left ) . Right panel , bar graphs displaying of GFP mean relative to mock control ( D ) mESCs stably expressing d2GFP ( mESCs - d2GFP ) were mock transfected or transfected with indicated Cy5 - labeled dsRNA . level of d2GFP was analysed at indicated timepoints post - transfection displayed as in ( C ) .

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

(C) mESCs stably expressing GFP (mESCs-GFP) were mock transfected or transfected with the indicated Cy5-labeled dsRNA and the level of GFP in Cy5+cells was analysed 3 days later and is represented as histograms (left). Right panel, bar graphs displaying the percentage of GFP mean fluorescence intensity relative to mock control (D) mESCs stably expressing d2GFP (mESCs-d2GFP) were mock transfected or transfected with the indicated Cy5-labeled dsRNA. The level of d2GFP was analysed at the indicated timepoints post-transfection and displayed as in (C).


( E ) L929 stably expressing d2GFP were mock transfected or transfected with indicated Cy5 - labeled dsRNA d2GFP level was measured as in ( D ) .

O O O O 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) L929 cells stably expressing d2GFP were mock transfected or transfected with the indicated Cy5-labeled dsRNA and d2GFP level was measured as in (D).


( A ) Mavs + / - Mavs - / - MEFs stably expressing GFP were transfected or not ( mock ) with indicated Cy5 - labeled dsRNAs ( top panels ) or siRNAs ( bottom panels ) . GFP level was measured in Cy5 + by flow cytometry 72 hours ( hrs ) post - transfection .

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

(A) Mavs+/- and Mavs-/- MEFs stably expressing GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs (top panels) or siRNAs (bottom panels). GFP level was measured in Cy5+ cells by flow cytometry 72 hours (hrs) post-transfection.


( B ) Mavs + / - Mavs - / - MEFs stably expressing d2GFP were transfected or not ( mock ) with indicated Cy5 - labeled dsRNAs . level of d2GFP in Cy5 + was measured at various timepoints post - transfection as indicated . Histograms plots show d2GFP level 48 hrs post - transfection . Bar graphs show 3 timepoints as indicated .

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

(B) Mavs+/- and Mavs-/- MEFs stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs. The level of d2GFP in Cy5+ cells was measured at various timepoints post-transfection as indicated. Histograms plots show d2GFP level 48 hrs post-transfection. Bar graphs show 3 timepoints as indicated.


( C ) WT , Ifnar1 + / - or Ifnar1 - / - MEFs stably expressing d2GFP were transfected or not ( mock ) with indicated Cy5 - labeled dsRNAs d2GFP level in Cy5 + was monitored by flow cytometry at 48 hrs post - transfection .

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

(C) WT, Ifnar1+/- or Ifnar1-/- MEFs stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry at 48 hrs post-transfection.


( A ) Mavs + / - Mavs - / - MEFs stably expressing GFP were incubated with either a neutralising antibody against IFNAR ( α - IFNAR Ab ) , an isotype control antibody ( IgG1 ) or no antibody ( no Ab ) . were then were transfected or not ( mock ) with Cy5 - labeled dsRNA as indicated GFP level monitored in Cy5 + 72 hrs post - transfection .

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

(A) Mavs+/- and Mavs-/-MEFs stably expressing GFP were incubated with either a neutralising antibody against IFNAR (α-IFNAR Ab), an isotype control antibody (IgG1) or no antibody (no Ab). Cells were then were transfected or not (mock) with Cy5-labeled dsRNA as indicated and GFP level monitored in Cy5+cells 72 hrs post-transfection.


( B ) Mavs - / - MEFs stably expressing d2GFP were treated with recombinant IFN ( 200 U / ml ) for 24 hrs prior to transfection or not ( mock ) with Cy5 - labeled dsRNA , as indicated . IFN treatment was maintained for 2 days post - transfection then GFP level monitored in Cy5 + .

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

(B) Mavs-/-MEFs stably expressing d2GFP were treated with recombinant IFN (200 U/ml) for 24 hrs prior to transfection or not (mock) with Cy5-labeled dsRNA, as indicated. IFN treatment was maintained for 2 days post-transfection and then GFP level monitored in Cy5+cells.


( A ) Northern blot of Ifnar1 - / - MEFs at 24 h . p . t with indicated dsRNA or transfection reagent alone ( mock ) using a probe specific for dsRNA - GFP ( top ) or dsRNA - RL ( bottom ) . Reactions from a dicing using indicated dsRNA incubated in vitro with immunoprecipitated FLAG - human Dicer were loaded in parallel . Two lanes ( delimited by dashed lines ) were left empty between Ifnar1 - / - MEFs transfected samples in vitro dicing reactions . A miRNA marker containing 3 synthetic ssRNA oligonucleotides of 17 , 21 25 - nt in was run in parallel . Endogenous U6 was used as a loading control . Signals corresponding to dsRNA - derived siRNAs are indicated with an arrow . was first probed for dsRNA - GFP ( top ) , then probed for miRNA marker ( top left ) subsequently stripped reprobed for dsRNA - RL ( middle ) finally stripped reprobed for both U6 ( bottom ) for miRNA marker again ( middle left ) .

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

(A) Northern blot analysis of Ifnar1-/- MEFs at 24 h.p.t with the indicated dsRNA or transfection reagent alone (mock) using a probe specific for dsRNA-GFP (top) or dsRNA-RL (bottom). Reactions from a dicing assay using the indicated dsRNA incubated in vitro with immunoprecipitated FLAG-human Dicer were loaded in parallel. Two lanes (delimited by dashed lines) were left empty between the Ifnar1-/- MEFs transfected samples and the in vitro dicing assay reactions. A miRNA marker containing 3 synthetic ssRNA oligonucleotides of 17, 21 and 25-nt in length was run in parallel. Endogenous U6 was used as a loading control. Signals corresponding to the dsRNA-derived siRNAs are indicated with an arrow. The membrane was first probed for dsRNA-GFP (top), then probed for the miRNA marker (top left) and subsequently stripped and reprobed for dsRNA-RL (middle) and finally stripped and reprobed for both U6 (bottom) and for the miRNA marker again (middle left).


( B ) Western blot of Dicer in d2GFP - expressing Mavs - / - MEFs either not transduced ( NT ) or transduced to express a non - targeting shRNA control ( CTL shRNA ) or two different Dicer - specific targeting shRNAs ( DICER shRNA 1 2 ) . P97 served as loading control .

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

(B) Western blot of Dicer in d2GFP-expressing Mavs-/- MEFs either not transduced (NT) or transduced to express a non-targeting shRNA control (CTL shRNA) or two different Dicer-specific targeting shRNAs (DICER shRNA 1 and 2). P97 served as loading control.


( C ) described in ( B ) were transfected or not ( mock ) with indicated Cy5 - labeled dsRNAs d2GFP level in Cy5 + was monitored by flow cytometry 48 hrs later . Please note that biphasic pattern of d2GFP level observed in dsRNA - GFP transfected is caused by a lower transfection efficiency of dsRNA in these giving rise to significant amounts of lowly transfected that do not display a decrease level of

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

(C) Cells described in (B) were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later. Please note that the biphasic pattern of d2GFP level observed in dsRNA-GFP transfected cells is caused by a lower transfection efficiency of dsRNA in these cells giving rise to significant amounts of lowly transfected cells that do not display a decrease level of d2GFP


( D ) Western blots of Ago2 in parental Mavs - / - MEFs stably expressing d2GFP three Mavs - / - Ago2 - / - clones generated by CRISPR / CAS9 - mediated genome engineering . P97 served as loading control .

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

(D) Western blots of Ago2 in parental Mavs-/- MEFs stably expressing d2GFP and three Mavs-/- Ago2-/- clones generated by CRISPR/CAS9-mediated genome engineering. P97 served as loading control.


( E ) Mavs - / - ( parental ) Mavs - / - Ago2 - / - MEFs ( clone 5 . 1 ) stably expressing d2GFP were transfected or not ( mock ) with indicated Cy5 - labeled dsRNAs d2GFP level in Cy5 + was monitored by flow cytometry 48 hrs later .

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

(E) Mavs-/- (parental line) and Mavs-/- Ago2-/- MEFs (clone 5.1) stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later.


( A ) Western blots for Ago2 ( top ) HA tag ( bottom ) of parental Mavs - / - MEFs two Mavs - / - Ago2 - / - clones ( clone 5 . 2 5 . 3 ; see Fig 4A ) stably expressing d2GFP transduced with an empty vector or a vector encoding HA - tagged wild - type ( HA - mAgo2 WT ) or catalytic mutant ( HA - mAgo2 D597A ) versions of mouse Ago2 . β - actin served as loading control . was first analysed for Ago2 β - actin , stripped then analysed for HA tag .

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

(A) Western blots for Ago2 (top) and HA tag (bottom) of parental Mavs-/- MEFs and two Mavs-/- Ago2-/- clones (clone 5.2 and 5.3; see Fig 4A) stably expressing d2GFP and transduced with an empty vector or a vector encoding HA-tagged wild-type (HA-mAgo2 WT) or catalytic mutant (HA-mAgo2 D597A) versions of mouse Ago2. β-actin served as loading control. The membrane was first analysed for Ago2 and β-actin, stripped and then analysed for HA tag.


( B ) Parental non - transduced ( NT ) Mavs - / - MEFs complemented Mavs - / - Ago2 - / - MEFs as described in ( A ) were transfected or not ( mock ) with indicated Cy5 - labeled dsRNAs d2GFP level in Cy5 + was monitored by flow cytometry 48 hrs later .

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

(B) Parental non-transduced (NT) Mavs-/- MEFs and complemented Mavs-/- Ago2-/- MEFs as described in (A) were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later.


( A ) Ifnar1 - / - MEFs were transfected with indicated Cy5 - labeled dsRNA next day were infected with SFV - Rluc at indicated multiplicities of infection ( MOI ) . Renilla luciferase activity was measured 24 hours later . Each bar represents mean + SD of biological duplicates numbers above each pair of bars depict fold - reduction in viral activity achieved with dsRNA - RL vs . dsRNA - GFP treatment .

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

(A) Ifnar1-/- MEFs were transfected with the indicated Cy5-labeled dsRNA and the next day were infected with SFV-Rluc at the indicated multiplicities of infection (MOI). Renilla luciferase activity was measured 24 hours later. Each bar represents mean + SD of biological duplicates and numbers above each pair of bars depict the fold-reduction in viral activity achieved with dsRNA-RL vs. dsRNA-GFP treatment.


( B ) As in ( a ) but SFV - Rluc titre was measured by plaque expressed as plaque forming units ( PFU ) per ml of supernatant at 24 h post - infection ( MOI = 0 . 1 ) .

O O O O O O O O O O O O O O O O O O O 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) As in (a) but SFV-Rluc virus titre was measured by plaque assay and expressed as plaque forming units (PFU) per ml of supernatant at 24 h post-infection (MOI=0.1).


( C ) As in ( a ) but with Ifnar1 - / - or Mavs - / - MEFs a single dose of SFV - Rluc ( MOI = 0 . 1 ) .

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

(C) As in (a) but with Ifnar1-/- or Mavs-/- MEFs and a single dose of SFV-Rluc (MOI=0.1).


( D ) As in ( a ) but with two Mavs - / - Ago2 - / - clones ( clone 5 . 2 5 . 3 ) individually complemented to express wild - type ( HA - mAgo2 WT ) or a catalytic mutant ( HA - mAgo2 D597A ) version of mAgo2 or transduced with a control vector ( as described in Fig 5 ) . SFV - Rluc was used at an MOI = 0 . 1 .

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

(D) As in (a) but with two Mavs-/- Ago2-/- clones (clone 5.2 and 5.3) individually complemented to express wild-type (HA-mAgo2 WT) or a catalytic mutant (HA-mAgo2 D597A) version of mAgo2 or transduced with a control vector (as described in Fig 5). SFV-Rluc was used at an MOI=0.1.


( C ) Relative mRNA expression from real - time PCR of GnRH transcript in GFP - positive , in comparison with GFP - negative . GnRH transcript appears to be selectively expressed in GFP - positive . Mann - Whitney U , n = 4 mice from 2 litters .

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 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

(C) Relative mRNA expression from real-time PCR analysis of the GnRH transcript in GFP-positive cells, in comparison with GFP-negative cells. The GnRH transcript appears to be selectively expressed in GFP-positive cells. Mann-Whitney U test, n=4 mice from 2 litters.


( D ) Relative mRNA expression from real - time PCR of Neuropilin - 1 ( Nrp1 ) , Nrp2 , Plexin A1 ( PlxnA1 ) , PlxnA4 transcripts in FACS - isolated GFP - positive GnRH neurons from control ( Nrp1loxP / loxP ; grey ) mutant ( Gnrh : : cre ; Nrp1loxP / loxP ; red ) P0 mice . Unpaired t - , n = 5 to 6 mice per group from 3 litters .

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

(D) Relative mRNA expression from real-time PCR analysis of Neuropilin-1 (Nrp1), Nrp2, Plexin A1 (PlxnA1), and PlxnA4 transcripts in FACS-isolated GFP-positive GnRH neurons from control (Nrp1loxP/loxP; grey) and mutant (Gnrh::cre; Nrp1loxP/loxP; red) P0 mice. Unpaired t-test, n=5 to 6 mice per group from 3 litters.


( E ) Representative immunofluorescence showing GnRH neurons migrating along Nrp1 - immunoreactive vomeronasal / terminal nerve ( empty arrowheads ) at nose - brain junction in sagittal slices from E14 . 5 control mutant . GnRH neurons ( green ) themselves express Nrp1 ( red , arrows ) in control Nrp1loxP / loxP mice whereas Nrp1 is undetectable in GnRH neurons from mutant Gnrh : : cre ; Nrp1loxP / loxP ( black arrows ) . Scale bar : 50μm .

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

(E) Representative immunofluorescence images showing GnRH neurons migrating along Nrp1-immunoreactive vomeronasal/terminal nerve fibers (empty arrowheads) at the nose-brain junction in sagittal slices from E14.5 control and mutant embryos. GnRH neurons (green) themselves express Nrp1 (red, white arrows) in control Nrp1loxP/loxP mice whereas Nrp1 is undetectable in GnRH neurons from mutant Gnrh::cre; Nrp1loxP/loxP littermates (black arrows). Scale bar: 50μm.


( A ) of total number of GnRH neurons during embryogenesis , at E12 . 5 , E14 . 5 E18 . 5 . More GnRH neurons were found in E14 . 5 E18 . 5 mutant . Two - way ANOVA , Fisher ' s LSD multiple - comparison , n = 4 to 6 mouse from at least 3 litters .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of total number of GnRH neurons during embryogenesis, at E12.5, E14.5 and E18.5. More GnRH neurons were found in E14.5 and E18.5 mutant embryos. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=4 to 6 mouse embryos from at least 3 litters.


( B ) Schematic representation of areas containing GnRH migrating during embryogenesis at E14 . 5 representative immunofluorescence showing migrating GnRH neurons at level of nose , olfactory bulb ( OB ) ventral forebrain ( VFB ) in control mutant . Scale bar : 50μm . VNO , vomeronasal organ ; OE , olfactory epithelium ; OB , olfactory bulbs ; VFB , ventral forebrain .

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

(B) Schematic representation of the areas containing GnRH migrating cells during embryogenesis at E14.5 and representative immunofluorescence showing migrating GnRH neurons at the level of the nose, the olfactory bulb (OB) and the ventral forebrain (VFB) in control and mutant embryos. Scale bar: 50μm. VNO, vomeronasal organ; OE, olfactory epithelium; OB, olfactory bulbs; VFB, ventral forebrain.


( C ) Quantitative showing distribution of GnRH neurons in nose , OB VFB of mutant control . Two - way ANOVA , Fisher ' s LSD multiple - comparison , n = 5 6 mouse for mutants controls , respectively , from at least 3 litters .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitative analysis showing distribution of GnRH neurons in the nose, OB and VFB of mutant and control embryos. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=5 and 6 mouse embryos for mutants and controls, respectively, from at least 3 litters.


( D ) Representative image of cleared brains immunolabeling for GnRH at level of olfactory bulbs , in a control ( left panel ) a mutant ( right panel ) postnatal brain . Knock - out show an accumulation of GnRH neurons in accessory olfactory bulb ( circle formed by GnRH labelling , right panel ) . AOB , accessory olfactory bulb . Scale bar : 300μm .

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

(D) Representative image of cleared brains and immunolabeling for GnRH at the level of the olfactory bulbs, in a control (left panel) and a mutant (right panel) postnatal brain. Knock-out animals show an accumulation of GnRH neurons in the accessory olfactory bulb (circle formed by the GnRH labelling, right panel). AOB, accessory olfactory bulb. Scale bar: 300μm.


( E ) Representative image of cleared brains immunolabeling for GnRH at level of organum vasculosum of lamina terminalis ( OVLT ) in median preoptic , from a control ( left ) a mutant ( right ) postnatal brain . Scale bar : 500μm . Oc , optic chiasma .

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

(E) Representative image of cleared brains and immunolabeling for GnRH at the level of the organum vasculosum of the lamina terminalis (OVLT) in the median preoptic area, from a control (left) and a mutant (right) female postnatal brain. Scale bar: 500μm. Oc, optic chiasma.


( F ) Quantitative of distribution of GnRH neurons in cleared brains of mutant control . OB , olfactory bulb ; DBB , diagonal band of Broca ; 3V , periventricular region of preoptic ; Tot , total . Two - way ANOVA , Fisher ' s LSD multiple - comparison , n = 3 adult mice from 3 litters .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitative analysis of the distribution of GnRH neurons in cleared brains of mutant and control littermates. OB, olfactory bulb; DBB, diagonal band of Broca; 3V, periventricular region of the preoptic area; Tot, total. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=3 adult mice from 3 litters.


( A ) Schematic representation of transwell used to assess effect of Sema3A , which was placed in lower chamber , on GN11 migration representative microphotograph of nuclei stained with Hoechst ( grey ) in lower part of transwell after overnight incubation in SFM ( left panel ) , SFM with 100ng / ml Sema3A ( middle panel ) SFM with 100ng / ml Sema3A + 1μg Nrp1Ab ( right panel ) . Scale bar : 100μm .

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

(A) Schematic representation of the transwell assay used to assess the effect of Sema3A, which was placed in the lower chamber, on GN11 cell migration and representative microphotograph of cell nuclei stained with Hoechst (grey) in the lower part of the transwell membrane after overnight incubation in SFM (left panel), SFM with 100ng/ml Sema3A (middle panel) and SFM with 100ng/ml Sema3A + 1μg Nrp1Ab (right panel). Scale bar: 100μm.


( B ) Quantitative analyses of mean number of GN11 that migrated from upper chamber ( SFM ) through perforated in presence or absence of indicated doses of Sema3A Nrp1Ab in lower chamber . Kruskal - Wallis , n = 5 to 6 wells in two 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

(B) Quantitative analyses of the mean number of GN11 cells that migrated from the upper chamber (SFM) through the perforated membrane in the presence or absence of the indicated doses of Sema3A and Nrp1Ab in the lower chamber. Kruskal-Wallis test, n= 5 to 6 wells in two independent experiments.


( C ) Schematic illustration representative photomicrograph of an organotypic culture of a nasal explant from E11 . 5 mouse after 8 days in vitro . NE , nasal epithelium ; NMC , nasal midline cartilage . Scale bar 500μm ( 150μm in inset ) .

O O O O O O O O O O O O O O O O O O O O O O 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) Schematic illustration and representative photomicrograph of an organotypic culture of a nasal explant from E11.5 mouse embryos after 8 days in vitro. NE, nasal epithelium; NMC, nasal midline cartilage. Scale bar 500μm (150μm in inset).


( D ) Total number of GnRH neurons that migrated out of nasal explants isolated from control mutant , quantified after 8 days in culture . Mann - Whitney U , n = 3 to 5 from at least 3 litters .

O O O O O O O O O O O O O O O O O O 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) Total number of GnRH neurons that migrated out of nasal explants isolated from control and mutant embryos, quantified after 8 days in culture. Mann-Whitney U test, n=3 to 5 embryos from at least 3 litters.


( E ) travelled by GnRH neurons from olfactory epithelia ; were quantified within a radius of 1 to 6 mm . Two - way ANOVA , Fisher ' s LSD multiple - comparison , n = 3 to 5 explants .

(E) Distance travelled by GnRH neurons from the olfactory epithelia; Cells were quantified within a radius of 1 to 6 mm. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=3 to 5 explants.


( F ) Representative immunofluorescence for GnRH - ( red ) BrdU - ( green ) positive in control mutant E14 . 5 arising from progenitors born between E9 . 5 E10 . 5 . BrdU - positive GnRH neurons ( arrowheads ) were more abundant in mutant than in control . Scale bar : 20μm .

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

(F) Representative immunofluorescence for GnRH- (red) and BrdU- (green) positive cells in control and mutant E14.5 embryos arising from progenitors born between E9.5 and E10.5. BrdU-positive GnRH neurons (white arrowheads) were more abundant in mutant than in control embryos. Scale bar: 20μm.


( G ) Number of BrdU - positive GnRH neurons in nose , olfactory bulb ( OB ) ventral forebrain ( VFB ) at E14 . 5 total number of in control mutant . Double - labeled were more numerous in mutant VFB . Two - way ANOVA , Fisher ' s LSD multiple - comparison , n = 4 from 2 litters .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Number of BrdU-positive GnRH neurons in the nose, olfactory bulb (OB) and ventral forebrain (VFB) at E14.5 and total number of cells in control and mutant embryos. Double-labeled cells were more numerous in the mutant VFB. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=4 embryos from 2 litters.


( I ) Number of GnRH neurons in E14 . 5 injected with neutralizing antibodies to Neuropilin - 1 ( Nrp1Ab ) compared to an isotype goat antibody , or antiapoptotic compound ZVAD compared to its vehicle , together with fold change in GnRH neuron number as well as mutant control at same embryonic . Note that data are represented as fold change , as raw values are not comparable with transgenic that did not undergo surgery . Unpaired t - , n = 5 to 7 from at least 2 litters .

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

(I) Number of GnRH neurons in E14.5 embryos injected with neutralizing antibodies to Neuropilin-1 (Nrp1Ab) compared to an isotype goat antibody, or the antiapoptotic compound ZVAD compared to its vehicle, together with fold change in GnRH neuron number as well as mutant and control embryos at the same embryonic age. Note that the data are represented as fold change, as raw values are not comparable with transgenic animals that did not undergo surgery. Unpaired t-test, n=5 to 7 embryos from at least 2 litters.


( A ) Olfactory sex preference in P20 ( left panel ) adult ( right panel ) control mutant mice . Half of mutant at P20 showed a preference for , similar to adults of both genotypes , whereas other half showed an even more pronounced preference for than their control at P20 . One - way ANOVA , Fisher ' s LSD multiple - comparison for P20 mice unpaired t - for adult mice ; n = 4 to 6 from at least 3 litters .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Olfactory sex preference test in P20 (left panel) and adult (right panel) control and mutant female mice. Half of the mutant females at P20 showed a preference for males, similar to adults of both genotypes, whereas the other half showed an even more pronounced preference for females than their control littermates at P20. One-way ANOVA, Fisher's LSD multiple-comparison test for P20 mice and unpaired t-test for adult mice; n=4 to 6 from at least 3 litters.


( B ) Time spent sniffing after introduction of both odors into cage of control mutant . One - way ANOVA , Fisher ' s LSD multiple - comparison for P20 mice unpaired t - for adult mice ; n = 4 to 6 from at least 3 litters .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Time spent sniffing after the introduction of both male and female odors into the cage of control and mutant littermates. One-way ANOVA, Fisher's LSD multiple-comparison test for P20 mice and unpaired t-test for adult mice; n=4 to 6 from at least 3 litters.


( C ) Representative traces of spontaneous firing in GnRH neurons from control ( left , black ) mutant ( right , red ) at week 2 ( upper panels ) week 4 ( lower panels ) ; lower traces show an enlargement of upper trace .

O O O O O O O O O O O O O O O O O O O O O O O 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) Representative traces of spontaneous firing in GnRH neurons from control (left, black) and mutant (right, red) animals at week 2 (upper panels) and week 4 (lower panels); lower traces show an enlargement of the upper trace.


( D ) Average spontaneous firing frequency in GnRH neurons from control mutant mice at 2 , 3 4 . used for this are represented by blue grey dots , respectively . Two - way ANOVA , Fisher ' s LSD multiple - comparison , n = 8 to 14 neurons from at least 3 from different litters . Note that neurons from ( n = 3 to 8 ) mice ( n = 5 to 8 ) used for this are represented by blue grey dots , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Average spontaneous firing frequency in GnRH neurons from control and mutant mice at weeks 2, 3 and 4. Individual males and females used for this analysis are represented by blue and grey dots, respectively. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=8 to 14 neurons from at least 3 animals from different litters. Note that individual neurons from male (n=3 to 8) and female mice (n=5 to 8) used for this analysis are represented by blue and grey dots, respectively.


( E ) of evoked firing in GnRH neurons from control mutant mice in response to current steps . Two - way repeated - measures ANOVA , n = 10 to 11 neurons from at least 3 mice from different litters .

(E) Quantification of evoked firing in GnRH neurons from control and mutant mice in response to current steps. Two-way repeated-measures ANOVA, n=10 to 11 neurons from at least 3 mice from different litters.


( A ) Evolution of in control mutant from 6 to 100 days of . Mutant show a significantly higher from P21 . grey lines mark at puberty onset . Two - way ANOVA , Fisher ' s LSD multiple - comparison , n = 7 to 17 mice from at least 4 litters .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Evolution of body weight in control and mutant female littermates from 6 to 100 days of age. Mutant females show a significantly higher body weight from P21. The grey lines mark body weight at puberty onset. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=7 to 17 mice from at least 4 litters.


( B ) Fat mass or volume of adipose tissue ( WAT ) . Adult mutant showed higher WAT volumes . Unpaired t - , n = 8 mice .

O O O O O O 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) Fat mass or volume of white adipose tissue (WAT). Adult mutant females showed higher WAT volumes. Unpaired t-test, n=8 mice.


( C ) Volume of lean tissue . Mann - Whitney U , n = 8 mice .

(C) Volume of lean tissue. Mann-Whitney U test, n=8 mice.


( D ) Cumulative of at vaginal opening , n = 13 mice .

(D) Cumulative percentage of the age at vaginal opening, n=13 mice.


( E ) Cumulative of undergoing first ovulation as assessed by occurrence of first estrus at different ages n = 13 mice .

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

(E) Cumulative percentage of animals undergoing first ovulation as assessed by the occurrence of first estrus at different ages n=13 mice.


( F ) Bar graph showing at P45 , which is mean at puberty in mutants . Unpaired t - , n = 9 to 14 mice .

O O O O 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) Bar graph showing body weight at P45, which is the mean age at puberty in mutants. Unpaired t-test, n=9 to 14 mice.


( G ) Plasma LH at P45 in control mutant mice . Mann - Whitney U , n = 5 to 6 mice .

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

(G) Plasma LH levels at P45 in control and mutant mice. Mann-Whitney U test, n=5 to 6 mice.


( H ) Mean uterine normalized to ( mg / g BW ) . Unpaired t - , n = 6 to 10 mice .

O 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) Mean uterine weight normalized to body weight (mg/g BW). Unpaired t-test, n=6 to 10 mice.


PMO was administered intravenously into adult mdx mice at 25 or 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration . ( B ) Immunohistochemistry for dystrophin expression in - wide from mdx mice treated with PMO in saline ( PMO - S ) , PMO - G ( IV ) or PMO - G ( D ) ( scale bar = 100μm ) . PMO - G ( IV ) refers to intravenous injection of PMO in combination with intravenous injection of 5 % glycine every other day for 3 . PMO - G ( D ) represents intravenous injection of PMO supplemented with 1 % glycine orally .

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

PMO was administered intravenously into adult mdx mice at 25 or 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration. (B) Immunohistochemistry for dystrophin expression in body-wide muscles from mdx mice treated with PMO in saline (PMO-S), PMO-G(IV) or PMO-G (D)(scale bar=100μm).PMO-G (IV) refers to intravenous injection of PMO in combination with intravenous injection of 5% glycine every other day for 3 weeks. PMO-G (D) represents intravenous injection of PMO supplemented with 1% glycine orally.


PMO was administered intravenously into adult mdx mice at 25 or 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration . ( C ) Western blot for dystrophin expression in - wide from mdx mice treated with PMO - G ( IV ) , PMO - G ( D ) or PMO - S . 0 . 5µg , 2 . 5 µg , 5µg , 10µg 15µg total from C57BL / 6 50 µg of muscle samples from untreated treated mdx mice were loaded . α - actinin was used as loading control . TA - tibialis anterior , Q - quadriceps , G - gastrocnemius , T - triceps , D - diaphragm A - abdominal muscle . ( D ) Quantitative of dystrophin expression in - wide from mdx mice treated with PMO - G ( i . v . , n = 4 ) , PMO - G ( D25 D50 , n = 3 ) PMO - S ( n = 3 ) ( * P < 0 . 05 , * * P < 0 . 001 ; One way - ANOVA post hoc Student - Newman - Keuls ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into adult mdx mice at 25 or 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration. (C) Western blot for dystrophin expression in body-wide muscles from mdx mice treated with PMO-G (IV), PMO-G (D) or PMO-S. 0.5µg, 2.5 µg, 5µg, 10µg and 15µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded. α-actinin was used as the loading control. TA-tibialis anterior, Q-quadriceps, G-gastrocnemius, T-triceps, D-diaphragm and A-abdominal muscle. (D) Quantitative analysis of dystrophin expression in body-wide muscles from mdx mice treated with PMO-G (i.v., n=4), PMO-G (D25 and D50, n=3) and PMO-S (n=3) (*P<0.05,**P<0.001; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( A ) Muscle function was assessed to determine physical improvement with grip strength ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (A) Muscle function was assessed to determine the physical improvement with grip strength test (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( B ) Measurement of bodyweight changes of DKO mice treated with PMO - G or PMO - S ( n = 3 ; * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (B) Measurement of bodyweight changes of DKO mice treated with PMO-G or PMO-S (n=3; *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( C ) Measurement of TA muscle mass of DKO mice treated with PMO - G or PMO - S ( n = 3 , * P < 0 . 05 ; one - way ANOVA post - hoc Student - Newman - Keuls ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (C) Measurement of TA muscle mass of DKO mice treated with PMO-G or PMO-S (n = 3, *P < 0.05; one-way ANOVA post-hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( D ) Immunohistochemistry for dystrophin expression in treated DKO mice ( scale bar = 100 μm ) . TA - tibialis

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (D) Immunohistochemistry for dystrophin expression in treated DKO mice (scale bar=100 μm). TA-tibialis anterior


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( E , F ) Western blot quantitative for dystrophin in treated DKO mice ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls ) . TA - tibialis anterior , Q - quadriceps , G - gastrocnemius , T - triceps , A - abdominal muscle D - diaphragm . 2 . 5 µg , 5µg 15µg total from C57BL / 6 50 µg of muscle samples from untreated treated DKO mice were loaded . α - actinin was used as loading control . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (E, F) Western blot and quantitative analysis for the dystrophin protein in treated DKO mice (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman -Keuls test). TA-tibialis anterior, Q-quadriceps, G-gastrocnemius, T-triceps, A-abdominal muscle and D-diaphragm. 2.5 µg, 5µg and 15µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded. α-actinin was used as the loading control. Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) metformin daily via drinking water ( PMO - GM ) . ( B ) Measurement of Ejection Fraction ( EF ) Fractional Shortening ( FS ) in treated DKO mouse hearts ( n = 3 ) C57BL / 6 mice ( n = 6 ) with echocardiography ( * P < 0 . 05 ; one - way ANOVA post hoc Student - Newman - Keuls ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). (B) Measurement of Ejection Fraction (EF) and Fractional Shortening (FS) in treated DKO mouse hearts (n=3) and C57BL/6 mice (n=6) with echocardiography (*P<0.05; one-way ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) metformin daily via drinking water ( PMO - GM ) . ( C ) Morphological examination of treated DKO mouse hearts with H & E ( scale bar = 800μm ) . Boxed areas are magnified . ( n = 3 , * P < 0 . 05 ; one - way ANOVA post hoc Student - Newman - Keuls ) Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). (C) Morphological examination of treated DKO mouse hearts with H&E staining (scale bar=800μm). Boxed areas are magnified. (n=3, *P<0.05; one-way ANOVA post hoc Student-Newman-Keuls test) Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) metformin daily via drinking water ( PMO - GM ) . Immunohistochemistry for immunoglobin G ( IgG ) in treated DKO mouse hearts ( D , scale bar = 100 μm ) .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). Immunohistochemistry for immunoglobin G (IgG) in treated DKO mouse hearts (D, scale bar=100 μm).


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) metformin daily via drinking water ( PMO - GM ) . Muscle function was evaluated to determine physical improvement with grip strength ( E ) or running wheel ( F ) for treated DKO ( n = 3 ) or C57BL / 6 mice ( n = 4 ) ( * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). Muscle function was evaluated to determine the physical improvement with grip strength (E) or running wheel tests (F) for treated DKO (n=3) or C57BL/6 mice (n=4) (*P<0.05; One way-ANOVA post hoc Student-Newman -Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) metformin daily via drinking water ( PMO - GM ) . ( G ) Immunohistochemistry for dystrophin expression in - wide from treated DKO mice ( scale bar = 100 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). (G) Immunohistochemistry for dystrophin expression in body-wide muscles from treated DKO mice (scale bar=100 μm).


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) metformin daily via drinking water ( PMO - GM ) . ( H ) Western blot quantitative for dystrophin in treated DKO mice ( n = 3 ) . 5µg , 10µg 20µg total from C57BL / 6 50 µg of muscle samples from untreated treated DKO mice were loaded . α - actinin was used as loading control . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). (H) Western blot and quantitative analysis for dystrophin protein in treated DKO mice (n=3). 5µg, 10µg and 20µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded. α-actinin was used as the loading control. Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( B ) Survival rate of DKO mice treated with PMO - GM ( n = 6 ) . number of untreated DKO controls is 15 .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (B) Survival rate of DKO mice treated with PMO-GM (n=6).The number of untreated DKO controls is 15.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( C ) Measurement of kyphosis index of DKO mice treated with PMO - GM ( scale bar = 5mm ) wild - type C57BL / 6 mice ( scale bar = 8mm ) ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (C) Measurement of kyphosis index of DKO mice treated with PMO-GM (scale bar=5mm) and wild-type C57BL/6 mice (scale bar=8mm)(n=3, *P<0.05; One way- ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( D ) Measurement of tidal volume in treated DKO mice with a cardio - respiratory gated technique ( n = 3 , * P < 0 . 05 ; one - way ANOVA post hoc Student - Newman - Keuls ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (D) Measurement of tidal volume in treated DKO mice with a cardio-respiratory gated technique (n=3, *P<0.05; one-way ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( E - F ) Examination of EF FS ( E ) stroke volume cardiac output ( F ) in treated DKO mouse hearts ( n = 3 ) C57BL / 6 ( n = 5 ) with echocardiography ( * P < 0 . 05 , * * P < 0 . 001 ; one - way ANOVA post hoc Student - Newman - Keuls ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (E-F) Examination of EF and FS (E) and stroke volume and cardiac output (F) in treated DKO mouse hearts (n=3) and C57BL/6(n=5) with echocardiography (*P<0.05, **P<0.001; one-way ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( G ) Masson trichrome ' s for cardiac fibrosis quantitative of fibrotic areas in treated DKO mouse hearts ( scale bar = 800 μm ) . Boxed areas are magnified ( n = 3 , * * P < 0 . 001 ; One way - ANOVA post hoc Student - Newman - Keuls ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (G) Masson trichrome's staining for cardiac fibrosis and quantitative analysis of fibrotic areas in treated DKO mouse hearts (scale bar=800 μm). Boxed areas are magnified (n=3, **P<0.001;One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( H ) Measurement of serum creatine kinase - MB ( CK - MB ) in treated DKO mice ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (H) Measurement of serum creatine kinase-MB (CK-MB) levels in treated DKO mice (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( I - J ) Muscle function was assessed to determine physical improvement with grip strength ( I ) or running wheel ( J ) in treated DKO ( n = 4 ) C57BL / 6 mice ( n = 6 ) ( * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (I-J) Muscle function was assessed to determine the physical improvement with grip strength (I) or running wheel tests (J) in treated DKO (n=4) and C57BL/6 mice (n=6) (*P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( A ) Immunohistochemistry for dystrophin expression in treated DKO mice ( scale bar = 100 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 O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (A) Immunohistochemistry for dystrophin expression in treated DKO mice (scale bar=100 μm).


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( B ) Western blot quantitative for dystrophin in treated DKO mice ( n = 3 ) . 5µg , 10µg 25µg total from C57BL / 6 50 µg of muscle samples from untreated treated DKO mice were loaded . α - actinin was used as loading control . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (B) Western blot and quantitative analysis for dystrophin protein in treated DKO mice (n=3).5µg, 10µg and 25µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded. α-actinin was used as the loading control. Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( C ) Re - localization of DAPC components in treated DKO mice to assess dystrophin function recovery of normal myoarchitecture ( scale bar = 50μm ) . arrowheads point to identical myofibers .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (C) Re-localization of DAPC components in treated DKO mice to assess dystrophin function and recovery of normal myoarchitecture (scale bar=50μm). The arrowheads point to identical myofibers.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( D ) Measurement of serum CK in treated DKO mice ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls ) . Measurement of serum indices from treated DKO mice to reflect liver ( E ) functions ( n = 3 ; * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (D) Measurement of serum CK levels in treated DKO mice (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Measurement of serum indices from treated DKO mice to reflect liver (E) functions (n=3; *P<0.05; One way-ANOVA post hoc Student-Newman- Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . Measurement of serum indices from treated DKO mice to reflect kidney ( F ) functions ( n = 3 ; * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. Measurement of serum indices from treated DKO mice to reflect kidney (F) functions (n=3; *P<0.05; One way-ANOVA post hoc Student-Newman- Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( G ) Morphological examination of liver kidney from treated DKO mice ( scale bar = 50 μm ) .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (G) Morphological examination of liver and kidney from treated DKO mice (scale bar=50 μm).


e . Retrospective immunohistochemistry DAB of IgG4 IgG on archival temporal lobe resection tissue . As suggested by consensus statement on pathology of IgG4 - RD ( Deshpande et al , 2012 ) , three 40x fields with highest number of IgG4 + IgG + were selected , counted averaged within these fields . as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Retrospective immunohistochemistry DAB staining of IgG4 and IgG on archival temporal lobe resection tissue. As suggested by the consensus statement on the pathology of IgG4-RD (Deshpande et al, 2012), three 40x fields with the highest number of IgG4+ and IgG+ cells were selected, counted and averaged within these fields. Cell counts as indicated.


f . C - X - C motif chemokine 13 ( CXCL13 ) concentrations measured by ELISA from inflammatory pseudotumor ( IPT ) CSF , control CSF , MS - derived CSF . values , mean ± ; n = 4 experiment repeats with technical replicates .

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

f. C-X-C motif chemokine 13 (CXCL13) concentrations measured by ELISA from inflammatory pseudotumor (IPT) CSF, control CSF, and MS patient-derived CSF. Individual values, mean ± SEM; n=4 experiment repeats with technical replicates.


g . Stacked bar chart depicting mean relative expression of T helper - associated indicated cytokines in T subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF as in ( a ) . , T effector memory ; TCM , T central memory ; Treg , regulatory T .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Stacked bar chart depicting mean relative expression levels of T helper cell-associated indicated cytokines in T cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF as in (a). TEM, T effector memory; TCM, T central memory; Treg, regulatory T cell.


h . Violin plot depicting relative expression of IL4R ( left ) IL10RA ( right ) in B subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF .

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

h. Violin plot depicting relative expression levels of IL4R (left) and IL10RA (right) in B cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF.


a . Relative abundances of types identified by Seurat v4 reference mapping as in Figure 1a in IPT CSF compared to control or MS - derived CSF . Boxplot depicting 25th - 75th percentiles with median shown as central band whiskers extending from minimum to maximum 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

a. Relative abundances of cell types identified by Seurat v4 reference mapping as in Figure 1a in IPT CSF compared to control or MS patient-derived CSF. Boxplot depicting 25th-75th percentiles with median shown as central band and whiskers extending from minimum to maximum values.


A . Confocal of live control ( top ) U18 - treated ( bottom ) tsA201 expressing P4M - YFP . Black orange rectangles show expanded views of PtdIns4P in perinuclear endo / lysosomal compartments , respectively . B . of perinuclear P4M - YFP mean normalized to cytoplasm . Control : n = 19 ; U18 : n = 16 .

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

A. Confocal images of live control (top) and U18-treated (bottom) tsA201 cells expressing P4M-YFP. Black and orange rectangles show expanded views of PtdIns4P in perinuclear and endo/lysosomal compartments, respectively. B. Quantification of perinuclear P4M-YFP mean fluorescence intensity normalized to the cytoplasm. Control: n=19; U18: n=16.


K . Heat map of PtdIns , PtdInsP PtdInsP2 from 4 different murine brain regions . colors from green to red indicate relative contents of lipid isoforms in different brain regions from NPC1I1061T mice relative to wild - type brains ; n = 3 for both WT NPC1I1061T .

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

K. Heat map of PtdIns, PtdInsP and PtdInsP2 levels from 4 different murine brain regions. The colors from green to red indicate the relative contents of lipid isoforms in different brain regions from NPC1I1061T mice relative to wild-type brains; n=3 for both WT and NPC1I1061T animals.


C . Confocal of control U18 - treated tsA201 fixed stained for OSBP ( left ) , TGN46 ( middle ) , merge ( right ) . Black rectangles show expanded views of OSBP TGN46 regions . D . of OSBP signal in TGN46 regions ( OSBPTGN46 ) . Control : n = 40 ; U18 : n = 39 .

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

C. Confocal images of control and U18-treated tsA201 cells fixed and stained for OSBP (left), TGN46 (middle), and merge (right). Black rectangles show expanded views of OSBP and TGN46 regions. D. Quantification of OSBP signal in TGN46 regions (OSBPTGN46). Control: n=40; U18: n=39.


J . Confocal of control U18 - treated GFP11 - SAC1 HEK293 ( top ) control NPC1I1061T fibroblasts immunolabeled with anti - SAC1 antibody ( bottom ) . Insets show an enlarged view of SAC1 centered on a perinuclear region . K . analyses of GFP11 - SAC1 ( top ; Control : n = 10 ; U18 : n = 10 ) anti - SAC1 ( bottom ; Control : n = 7 ; NPC1I1061T : n = 8 ) mean intensities in Golgi normalized to cytoplasm .

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

J. Confocal images of control and U18-treated GFP11-SAC1 HEK293 cells (top) and control and NPC1I1061T patient fibroblasts immunolabeled with anti-SAC1 antibody (bottom). Insets show an enlarged view of SAC1 centered on a perinuclear region. K. Quantification analyses of GFP11-SAC1 (top; Control: n=10; U18: n=10) and anti-SAC1 (bottom; Control: n=7; NPC1I1061T: n=8) mean fluorescence intensities in Golgi normalized to the cytoplasm.


C . Left : Confocal of control ( left ) U18 - treated ( right ) tsA201 expressing PI4KIIα - GFP . Bottom panels show an enlarged view of probe centered on a perinuclear region . Right : of perinuclear PI4KIIα relative to cytoplasm . Control : n = 12 ; U18 : n = 14 . D . Same as ( C ) only PI4KIIIβ . Control : n = 12 ; U18 : n = 15 .

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

C. Left: Confocal images of control (left) and U18-treated (right) tsA201 cells expressing PI4KIIα -GFP. Bottom panels show an enlarged view of the probe centered on a perinuclear region. Right: Quantification of perinuclear PI4KIIα intensity relative to the cytoplasm. Control: n=12; U18: n=14. D. Same as (C) only PI4KIIIβ. Control: n=12; U18: n=15.


E . Confocal of control U18 - treated tsA201 fixed stained for PI4KIIα ( left ) , TGN46 ( middle ) , merge ( right ) . Black rectangles show expanded views of PI4KIIα TGN46 regions . F . of PI4KIIα signal in TGN46 regions ( PI4KIIα TGN46 ) . Control : n = 36 ; U18 : n = 36 .

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

E. Confocal images of control and U18-treated tsA201 cells fixed and stained for PI4KIIα (left), TGN46 (middle), and merge (right). Black rectangles show expanded views of PI4KIIα and TGN46 regions. F. Quantification of PI4KIIα signal in TGN46 regions (PI4KIIα TGN46). Control: n=36; U18: n=36.


B , Confocal of wild - type PI4KIIα - depleted ( sgPI4K2A ) HEK293 - CAS9 expressing P4M - YFP , treated with or without U18 . Bottom panels show enlarged views of PtdIns4P in a Golgi region . C , Quantitative analyses of P4M - YFP mean distributed in Golgi normalized to cytoplasm under different conditions . Control : n = 19 ; U18 : n = 13 , sgPI4K2A : n = 19 ; sgPI4K2A + U18 : n = 17 ; sgDHHC3 : n = 11 ; sgDHHC3 + U18 : n = 11 . * represents a significant difference ( P < 0 . 05 ) between control other non - U18 treated groups ; # represents a significant difference ( P < 0 . 05 ) between U18 - treated other U18 - treated sgRNA groups .

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

B, Confocal images of wild-type and PI4KIIα-depleted (sgPI4K2A) HEK293-CAS9 cells expressing P4M-YFP, treated with or without U18. Bottom panels show enlarged views of PtdIns4P in a Golgi region. C, Quantitative analyses of P4M-YFP mean fluorescence intensity distributed in Golgi normalized to the cytoplasm under different conditions. Control: n=19; U18: n=13, sgPI4K2A: n=19; sgPI4K2A+U18: n=17; sgDHHC3: n=11; sgDHHC3+U18: n=11. * represents a significant difference (P<0.05) between control and other non-U18 treated groups; # represents a significant difference (P<0.05) between U18-treated and other U18-treated sgRNA groups.


H . Control or GFP - DHHC3 expressing tsA201 fixed stained for PI4KIIα or PI4KIIIβ . Bottom panels show enlarged views of DHHC3 , PI4KIIα , or PI4KIIIβ at Golgi regions . I . of PI4KIIα ( left ; control : n = 42 , DHHC3 : n = 24 ) , or PI4KIIIβ ( right ; control : n = 39 , DHHC3 : n = 26 ) relative to cytoplasm .

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

H. Control or GFP-DHHC3 expressing tsA201 cells fixed and stained for PI4KIIα or PI4KIIIβ. Bottom panels show enlarged views of DHHC3, PI4KIIα, or PI4KIIIβ at Golgi regions. I. Quantification of PI4KIIα (left; control: n=42, DHHC3: n=24), or PI4KIIIβ (right; control: n=39, DHHC3: n=26) intensity relative to cytoplasm.


M . Confocal of control ( top row ) , NPC1I1061T ( middle row ) , NPC1I1061T treated with sgDHHC3 ( bottom row ) expressing P4M , LAMP1 CAS9 . Insets are zoomed regions from LAMP1 positive areas . N . of P4M changes at endomembranes . Control : n = 149 , U18 : n = 251 , sgPI4KIIa + U18 : n = 131 , sgDHHC3 + U18 : n = 271 . Each n represents a single enodolysosome .

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

M. Confocal images of control (top row), NPC1I1061T (middle row), and NPC1I1061T treated with sgDHHC3 (bottom row) patient cells expressing P4M, LAMP1 and CAS9. Insets are zoomed regions from LAMP1 positive areas. N. Quantification of P4M changes at endomembranes. Control: n=149, U18: n=251, sgPI4KIIa +U18: n=131, sgDHHC3+U18: n= 271. Each n represents a single enodolysosome.


C . Confocal of control NPC1I1061T fibroblasts immunolabeled for ACBD3 . Black orange squares show expanded views of anti - ACBD3 in Golgi endomembrane compartments , respectively . D . Analyses of ACBD3 distributed in Golgi ( left graph ; Control : n = 9 ; NPC1I1061T : n = 9 ) or in endomembranes ( right graph ; Control : n = 1360 ; NPC1I1061T : n = 1645 from 15 ) , normalized to cytoplasm .

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

C. Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for ACBD3. Black and orange squares show expanded views of anti-ACBD3 in Golgi and endomembrane compartments, respectively. D. Analyses of ACBD3 intensity distributed in Golgi (left graph; Control: n=9; NPC1I1061T: n=9) or in endomembranes (right graph; Control: n=1360; NPC1I1061T: n=1645 from 15 cells), normalized to the cytoplasm.