mitochondrial potential of 293 transfected with indicated shRNA in normal medium or after 18 hr glucose starvation ( G ) results are from three independent experiments . quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 ( Mann - Whitney ) .

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The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or after 18 hr glucose starvation (G) All results are from three independent experiments. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 (Mann-Whitney test).


mitochondrial potential of 293 transfected with indicated shRNA in normal medium or 48 hr hypoxia ( H ) . results are from three independent experiments . quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 ( Mann - Whitney ) .

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The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or 48 hr hypoxia (H). All results are from three independent experiments. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 (Mann-Whitney test).


H1299 transfected with indicated shRNA in normal medium or after 12h glucose starvation ( A ) Representative FACS of apoptosis . Data are presented as mean ± s . e . m . from 3 independent experiments ; * p < 0 . 05 compared to NC glucose starvation ( A ) ( Student ' s t - ) .

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H1299 cells transfected with the indicated shRNA in normal medium or after 12h glucose starvation (A) Representative FACS analysis of apoptosis. Data are presented as mean ± s.e.m. from 3 independent experiments; *p < 0.05 compared to NC glucose starvation (A) (Student's t-test).


H1299 transfected with indicated shRNA in normal medium or 48h hypoxia stimulation ( B ) . Representative FACS of apoptosis . Data are presented as mean ± s . e . m . from 3 independent experiments * * p < 0 . 01 compared to NC hypoxia ( B ) ( Student ' s t - ) .

B-CELL_LINE O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H1299 cells transfected with the indicated shRNA in normal medium or 48h hypoxia stimulation (B). Representative FACS analysis of apoptosis. Data are presented as mean ± s.e.m. from 3 independent experiments **p < 0.01 compared to NC hypoxia (B) (Student's t-test).


H1299 transfected with indicated plasmids in normal medium or after 12h glucose starvation ( C ) Representative FACS of apoptosis . Results from three independent experiments are presented as a histogram . Data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 compared to Beclin 1 S9093A ( AA ) treated with H2O2 or hypoxia ( Mann - Whitney ) .

B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O

H1299 cells transfected with the indicated plasmids in normal medium or after 12h glucose starvation (C) Representative FACS analysis of apoptosis. Results from three independent experiments are presented as a histogram. Data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 compared to Beclin 1 S9093A (AA) treated with H2O2 or hypoxia (Mann-Whitney test).


H1299 transfected with indicated plasmids in normal medium or 48h hypoxia stimulation ( D ) . Representative FACS of apoptosis . Results from three independent experiments are presented as a histogram . Data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 compared to Beclin 1 S9093A ( AA ) treated with H2O2 or hypoxia ( Mann - Whitney ) .

B-CELL_LINE O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O

H1299 cells transfected with the indicated plasmids in normal medium or 48h hypoxia stimulation (D). Representative FACS analysis of apoptosis. Results from three independent experiments are presented as a histogram. Data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 compared to Beclin 1 S9093A (AA) treated with H2O2 or hypoxia (Mann-Whitney test).


( E ) CHK2 + / + CHK2 - / - mice were subjected to MCAO for 1 hr reperfusion for 12 h . ( C ) ( I ) tissues of mouse brain were coronally sectioned stained with 2 % TTC .

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(E) CHK2+/+ and CHK2-/- mice were subjected to MCAO for 1 hr and reperfusion for 12 h. Contralateral (C) and ipsilateral (I) tissues of the mouse brain were coronally sectioned and stained with 2 % TTC.


( F ) infarct volume was determined by measuring infarct relative to normal in slice . ( n = 5 mice ) . Data are presented as mean ± s . e . m . ; * * P < 0 . 01 ( Student ' s t - ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O O O O O 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) The infarct volume was determined by measuring infarct size relative to normal in the slice. (n = 5 mice). Data are presented as mean ± s.e.m.; **P < 0.01 (Student's t-test).


( G - J ) Immunoblots for p62 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 Beclin 1 in cortical extracts from ischemia reperfusion - treated CHK2 + / + CHK2 - / - mice . of p62 , p - Beclin 1 Ser90 p - Beclin 1 Ser93 ( n = 3 mice ) . Data are presented as mean ± s . e . m . ; * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O B-TISSUE O O B-DISEASE I-DISEASE O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O 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-J) Immunoblots for p62, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in the cortical extracts from ischemia and reperfusion-treated CHK2+/+ and CHK2-/- mice. Quantification of p62, p-Beclin 1 Ser90 and p-Beclin 1 Ser93 protein levels (n = 3 mice). Data are presented as mean ± s.e.m.; *P < 0.05, **P < 0.01 (Student's t-test).


( A ) NPM1 expression in CD34 + Lin - from myelodysplastic syndrome ( MDS ) compared to healthy volunteers ( n = 29 ) .

O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O B-DISEASE I-DISEASE O B-DISEASE O O O O O O O O O O O

(A) NPM1 expression in CD34+Lin- cells from myelodysplastic syndrome (MDS) patients compared to healthy volunteers (n = 29).


( B ) Representative of NPM1 in bone marrow samples of MDS or acute myeloid leukemia ( AML ) with multi - lineage dysplasia ( MLD ) . Insets show representative in blasts [ B ] in differentiated myeloid [ e . g . , neutrophil , M ] with a high magnification .

O O O O O B-GENEPROD O B-TISSUE I-TISSUE O O B-DISEASE O B-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O O O O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O O O O

(B) Representative staining of NPM1 protein levels in bone marrow samples of MDS or acute myeloid leukemia (AML) with multi-lineage dysplasia (MLD). Insets show representative staining in blasts [B] and in differentiated myeloid cells [e.g., neutrophil, M] with a high magnification.


( C ) Percentages of samples with NPM1 - low NPM1 - normal in healthy , MDS MLD in AML groups .

O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-DISEASE I-DISEASE O B-DISEASE O O

(C) Percentages of patient samples with NPM1-low and NPM1-normal in healthy, MDS and MLD in AML groups.


( C ) Representative distribution of Tubulin CDC42 in Ctrl , 4 - months old Npm1 cKO 2 - years old WT HSCs determined by IF . Scale bars , 5 μm .

O O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O B-EXP_ASSAY O O O O O O O

(C) Representative distribution of Tubulin and CDC42 in Ctrl, 4-months old Npm1 cKO and 2-years old WT HSCs determined by IF. Scale bars, 5 μm.


( F ) HSCs of Ctrl , 4 - months old Npm1 cKO 2 - years old WT mice were sorted stained for TOM20 . Representative ( top ) of mitochondrial volume number ( bottom ) in single ( n = 58 in Ctrl , n = 70 in cKO n = 23 in Old , from 3 mice per group ) , identified by volume rendering of TOM20 . Scale bars , 2 μm .

O O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-SUBCELLULAR O O O 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-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O

(F ) HSCs of Ctrl, 4-months old Npm1 cKO and 2-years old WT mice were sorted and stained for TOM20. Representative images (top) and quantification of mitochondrial volume and number (bottom) in single cells (n = 58 cells in Ctrl, n = 70 cells in cKO and n = 23 cells in Old, from 3 mice per group), identified by volume rendering of TOM20. Scale bars, 2 μm.


( J ) Representative ( top ) of mitochondrial volume number ( bottom ) , identified by volume rendering of TOM20 , in MDS - L transfected with siNPM1 or control ( siCTRL ) ( n = 58 per group , from 3 transfected MDS - L cultures ) . Scale bars , 2 μm .

O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O

(J) Representative images (top) and quantification of mitochondrial volume and number (bottom), identified by volume rendering of TOM20, in MDS-L cells transfected with siNPM1 or control (siCTRL) (n = 58 cells per group, from 3 transfected MDS-L cultures). Scale bars, 2 μm.


( F ) Representative peripheral blood ( PB ) smears . Insets show differentiated granulocytes ( a , b ) in control mice , anisocytosis ( c ) , dysplastic myeloid ( d - g ) in Npm1 cKO mice ( Npm1F / FVav1Cre + ) . Npm1 cKO mice show dysplastic erythroid ( polychromatophilic , blue arrowhead ; schistocyte , blue - outlined arrowhead ; tear drop , Howell - Jolly bodies , black arrowhead ; black - outlined arrowhead ) , dysplastic monocytes ( black arrow ) , dysplastic neutrophils ( d - f ) . Scale bars , 10 μm .

O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O B-CELL_TYPE O O O O O O O B-ORGANISM O B-DISEASE O O O O O B-CELL_TYPE O O O O O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O B-GENEPROD O B-ORGANISM O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O O O O O O O O

(F) Representative peripheral blood (PB) smears. Insets show the differentiated granulocytes (a, b) in control mice, and anisocytosis (c), dysplastic myeloid cells (d-g) in Npm1 cKO mice (Npm1F/FVav1Cre+). Npm1 cKO mice show dysplastic erythroid cells (polychromatophilic, blue arrowhead; schistocyte, blue-outlined arrowhead; tear drop, Howell-Jolly bodies, black arrowhead; black-outlined arrowhead), dysplastic monocytes (black arrow), and dysplastic neutrophils (d-f). Scale bars, 10 μm.


( C ) Npm1 p53 in KSL of Ctrl ( Npm1F / FVav1Cre - ) , cKO ( Npm1F / FVav1Cre + ) mice .

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(C) Npm1 and p53 protein levels in KSL cells of Ctrl (Npm1F/FVav1Cre-), and cKO (Npm1F/FVav1Cre+) mice.


( F ) Representative PB smears of DKO mice . Insets show anisocytosis ( a ) , poikilocytosis ( b ) dysplastic myeloid ( c ) . DKO mice show dysplastic erythroid ( polychromatophilic , blue arrowhead ) , Howell - Jolly bodies , ( black arrowhead in insert a ) , dysplastic monocytes ( black arrow , i - v ) , dysplastic neutrophils ( vi ) , a pseudo - Pelger - Huet anomaly ( ii ) , dysplastic platelets ( giant platelet , iv vii ) . Scale bars , 10μm .

O O O O B-TISSUE O O O B-ORGANISM O O O B-DISEASE O O O O B-DISEASE O O O O B-CELL_TYPE O O O O O B-ORGANISM O O B-CELL_TYPE O B-CELL_TYPE O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-CELL_TYPE O O O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE I-DISEASE I-DISEASE O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O

(F) Representative PB smears of DKO mice. Insets show anisocytosis (a), poikilocytosis (b) and dysplastic myeloid cell (c). DKO mice show dysplastic erythroid cells (polychromatophilic, blue arrowhead), Howell-Jolly bodies, (black arrowhead in insert a), dysplastic monocytes (black arrow, and i-v), dysplastic neutrophils (vi), a pseudo-Pelger-Huet anomaly (ii), and dysplastic platelets (giant platelet, iv and vii). Scale bars, 10μm.


( I ) Representative H & E of sections of spleen of 2 - month - old DKO mice with myeloid disorders . Arrowheads ( right ) indicate infiltrating myeloid blasts . Scale bars , 250 μm ( left ) 10μm ( right ) , respectively .

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(I) Representative H&E staining of sections of the spleen of 2-month-old DKO mice with myeloid disorders. Arrowheads (right) indicate the infiltrating myeloid blasts. Scale bars, 250 μm (left) and 10μm (right), respectively.


B . Growth comparisons of selected kinases with kinase ‑ dead mutant match . 24h growth curves ( OD595 ) from liquid culture of yeast strains ( W303 ) expressing human kinase in presence or absence of 3 . 2 % ( v / v ) DMSO . Upper panel : BUB1 : BUB1 wild type , BUB1 - KD : BUB1 ( K821M ) ; upper middle panel : NEK6 : NEK6 wild type , NEK6 - KD : NEK6 ( K74M & K75M ) ; lower middle panel : PKCα - ac : PKCα ( A25E ) , PKCα - KD : PKCα ( K368R ) ; lower panel : Control with empty plasmid . Error bars represent SD from three biological replicates .

O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

B. Growth comparisons of selected kinases with the kinase‑dead mutant match. 24h growth curves (OD595) from liquid culture of yeast strains (W303) expressing human kinase in the presence or absence of 3.2% (v/v) DMSO. Upper panel: BUB1: BUB1 wild type, BUB1-KD: BUB1(K821M); upper middle panel: NEK6: NEK6 wild type, NEK6-KD: NEK6(K74M&K75M); lower middle panel: PKCα-ac: PKCα(A25E), PKCα-KD: PKCα(K368R); lower panel: Control with empty plasmid. Error bars represent SD from three biological replicates.


C . Human kinases phosphorylate large sets of yeast . Upper panel : In total , 27 human kinases showed activity towards yeast visible on western blots through enhanced reactivity with phospho - motif antibodies on yeast in comparison to other human kinases , kinase ‑ dead mutant versions or vector control samples . Commercially available pS / T - antibodies with relatively low specificities were chosen to enable detection of phosphorylation of yeast proteome by human kinases . Lower panel : Examples of western blots with equal amount of whole yeast lysate from strains expressing human kinases loaded . When developed with indicated phospho - motif recognizing antibodies , immunoreactive bands indicated phosphorylation of yeast .

O O B-ORGANISM O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O B-ORGANISM O O O O O 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-EXP_ASSAY O B-ORGANISM O O B-ORGANISM O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-ORGANISM O

C. Human protein kinases phosphorylate large sets of yeast proteins. Upper panel: In total, 27 human protein kinases showed activity towards yeast proteins visible on western blots through enhanced reactivity with phospho-motif antibodies on yeast proteins in comparison to other human kinases, kinase‑dead mutant versions or vector control samples. Commercially available pS/T-antibodies with relatively low sequence specificities were chosen to enable detection of phosphorylation of the yeast proteome by human kinases. Lower panel: Examples of western blots with equal amount of whole yeast cell lysate from strains expressing human kinases loaded. When developed with the indicated phospho-motif recognizing antibodies, immunoreactive bands indicated phosphorylation of yeast proteins.


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 B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-ORGANISM O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-ORGANISM O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-ORGANISM B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-CELL_LINE B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O B-CELL_LINE O O O O O O B-GENEPROD 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-GENEPROD O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O 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 B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 B-CELL_TYPE O O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE 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 B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY 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-GENEPROD O O O B-GENEPROD O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY 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-GENEPROD O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O 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-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY 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-GENEPROD O O O O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-EXP_ASSAY 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-GENEPROD O O O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_LINE O O O O O 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-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD 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 B-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O B-CELL_LINE O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-GENEPROD 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 B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-CELL_LINE O B-GENEPROD O O O B-GENEPROD O O O O O 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-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD 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 B-GENEPROD O O O B-CELL_LINE O B-GENEPROD O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY 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 B-GENEPROD O O O B-CELL_LINE O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O O O O O B-GENEPROD O O O O O O O O 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-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-GENEPROD 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 B-ORGANISM O O O O O O B-EXP_ASSAY O O 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-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O O O O B-ORGANISM 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 B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-ORGANISM 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 B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O B-ORGANISM 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 B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM 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 B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O B-GENEPROD O O B-TISSUE O B-TISSUE I-TISSUE O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-ORGANISM O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O B-GENEPROD O B-GENEPROD 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 .

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(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-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE 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 .

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(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 B-EXP_ASSAY O O B-TISSUE B-EXP_ASSAY O B-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-TISSUE O O O O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O B-TISSUE I-TISSUE I-TISSUE O O O O B-GENEPROD O O O O O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE 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 .

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(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 B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM 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 B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-GENEPROD 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 .

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(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 ) .

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(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 .

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(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 .

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(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 B-EXP_ASSAY O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE I-CELL_TYPE 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 B-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-TISSUE 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 B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O 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 B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O B-ORGANISM 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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-ORGANISM O O O O O B-ORGANISM 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 B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O 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 .

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(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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O B-ORGANISM O O O O

(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 .

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(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 .

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(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 .

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


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

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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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 .

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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-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE O B-EXP_ASSAY O O B-ORGANISM O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O B-TISSUE O B-TISSUE

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 .

B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-ORGANISM O 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-TISSUE O B-TISSUE I-TISSUE O O O B-TISSUE O O O B-TISSUE O O O B-TISSUE O O O B-TISSUE I-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM B-TISSUE O O O O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O B-ORGANISM B-TISSUE 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-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O O B-ORGANISM 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 .

B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-ORGANISM 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-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O B-SMALL_MOLECULE 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-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-DISEASE O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE B-DISEASE O O B-DISEASE O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O B-ORGANISM 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).