L CM - H2DCFDA ( green ) indicating cellular ROS levels of WT or TMEM175 KO HEK293T cells pre - treatment with vehicle or 10 μM HA14 - 1 for 20 min . Scale bars = 10 μm . M Statistics of CM - H2DCFDA fluorescence intensity of ( L ) . The n values represent the cell numbers counted within the same experiment . Data information : The data are presented as the mean ± SEM . Statistical significance was calculated with two - sided student ' s t - tests M ) and is indicated with NS for not significant ( P > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . In panel M , n value means the number of technical replicates .

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

L CM-H2DCFDA (green) indicating cellular ROS levels of WT or TMEM175 KO HEK293T cells pre-treatment with vehicle or 10 μM HA14-1 for 20 min. Scale bars = 10 μm. M Statistics of CM-H2DCFDA fluorescence intensity of (L). The n values represent the cell numbers counted within the same experiment. Data information: The data are presented as the mean ± SEM. Statistical significance was calculated with two-sided student's t-tests M) and is indicated with NS for not significant (P > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. In panel M, n value means the number of technical replicates.


F Fluorescence of G3BP1 excited with 488 nm in control ( upper ) and TMEM175 - overexpressing ( lower ) cells . Scale bars = 10 μm . G Statistics of the numbers of puncta per cell ( left ) and the percentage of cells with puncta in ( F ) . We counted 547 cells in Vector group from 37 different fields of view and 332 cells in TMEM175 group from 76 different fields of view . n value represents the number of different fields of view randomly selected in the same imaging experiment . Data information : The data are presented as the mean ± SEM . Statistical significance was calculated with two - sided student ' s t - test , and is indicated with * * for p < 0 . 01 , and * * * for p < 0 . 001 . In panels G n value means the number of technical replicates .

O O O B-GENEPROD 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fluorescence of G3BP1 excited with 488 nm in control (upper) and TMEM175-overexpressing (lower) cells. Scale bars = 10 μm. G Statistics of the numbers of puncta per cell (left) and the percentage of cells with puncta in (F). We counted 547 cells in Vector group from 37 different fields of view and 332 cells in TMEM175 group from 76 different fields of view. n value represents the number of different fields of view randomly selected in the same imaging experiment. Data information: The data are presented as the mean ± SEM. Statistical significance was calculated with two-sided student's t-test, and is indicated with ** for p < 0.01, and *** for p < 0.001. In panels G n value means the number of technical replicates.


H Co - localization of mitochondrial indicator TOM20 - mCherry and lysosomal indicator LysoTracker in WT ( upper row ) or TMEM175 KO ( lower row ) HEK293T cells . Co - localization of mitochondrion and lysosome is observed as a yellow colour in the merged image ( marked by white arrows ) . Scale bars = 10 μm . I The number of merged puncta per cell in ( H ) . We counted 139 cells in WT group from 38 different fields of view and 148 cells in TMEM175 KO group from 42 different fields of view . n value represents the number of different fields of view randomly selected in the same imaging experiment . Data information : The data are presented as the mean ± SEM . Statistical significance was calculated with two - sided student ' s t - test , and is indicated with * * for p < 0 . 01 , and * * * for p < 0 . 001 . In panels I , n value means the number of technical replicates .

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

H Co-localization of mitochondrial indicator TOM20-mCherry and lysosomal indicator LysoTracker in WT (upper row) or TMEM175 KO (lower row) HEK293T cells. Co-localization of mitochondrion and lysosome is observed as a yellow colour in the merged image (marked by white arrows). Scale bars = 10 μm. I The number of merged puncta per cell in (H). We counted 139 cells in WT group from 38 different fields of view and 148 cells in TMEM175 KO group from 42 different fields of view. n value represents the number of different fields of view randomly selected in the same imaging experiment. Data information: The data are presented as the mean ± SEM. Statistical significance was calculated with two-sided student's t-test, and is indicated with ** for p < 0.01, and *** for p < 0.001. In panels I, n value means the number of technical replicates.


A Representative immunoblots for TMEM175 expression levels in the midbrains of mice of different ages ( W : weeks ; M : months ) . B Statistics of the relative TMEM175 protein levels normalized to GAPDH in ( A ) . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

O O B-EXP_ASSAY O B-GENEPROD O O O O B-TISSUE O B-ORGANISM O O O O O O O O O O O O O O O O O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Representative immunoblots for TMEM175 expression levels in the midbrains of mice of different ages (W: weeks; M: months). B Statistics of the relative TMEM175 protein levels normalized to GAPDH in (A). Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


D TMEM175 mRNA levels in the midbrains of WT or TMEM175 KO mice were detected by RT - qPCR using SYBR Green PCR Master Mix . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

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

D TMEM175 mRNA levels in the midbrains of WT or TMEM175 KO mice were detected by RT-qPCR using SYBR Green PCR Master Mix. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


E The knockout of TMEM175 in mouse was verified by Western blot of brain tissues .

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E The knockout of TMEM175 in mouse was verified by Western blot of brain tissues.


F Representative TMEM175 basal lysosomal currents recorded in macrophages of WT or TMEM175 - KO mice . Ψ is lysosomal membrane potential ( deﬁned as Vcytosol - Vlumen ) . The currents were elicited with ramp voltage protocols ( - 100 to + 100 mV in 1 s , Vh = 0 mV ) . G Statistics of the current amplitudes of ( F ) . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

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

F Representative TMEM175 basal lysosomal currents recorded in macrophages of WT or TMEM175-KO mice. Ψ is lysosomal membrane potential (deﬁned as Vcytosol - Vlumen). The currents were elicited with ramp voltage protocols (-100 to +100 mV in 1 s, Vh = 0 mV). G Statistics of the current amplitudes of (F). Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


H , Quantification of rears in the cylinder ( H ) pre - and post - MPTP treatment . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

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

H, Quantification of rears in the cylinder (H) pre- and post-MPTP treatment. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


I Quantification of the latency to fall from a wire hang apparatus ( I ) pre - and post - MPTP treatment . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

O 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 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 O O O O O O O O O O O O O O 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 Quantification of the latency to fall from a wire hang apparatus (I) pre- and post-MPTP treatment. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


J Swing Duration CV ( CV % ) ( Left panel ) and Ataxia Coefficient in Gait tests 7 days post - MPTP . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

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

J Swing Duration CV (CV%) (Left panel) and Ataxia Coefficient in Gait tests 7 days post-MPTP. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


K Latency to fall in rotarod tests 7 days post - MPTP . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K Latency to fall in rotarod tests 7 days post-MPTP. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


L Immunoblots of the TH levels post - MPTP treatment in WT and TMEM175 KO mouse midbrains . M Quantification of the relative TH levels normalized to GAPDH post - MPTP . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

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

L Immunoblots of the TH levels post-MPTP treatment in WT and TMEM175 KO mouse midbrains. M Quantification of the relative TH levels normalized to GAPDH post-MPTP. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


N TH immunofluorescent labeling on substantia nigra slices of WT or TMEM175 - KO mice post - MPTP treatment . Scale bars = 100 μm . O Statistics of the TH - positive neurons in ( N ) . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

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

N TH immunofluorescent labeling on substantia nigra slices of WT or TMEM175-KO mice post-MPTP treatment. Scale bars = 100 μm. O Statistics of the TH-positive neurons in (N). Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


factor . a , Duplicate screens depicting HCoV - 229E infection ( 24 hours post - infection ) in Huh7 cells ectopically expressing ISGs .

O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-CELL_LINE O O O B-GENEPROD O

factor.a, Duplicate screens depicting HCoV-229E infection (24 hours post-infection) in Huh7 cells ectopically expressing ISGs.


b , HCoV - 229E infection of Huh7 cells expressing LY6E or control vector ( Empty ) . Blue : DAPI , green : HCoV - 229E N protein , red : TagRFP encoded in SCRPSY vector .

O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O

b, HCoV-229E infection of Huh7 cells expressing LY6E or control vector (Empty). Blue: DAPI, green: HCoV-229E N protein, red: TagRFP encoded in SCRPSY vector.


c - h , Effect of LY6E expression on diverse coronaviruses . Stably transduced cells were infected with HCoV - 229E ( c ) , HCoV - OC43 ( d ) , MERS - CoV ( e ) , SARS - CoV ( f ) , SARS - CoV - 2 ( g ) , MHV - Gluc ( h ) . Viral replication readouts included : titer determination by plaque assay ( c ) , infectivity by nucleoprotein staining and quantitation by flow cytometry ( d ) , limiting dilution assay ( e , f , g ) , luciferase activity shown as relative light units ( RLU ) ( h , j , l ) .

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

c-h, Effect of LY6E expression on diverse coronaviruses. Stably transduced cells were infected with HCoV-229E (c), HCoV-OC43 (d), MERS-CoV (e), SARS-CoV (f), SARS-CoV-2 (g), MHV-Gluc (h). Viral replication readouts included: titer determination by plaque assay (c), infectivity by nucleoprotein staining and quantitation by flow cytometry (d), limiting dilution assay (e,f,g), luciferase activity shown as relative light units (RLU) (h,j,l).


i , Western blot of WT A549 or two LY6E KO clones ( # 3 , # 4 ) .

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i, Western blot of WT A549 or two LY6E KO clones (#3, #4).


j , HCoV - 229E infection of WT or LY6E KO A549 .

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j, HCoV-229E infection of WT or LY6E KO A549.


k , Western blot of WT or LY6E KO A549 reconstituted with CRISPR - resistant LY6E ( CR LY6E ) or control vector ( Empty ) .

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k, Western blot of WT or LY6E KO A549 reconstituted with CRISPR-resistant LY6E (CR LY6E) or control vector (Empty).


l , HCoV - 229E infection of CR LY6E - reconstituted WT or LY6E KO A549 .

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l, HCoV-229E infection of CR LY6E-reconstituted WT or LY6E KO A549.


formation . a - d , VSV pseudoparticles ( PP ) harboring spike proteins from HCoV - 229E ( a ) , MERS - CoV ( b ) , SARS - CoV ( c ) , SARS - CoV - 2 ( d ) were inoculated on LY6E - or empty vector - expressing cells . Virus entry efficiency was quantified by measuring virus - encoded luciferase reporter activity .

O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM 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 O

formation.a-d, VSV pseudoparticles (PP) harboring spike proteins from HCoV-229E (a), MERS-CoV (b), SARS-CoV (c), SARS-CoV-2 (d) were inoculated on LY6E- or empty vector-expressing cells. Virus entry efficiency was quantified by measuring virus-encoded luciferase reporter activity.


e , VSV pseudoparticles expressing VSV G protein on their surface and encoding CoV S protein and GFP ( VSV * ΔG ( CoV S ) ) were inoculated with LY6E - or empty vector - expressing Huh7 . Syncytia formation was analyzed by immunofluorescence microscopy . Blue : DAPI , green : GFP , red : TagRFP inserted in SCRPSY vector .

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

e, VSV pseudoparticles expressing VSV G protein on their surface and encoding CoV S protein and GFP (VSV*ΔG(CoV S)) were inoculated with LY6E- or empty vector-expressing Huh7. Syncytia formation was analyzed by immunofluorescence microscopy. Blue: DAPI, green: GFP, red: TagRFP inserted in SCRPSY vector.


f , Quantification of VSV * ΔG ( CoV S ) induced syncytia depicted as percentage syncytia area . Three independent areas were analyzed per biological replicate ( circle , square , triangle ) .

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f, Quantification of VSV*ΔG(CoV S) induced syncytia depicted as percentage syncytia area. Three independent areas were analyzed per biological replicate (circle, square, triangle).


h , Cell - cell fusion was measured by co - incubating cells transfected with plasmids encoding CoV S proteins and a split - luciferase construct ( DSP1 - 7 ) together with CoV permissive LY6E - expressing or control cells transfected with the second fragment of split - luciferase ( DSP8 - 11 ) .

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

h, Cell-cell fusion was measured by co-incubating cells transfected with plasmids encoding CoV S proteins and a split-luciferase construct (DSP1-7) together with CoV permissive LY6E-expressing or control cells transfected with the second fragment of split-luciferase (DSP8-11).


virus . a - b , Female Ly6efl / fl and Ly6eΔHSC mice were injected with PBS or MHV and monitored for survival ( a ) and weight loss ( b ) .

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virus.a-b, Female Ly6efl/fl and Ly6eΔHSC mice were injected with PBS or MHV and monitored for survival (a) and weight loss (b).


c - n , Mice were assessed at 3 ( c - h ) or 5 ( i - n ) days post - infection for serum ALT ( c , i ) , viral titers in liver ( d , j ) and spleen ( e , k ) , and liver necrosis and inflammation ( f - h , l - n ) .

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c-n, Mice were assessed at 3 (c-h) or 5 (i-n) days post-infection for serum ALT (c,i), viral titers in liver (d,j) and spleen (e,k), and liver necrosis and inflammation (f-h, l-n).


mice . a - e , Transcriptomic analysis from Ly6efl / fl ( WT ) and Ly6eΔHSC ( KO ) mice . a - b , Heat maps displaying significant changes ( mean RPKM > 0 . 5 ; fold change > 2 ; FDR ≤ 0 . 05 ) in liver ( a ) and spleen ( b ) . Dendrograms are normalized read data ( row z - score ) clustered with complete linkage method employing Spearman Rank correlation distance measurement .

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mice.a-e, Transcriptomic analysis from Ly6efl/fl (WT) and Ly6eΔHSC (KO) mice. a-b, Heat maps displaying significant changes (mean RPKM > 0.5; fold change > 2; FDR ≤ 0.05) in liver (a) and spleen (b). Dendrograms are normalized read data (row z-score) clustered with complete linkage method employing Spearman Rank correlation distance measurement.


d - e , Expression of select genes in liver ( d ) and spleen ( e ) .

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d-e, Expression of select genes in liver (d) and spleen (e).


f - g , Immune cell counts from liver ( f ) and spleen ( g ) .

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f-g, Immune cell counts from liver (f) and spleen (g).


h , Infection of cultured splenocytes . For a - e , n = 3 . For f - g , n = 8 MHV - injected , n = 4 PBS - injected from two pooled experiments .

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h, Infection of cultured splenocytes. For a-e, n=3. For f-g, n=8 MHV-injected, n=4 PBS-injected from two pooled experiments.


Schematic representation of the upstream region of myogenin and amplified regions by RT - PCR ( top ) . RT - PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes ( bottom ) . The presence or absence of reverse transcriptase ( RT ) is shown by ( + ) or ( - ) , respectively . The templates ( cDNA or genomic DNA ) are indicated by C or G , respectively .

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Schematic representation of the upstream region of myogenin and amplified regions by RT-PCR (top). RT-PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes (bottom). The presence or absence of reverse transcriptase (RT) is shown by (+) or (-), respectively. The templates (cDNA or genomic DNA) are indicated by C or G, respectively.


The primers used for RT - PCR ( top ) . Strand - specific RT - PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes using total RNA ( middle and bottom ) and poly ( A ) + RNA ( bottom ) .

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The primers used for RT-PCR (top). Strand-specific RT-PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes using total RNA (middle and bottom) and poly(A)+ RNA (bottom).


Relative expression of indicated RNAs in differentiating C2C12 cells . The presence or absence of RT is shown by ( + ) or ( - ) , respectively . PCR products were verified by sequencing . n = 3 , mean ± SD . The nuclear / cytoplasmic fractionation is confirmed by the expression of H2B and tubulin , respectively .

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Relative expression of indicated RNAs in differentiating C2C12 cells. The presence or absence of RT is shown by (+) or (-), respectively. PCR products were verified by sequencing. n = 3, mean ± SD. The nuclear/cytoplasmic fractionation is confirmed by the expression of H2B and tubulin, respectively.


Chromatin pellet extract ( CPE ) / soluble nuclear extract ( SNE ) ratio of SRA1 , Malat1 , and Myoparr in C2C12 myotubes on a log scale . n = 3 , mean ± SD .

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Chromatin pellet extract (CPE)/soluble nuclear extract (SNE) ratio of SRA1, Malat1, and Myoparr in C2C12 myotubes on a log scale. n = 3, mean ± SD.


Quantitative RT - PCR for myogenin ( A ) and Myoparr ( B ) during myogenesis of C2C12 cells , primary mouse myoblasts ( growth medium , GM ) , and mouse embryonic skeletal muscle ( S . K . ) . The x - axis shows days after differentiation induction or embryonic days . n = 3 , mean ± SD .

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Quantitative RT-PCR for myogenin (A) and Myoparr (B) during myogenesis of C2C12 cells, primary mouse myoblasts (growth medium, GM), and mouse embryonic skeletal muscle (S.K.). The x-axis shows days after differentiation induction or embryonic days. n = 3, mean ± SD.


qRT - PCR showing increased Myoparr expression by MyoD in C3H10T1 / 2 fibroblasts . n = 4 , mean ± SD . * * p < 0 . 01 ( unpaired two - tailed Welch ' s t - test ) .

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qRT-PCR showing increased Myoparr expression by MyoD in C3H10T1/2 fibroblasts. n = 4, mean ± SD. **p < 0.01 (unpaired two-tailed Welch's t-test).


Treatment of recombinant TGF - β for 24 h decreased Myoparr expression in differentiating C2C12 cells . n = 3 , mean ± SD . * p < 0 . 05 ( unpaired two - tailed Student ' s t - test ) .

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Treatment of recombinant TGF-β for 24 h decreased Myoparr expression in differentiating C2C12 cells. n = 3, mean ± SD. *p < 0.05 (unpaired two-tailed Student's t-test).


Relative luciferase activities of the indicated promoter in differentiating C2C12 cells by exogenous MyoD . n = 3 , mean ± SD . * * p < 0 . 01 . ( unpaired two - tailed Student ' s t - test ) .

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Relative luciferase activities of the indicated promoter in differentiating C2C12 cells by exogenous MyoD. n = 3, mean ± SD. **p < 0.01. (unpaired two-tailed Student's t-test).


Relative luciferase activities of the indicated promoter with / without E - box mutations in differentiating C2C12 cells . n = 3 , mean ± SD . * * p < 0 . 01 . ( unpaired two - tailed Student ' s t - test ) .

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Relative luciferase activities of the indicated promoter with/without E-box mutations in differentiating C2C12 cells. n = 3, mean ± SD. **p < 0.01. (unpaired two-tailed Student's t-test).


Relative luciferase activities of the indicated promoter with / without E - box mutations in differentiating C2C12 cells by TGF - β treatment . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 . n . s . , not significant ( unpaired two - tailed Student ' s t - test ) .

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Relative luciferase activities of the indicated promoter with/without E-box mutations in differentiating C2C12 cells by TGF-β treatment. n = 3, mean ± SD. *p < 0.05. **p < 0.01. n.s., not significant (unpaired two-tailed Student's t-test).


C2C12 cells were transfected with 50 nM of indicated siRNAs in growth medium . After additional 24 - h incubation in growth medium , cells were transferred to differentiation medium . The levels of Myoparr and myogenin expression were quantified by qRT - PCR 24 h after differentiation induction . n = 3 , mean ± SD . * p < 0 . 05 . n . s . , not significant . Statistical analyses were performed using unpaired two - tailed Student ' s t - test . In cases of unequal variances , unpaired two - tailed Welch ' s t - test was used .

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C2C12 cells were transfected with 50 nM of indicated siRNAs in growth medium. After additional 24-h incubation in growth medium, cells were transferred to differentiation medium. The levels of Myoparr and myogenin expression were quantified by qRT-PCR 24 h after differentiation induction. n = 3, mean ± SD. *p < 0.05. n.s., not significant. Statistical analyses were performed using unpaired two-tailed Student's t-test. In cases of unequal variances, unpaired two-tailed Welch's t-test was used.


Western blot showing decreased expression of myogenin in differentiating C2C12 cells 48 h after Myoparr KD . Tubulin expression served as an internal control .

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Western blot showing decreased expression of myogenin in differentiating C2C12 cells 48 h after Myoparr KD. Tubulin expression served as an internal control.


The expression levels of myogenin and Myoparr evaluated by qRT - PCR in myogenin - depleted C2C12 cells . n = 3 , mean ± SD . * * p < 0 . 01 . * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

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The expression levels of myogenin and Myoparr evaluated by qRT-PCR in myogenin-depleted C2C12 cells. n = 3, mean ± SD. **p < 0.01. ***p < 0.001 (unpaired two-tailed Student's t-test).


ChIP - qPCR detection of Pol II occupancy and histone modification status at the myogenin locus in Myoparr - depleted differentiating C2C12 cells . The data were normalized to input values . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 ( unpaired two - tailed Student ' s t - test or unpaired two - tailed Welch ' s t - test ) .

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ChIP-qPCR detection of Pol II occupancy and histone modification status at the myogenin locus in Myoparr-depleted differentiating C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05. **p < 0.01 (unpaired two-tailed Student's t-test or unpaired two-tailed Welch's t-test).


Retrieval rate of Myoparr using Myoparr - ChIRP probes from C2C12 cells quantified by qPCR is shown as percent of input values . GAPDH was amplified as a negative control . n = 3 , mean ± SD . ND , not detected .

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Retrieval rate of Myoparr using Myoparr-ChIRP probes from C2C12 cells quantified by qPCR is shown as percent of input values. GAPDH was amplified as a negative control. n = 3, mean ± SD. ND, not detected.


ChIRP - qPCR detection of the interaction between endogenous Myoparr and the myogenin promoter . The myogenin 3 ´ UTR and GAPDH promoter were amplified as negative controls . The data were normalized to input values . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 ( unpaired two - tailed Welch ' s t - test ) . ND , not detected .

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ChIRP-qPCR detection of the interaction between endogenous Myoparr and the myogenin promoter. The myogenin 3´UTR and GAPDH promoter were amplified as negative controls. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05. ** p < 0.01 (unpaired two-tailed Welch's t-test). ND, not detected.


Immunocytochemistry for the detection of myogenin at 24 h ( G ) or MHC at 72 h ( H ) in Myoparr - depleted C2C12 cells after differentiation induction . Nuclei were counterstained with DAPI . Bar , 100 μm . The percentage of myogenin - positive cells or fusion index is shown as percent of the control . n = 3 , mean ± SD . * * p < 0 . 01 . * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test or unpaired two - tailed Welch ' s t - test ) .

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Immunocytochemistry for the detection of myogenin at 24 h (G) or MHC at 72 h (H) in Myoparr-depleted C2C12 cells after differentiation induction. Nuclei were counterstained with DAPI. Bar, 100 μm. The percentage of myogenin-positive cells or fusion index is shown as percent of the control. n = 3, mean ± SD. **p < 0.01. ***p < 0.001 (unpaired two-tailed Student's t-test or unpaired two-tailed Welch's t-test).


Genes significantly differentially expressed in both Myoparr - and myogenin - depleted cells show correlated expression ( R = 0 . 63 , log 2 ratio scale ) .

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Genes significantly differentially expressed in both Myoparr- and myogenin-depleted cells show correlated expression (R = 0.63, log 2 ratio scale).


Following immunoprecipitation , the interaction between Myoparr and endogenous Ddx17 was confirmed by immunoblotting using a Ddx17 - specific antibody . Two different Ddx17 isoforms ( p72 and p82 ) were observed .

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Following immunoprecipitation, the interaction between Myoparr and endogenous Ddx17 was confirmed by immunoblotting using a Ddx17-specific antibody. Two different Ddx17 isoforms (p72 and p82) were observed.


Determination of the Ddx17 - binding region of Myoparr by RNA pull - down analyses . Schematic diagram of full - length or truncated Myoparr used for RNA pull - down ( top ) . In vitro transcribed / translated Ddx17 protein was pulled down by indicated Myoparr and then detected by western blot using a Ddx17 antibody ( bottom ) .

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Determination of the Ddx17-binding region of Myoparr by RNA pull-down analyses. Schematic diagram of full-length or truncated Myoparr used for RNA pull-down (top). In vitro transcribed/translated Ddx17 protein was pulled down by indicated Myoparr and then detected by western blot using a Ddx17 antibody (bottom).


Relative luciferase activities of indicated constructs in differentiating C2C12 cells .

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Relative luciferase activities of indicated constructs in differentiating C2C12 cells.


Relative luciferase activities of indicated constructs in differentiating C2C12 cells transfected with control or Myoparr shRNA ( Myoparr KD ) .

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Relative luciferase activities of indicated constructs in differentiating C2C12 cells transfected with control or Myoparr shRNA (Myoparr KD).


Significantly decreased MHC expression in Ddx17 - depleted C2C12 cells is shown by immunocytochemistry . Nuclei were counterstained with DAPI . Bar , 100 μm . Fusion index is shown as percent of the control . n = 3 , mean ± SD . * * p < 0 . 01 ( unpaired two - tailed Student ' s t - test ) . Immunocytochemistry for myogenin in Ddx17 - depleted C2C12 cells . Bar , 100 μm . Myogenin - positive cells are shown as percent of the control . n = 4 , mean ± SD . * * p < 0 . 01 ( unpaired two - tailed Student ' s t - test ) .

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Significantly decreased MHC expression in Ddx17-depleted C2C12 cells is shown by immunocytochemistry. Nuclei were counterstained with DAPI. Bar, 100 μm. Fusion index is shown as percent of the control. n = 3, mean ± SD. **p < 0.01 (unpaired two-tailed Student's t-test). Immunocytochemistry for myogenin in Ddx17-depleted C2C12 cells. Bar, 100 μm. Myogenin-positive cells are shown as percent of the control. n = 4, mean ± SD. **p < 0.01 (unpaired two-tailed Student's t-test).


Relative luciferase activities of the - 1650 - Luc by the combination of MyoD and Ddx17 or Ddx17 mutant ( K142R ) . Bars indicate the average of two independent experiments , and open circles represent the values of each experiment .

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Relative luciferase activities of the -1650-Luc by the combination of MyoD and Ddx17 or Ddx17 mutant (K142R). Bars indicate the average of two independent experiments, and open circles represent the values of each experiment.


Ddx17 or Ddx17 mutant ( K142R ) was pulled down by full - length Myoparr and then detected by western blot .

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Ddx17 or Ddx17 mutant (K142R) was pulled down by full-length Myoparr and then detected by western blot.


Reduced interaction between endogenous Ddx17 and PCAF in Myoparr - depleted C2C12 cells . After Myoparr KD , the cell lysates were subjected to immunoprecipitation ( IP ) with a Ddx17 - specific antibody 36 h after differentiation induction ( right panel ) . Each lysate was loaded as an input ( left panel ) . Relative quantification of ( E ) is shown as IP / input ratio . n = 3 , mean ± SD . * p < 0 . 05 . n . s . , not significant ( unpaired two - tailed Student ' s t - test ) .

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Reduced interaction between endogenous Ddx17 and PCAF in Myoparr-depleted C2C12 cells. After Myoparr KD, the cell lysates were subjected to immunoprecipitation (IP) with a Ddx17-specific antibody 36 h after differentiation induction (right panel). Each lysate was loaded as an input (left panel). Relative quantification of (E) is shown as IP/input ratio. n = 3, mean ± SD. *p < 0.05. n.s., not significant (unpaired two-tailed Student's t-test).


Increased interaction between Ddx17 and PCAF by Myoparr . PCAF protein was pulled down by 3 × Flag - Ddx17 in the presence or absence of Myoparr using a Flag antibody and then detected by western blot .

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Increased interaction between Ddx17 and PCAF by Myoparr. PCAF protein was pulled down by 3×Flag-Ddx17 in the presence or absence of Myoparr using a Flag antibody and then detected by western blot.


ChIP - qPCR detection of Ddx17 ( H ) and PCAF ( I ) occupancies at the myogenin locus in Myoparr - depleted differentiating C2C12 cells . The data were normalized to input values . n = 3 , mean ± SD . * p < 0 . 05 ( unpaired two - tailed Student ' s t - test ) .

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ChIP-qPCR detection of Ddx17 (H) and PCAF (I) occupancies at the myogenin locus in Myoparr-depleted differentiating C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05 (unpaired two-tailed Student's t-test).


Heatmap displaying expression changes of 754 genes significantly altered either in Myoparr - or Ddx17 - depleted cells ( log 2 ratio scale ) .

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Heatmap displaying expression changes of 754 genes significantly altered either in Myoparr- or Ddx17-depleted cells (log 2 ratio scale).


Genes significantly differentially expressed in both Myoparr - and Ddx17 - depleted cells show correlated expression ( R = 0 . 94 , log 2 ratio scale ) .

Genes significantly differentially expressed in both Myoparr- and Ddx17-depleted cells show correlated expression (R = 0.94, log 2 ratio scale).


Myoparr and Ddx17 are required for C2C12 cell cycle withdrawal . C2C12 cells transfected with each siRNA were cultured in growth medium for 24 h . After differentiation induction , cells were maintained in differentiation medium for 40 h and then treated with EdU for 6 h . EdU - positive cells are shown as percent of the control . Nuclei were counterstained with Hoechst 33342 . n = 3 , mean ± SD . * * p < 0 . 01 ( unpaired two - tailed Student ' s t - test ) . Bar , 100 μm .

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Myoparr and Ddx17 are required for C2C12 cell cycle withdrawal. C2C12 cells transfected with each siRNA were cultured in growth medium for 24 h. After differentiation induction, cells were maintained in differentiation medium for 40 h and then treated with EdU for 6 h. EdU-positive cells are shown as percent of the control. Nuclei were counterstained with Hoechst 33342. n = 3, mean ± SD. **p < 0.01 (unpaired two-tailed Student's t-test). Bar, 100 μm.


qRT - PCR for pri - miR - 133b , pri - miR - 206 , and H19 expression in differentiating C2C12 cells transfected with indicated siRNAs . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 .

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qRT-PCR for pri-miR-133b, pri-miR-206, and H19 expression in differentiating C2C12 cells transfected with indicated siRNAs. n = 3, mean ± SD. *p < 0.05. **p < 0.01.


ChIP - qPCR detection of Pol II occupancy at the indicated promoters in Myoparr - depleted C2C12 cells . The data were normalized to input values . n = 3 , mean ± SD . * p < 0 . 05 . Myoparr KD decreased Pol II occupancy at the miR - 133b promoter with a marginal trend toward significance ( p = 0 . 052 ) .

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ChIP-qPCR detection of Pol II occupancy at the indicated promoters in Myoparr-depleted C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05. Myoparr KD decreased Pol II occupancy at the miR-133b promoter with a marginal trend toward significance (p = 0.052).


ChIP - qPCR detection of Ddx17 ( C ) occupancies at the indicated promoters in Myoparr - depleted C2C12 cells . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 .

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ChIP-qPCR detection of Ddx17 (C) occupancies at the indicated promoters in Myoparr-depleted C2C12 cells. n = 3, mean ± SD. *p < 0.05. **p < 0.01.


ChIP - qPCR detection of PCAF ( D ) occupancies at the indicated promoters in Myoparr - depleted C2C12 cells . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 .

ChIP-qPCR detection of PCAF (D) occupancies at the indicated promoters in Myoparr-depleted C2C12 cells. n = 3, mean ± SD. *p < 0.05. **p < 0.01.


qRT - PCR showing decreased expression of miR - 133b and miR - 206 in both Myoparr - and Ddx17 - depleted C2C12 cells . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 .

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qRT-PCR showing decreased expression of miR-133b and miR-206 in both Myoparr- and Ddx17-depleted C2C12 cells. n = 3, mean ± SD. *p < 0.05. **p < 0.01.


Western blots showing increased ERK1 / 2 activity ( pERK1 / 2 ) and Cdc6 expression in differentiating C2C12 cells 48 h after Myoparr and Ddx17 KD . Tubulin expression served as an internal control .

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Western blots showing increased ERK1/2 activity (pERK1/2) and Cdc6 expression in differentiating C2C12 cells 48 h after Myoparr and Ddx17 KD. Tubulin expression served as an internal control.


Increased Pola1 expression detected by qRT - PCR . n = 3 , mean ± SD . * * p < 0 . 01 .

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Increased Pola1 expression detected by qRT-PCR. n = 3, mean ± SD. **p < 0.01.


Seven days after denervation , weights of innervated ( - ) and denervated ( + ) tibialis anterior ( TA ) muscles were measured . n = 4 , mean ± SEM . * p < 0 . 05 ( unpaired two - tailed Student ' s t - test ) .

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Seven days after denervation, weights of innervated (-) and denervated (+) tibialis anterior (TA) muscles were measured. n = 4, mean ± SEM. *p < 0.05 (unpaired two-tailed Student's t-test).


Expression of myogenin in innervated and denervated TA muscles detected by qRT - PCR 7 d after denervation . n = 4 , mean ± SD . * * * p < 0 . 001 ( unpaired two - tailed Welch ' s t - test ) . qRT - PCR showing increased Myoparr expression in denervated TA muscles 7 d after denervation . n = 3 , mean ± SD . * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

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Expression of myogenin in innervated and denervated TA muscles detected by qRT-PCR 7 d after denervation. n = 4, mean ± SD. ***p < 0.001 (unpaired two-tailed Welch's t-test). qRT-PCR showing increased Myoparr expression in denervated TA muscles 7 d after denervation. n = 3, mean ± SD. ***p < 0.001 (unpaired two-tailed Student's t-test).


Immunoblot showing decreased expression of myogenin by Myoparr depletion in denervated TA muscle 7 d after denervation . Expression of tubulin served as an internal control .

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Immunoblot showing decreased expression of myogenin by Myoparr depletion in denervated TA muscle 7 d after denervation. Expression of tubulin served as an internal control.


Weights of innervated and denervated TA muscles electroporated either with control or Myoparr shRNA . Muscle weights were measured 7 d after denervation . n = 5 for each group , mean ± SEM . * * * p < 0 . 001 . n . s . , not significant ( unpaired two - tailed Student ' s t - test ) .

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Weights of innervated and denervated TA muscles electroporated either with control or Myoparr shRNA. Muscle weights were measured 7 d after denervation. n = 5 for each group, mean ± SEM. ***p < 0.001. n.s., not significant (unpaired two-tailed Student's t-test).


Representative immunostaining images for laminin ( red ) and EmGFP ( green ) of innervated or denervated TA muscles electroporated with control or Myoparr shRNA , both containing EmGFP . Bar , 100 μm .

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Representative immunostaining images for laminin (red) and EmGFP (green) of innervated or denervated TA muscles electroporated with control or Myoparr shRNA, both containing EmGFP. Bar, 100 μm.


Cross - sectional area ( CSA ) of electroporated EmGFP - positive myofibers ( 250 fibers per sample ) was analyzed 7 d after denervation . n = 5 for each group , mean ± SEM . * * p < 0 . 01 . n . s . , not significant ( unpaired two - tailed Student ' s t - test ) .

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Cross-sectional area (CSA) of electroporated EmGFP-positive myofibers (250 fibers per sample) was analyzed 7 d after denervation. n = 5 for each group, mean ± SEM. **p < 0.01. n.s., not significant (unpaired two-tailed Student's t-test).


A Immunofluorescence staining of endothelium ( CD31 , in red ) , pericytes ( NG2 , in green ) , smooth muscle cells ( α - SMA , in cyan ) , and nuclei ( DAPI , in blue ) on frozen sections of limbmuscles injected with myoblast clones expressing different VEGF levels ( V Low , V Med , and V High , respectively ) and treated with VEGF - Trap or saline after 10 and 17 days . Analysis was performed after 4 days of treatment ( 2 and 3 weeks , respectively ) . All normal vessels induced by low and medium VEGF doses displayed a similar coverage by normal pericytes , whereas aberrant structures induced by high VEGF were covered with α - SMA - positive smooth muscle cells . Asterisks indicate muscle fibers , which were mostly sectioned in longitudinal orientation , although some cross sections are visible depending on the orientation of muscle bundles . Scale bar = 25 μm . B , C Quantification of vessel length density ( VLD ) on the same samples showed that stabilization started already by 10 days for vessels induced by low VEGF , but not until 17 days for those induced by medium VEGF , despite similar pericyte coverage , while aberrant structures induced by high VEGF always regressed . Data represent the mean ± SEM of individual images ( n ) acquired from three muscles / group ; 2 weeks ( B ) : V Low saline , n = 9 ; V Med saline , n = 13 ; V High saline , n = 8 ; V Low TRAP , n = 28 ; V Med TRAP , n = 22 ; V High TRAP , n = 30 ; 3 weeks ( C ) : V Low saline , n = 9 ; V Med saline , n = 11 ; V High saline , n = 19 ; V Low TRAP , n = 43 ; V Med TRAP , n = 14 ; V High TRAP , n = 19 ; * P < 0 . 05 and * * * P < 0 . 001 ( all versus Ctrl ) by one - way ANOVA with Bonferroni multiple comparisons test ; 2 weeks ( B ) : V Low TRAP versus Ctrl P < 0 . 0001 ; 3 weeks ( C ) : V Low TRAP versus Ctrl P < 0 . 0001 ; V Med TRAP versus Ctrl P = 0 . 0158 .

O B-EXP_ASSAY O O B-TISSUE O B-GENEPROD O O O O O B-CELL_TYPE O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O O O B-TISSUE O O B-CELL_TYPE O O O B-GENEPROD O O O O O 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 O O 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 B-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O 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 O O O B-TISSUE I-TISSUE 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 B-TISSUE O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Immunofluorescence staining of endothelium (CD31, in red), pericytes (NG2, in green), smooth muscle cells (α-SMA, in cyan), and nuclei (DAPI, in blue) on frozen sections of limbmuscles injected with myoblast clones expressing different VEGF levels (V Low, V Med, and V High, respectively) and treated with VEGF-Trap or saline after 10 and 17 days. Analysis was performed after 4 days of treatment (2 and 3 weeks, respectively). All normal vessels induced by low and medium VEGF doses displayed a similar coverage by normal pericytes, whereas aberrant structures induced by high VEGF were covered with α-SMA-positive smooth muscle cells. Asterisks indicate muscle fibers, which were mostly sectioned in longitudinal orientation, although some cross sections are visible depending on the orientation of muscle bundles. Scale bar = 25 μm.B, C Quantification of vessel length density (VLD) on the same samples showed that stabilization started already by 10 days for vessels induced by low VEGF, but not until 17 days for those induced by medium VEGF, despite similar pericyte coverage, while aberrant structures induced by high VEGF always regressed. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group; 2 weeks (B): V Low saline, n = 9; V Med saline, n = 13; V High saline, n = 8; V Low TRAP,n = 28; V Med TRAP,n = 22; V High TRAP,n = 30; 3 weeks (C): V Low saline, n = 9; V Med saline, n = 11; V High saline, n = 19; V Low TRAP,n = 43; V Med TRAP,n = 14; V High TRAP,n = 19; *P < 0.05 and ***P < 0.001 (all versus Ctrl) by one-way ANOVA with Bonferroni multiple comparisons test; 2 weeks (B): V Low TRAP versus Ctrl P < 0.0001; 3 weeks (C): V Low TRAP versus Ctrl P < 0.0001; V Med TRAP versus Ctrl P = 0.0158.


Muscles were harvested 7 days after implantation of V Low , V Med , and V High clones or control cells ( Ctrl ) . Gene expression of Pdgfb , Ang1 , Ang2 , Tgfb1 , and Sema3a was quantified by qRT - PCR and expressed as fold - change versus control muscles . Data represent individual values , with mean ± SEM ( n = 3 or 4 , as indicated ) ; * P < 0 . 05 and * * P < 0 . 01 by one - way ANOVA with Bonferroni multiple comparisons test , after data normalization by logarithmic transformation . TGF - β1 : V Low versus V Med P = 0 . 0047 ; V Low versus V High P = 0 . 0379 ; Sema3A : V Low versus V High P = 0 . 0042 ; V Med versus V High P = 0 . 0179 .

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Muscles were harvested 7 days after implantation of V Low, V Med, and V High clones or control cells (Ctrl).Gene expression of Pdgfb,Ang1,Ang2,Tgfb1, and Sema3a was quantified by qRT-PCR and expressed as fold-change versus control muscles. Data represent individual values, with mean ± SEM (n = 3 or 4, as indicated); *P < 0.05 and **P < 0.01 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation. TGF-β1: V Low versus V Med P = 0.0047; V Low versus V High P = 0.0379; Sema3A: V Low versus V High P = 0.0042; V Med versus V High P = 0.0179.


Muscles were harvested 7 days after implantation of V Low , V Med , and V High clones or control cells ( Ctrl ) . Immunohistochemistry for Sema3A on frozen muscle sections confirmed a decreasing protein expression in the areas implanted with myoblasts producing increasing VEGF doses ( n = 4 ) . The right panels represent higher - magnification views of the left panels , which encompass the majority of the implantation sites . Scale bar = 50 μm .

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Muscles were harvested 7 days after implantation of V Low, V Med, and V High clones or control cells (Ctrl). Immunohistochemistry for Sema3A on frozen muscle sections confirmed a decreasing protein expression in the areas implanted with myoblasts producing increasing VEGF doses (n = 4). The right panels represent higher-magnification views of the left panels, which encompass the majority of the implantation sites. Scale bar = 50 μm.


Muscles were harvested 7 days after implantation of V Low , V Med , and V High clones or control cells ( Ctrl ) . In situ hybridization for Sema3A ( green ) and CD31 ( red ) mRNA , with nuclei staining by DAPI , on frozen muscle sections . The three lower panels represent a higher - magnification of the area in the white square in the top panel , as a merged image and as individual channels : arrows indicate cells expressing both transcripts ( yellow ) , only Sema3A ( green ) , or only CD31 ( red ) . Scale bar = 30 μm . Quantification of the percentage of endothelial cells ( CD31 + ) that express Sema3A ( left group ) and of Sema3A - expressing cells that are endothelial ( CD31 + , right group ) . Data represent the mean ± SEM of 12 individual fields of view from three independent muscles ( 15 - 94 nuclei / image , 642 total nuclei ) . No statistics was applied , as data represent complementary sets .

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Muscles were harvested 7 days after implantation of V Low, V Med, and V High clones or control cells (Ctrl).In situ hybridization for Sema3A (green) and CD31 (red) mRNA, with nuclei staining by DAPI, on frozen muscle sections. The three lower panels represent a higher-magnification of the area in the white square in the top panel, as a merged image and as individual channels: arrows indicate cells expressing both transcripts (yellow), only Sema3A (green), or only CD31 (red). Scale bar = 30 μm.Quantification of the percentage of endothelial cells (CD31+) that express Sema3A (left group) and of Sema3A-expressing cells that are endothelial (CD31+, right group). Data represent the mean ± SEM of 12 individual fields of view from three independent muscles (15-94 nuclei/image, 642 total nuclei). No statistics was applied, as data represent complementary sets.


Immunofluorescence staining of endothelial cells ( CD31 , in red ) and NEM ( CD11b , in green ) on cryosections of limb muscles 1 week after injection with V Low , V Med , and V High myoblast clones . Scale bar = 100 μm . Quantification of the number of NEM / cm of vessel length in sites of new angiogenesis by increasing VEGF doses . Data represent the mean ± SEM of individual images ( n ) acquired from three muscles / group : V Low , n = 48 ; V Med , n = 39 ; V High , n = 94 ; * P < 0 . 05 and * * * P < 0 . 001 by one - way ANOVA with Bonferroni multiple comparisons test : V Low versus V Med P = 0 . 0008 ; V Low versus V High P < 0 . 0001 ; V Med versus V High P = 0 . 0304 .

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

Immunofluorescence staining of endothelial cells (CD31, in red) and NEM (CD11b, in green) on cryosections of limb muscles 1 week after injection with V Low, V Med, and V High myoblast clones. Scale bar = 100 μm.Quantification of the number of NEM/cm of vessel length in sites of new angiogenesis by increasing VEGF doses. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: V Low, n = 48; V Med, n = 39; V High, n = 94; *P < 0.05 and ***P < 0.001 by one-way ANOVA with Bonferroni multiple comparisons test: V Low versus V Med P = 0.0008; V Low versus V High P < 0.0001; V Med versus V High P = 0.0304.


Endothelial cells ( CD31 + quadrant ) and NEM ( CD11b + quadrant ) were sorted by FACS from muscles 1 week after injection with the same myoblast clones .

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Endothelial cells (CD31+ quadrant) and NEM (CD11b+ quadrant) were sorted by FACS from muscles 1 week after injection with the same myoblast clones.


Relative gene expression was quantified in the ex vivo FACS - purified endothelial cells ( CD31 + ) and NEM ( CD11b + ) and expressed as fold - change versus the V High group . Data represent individual values , with mean ± SEM ( n = 3 - 7 , as indicated ) ; * * P < 0 . 01 by one - way ANOVA with Bonferroni multiple comparisons test , after data normalization by logarithmic transformation ; Sema3A : V Low versus V High P = 0 . 0157 .

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Relative gene expression was quantified in the ex vivo FACS-purified endothelial cells (CD31+) and NEM (CD11b+) and expressed as fold-change versus the V High group. Data represent individual values, with mean ± SEM (n = 3-7, as indicated); **P < 0.01 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation; Sema3A: V Low versus V High P = 0.0157.


Mice , implanted with control cells ( Ctrl ) or myoblasts expressing low ( V Low ) or medium ( V Med ) VEGF levels , were treated with the anti - NRP1A antibody blocking Sema3A / NRP1 binding ( AbαNRP1A ) or with control IgG2a . Analyses were performed after 1 week . A , B Quantification of recruited NEM in sites of new angiogenesis by increasing VEGF doses , expressed as number of CD11b + cells / mm2 of area ( A ) or / cm of vessel length ( B ) . Data represent the mean ± SEM of individual images ( n ) acquired from three muscles / group . ( A ) Ctrl IgG2a , n = 11 ; Ctrl AbαNRP1A , n = 17 ; V Low IgG2a , n = 24 ; V Low AbαNRP1A , n = 28 ; V Med IgG2a , n = 25 ; V Med AbαNRP1A , n = 34 ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 by Kruskal - Wallis analysis with Dunn ' s multiple comparisons test : V Low IgG2a versus Ctrl IgG2a P = 0 . 0068 ; V Low IgG2a versus V Low AbαNRP1A P = 0 . 0068 ; V Med IgG2a versus V Med AbαNRP1A P = 0 . 0460 . ( B ) V Low IgG2a versus V Low AbαNRP1A P < 0 . 0001 ; V Low IgG2a versus V Med IgG2a P = 0 . 0193 ; V Med IgG2a versus V Med AbαNRP1A P = 0 . 0046 .

B-ORGANISM O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-CELL_TYPE 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 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 O O O O O O O B-GENEPROD 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 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 O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O 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 O O O O O B-GENEPROD O 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 B-GENEPROD O O O O O O O O O O

Mice, implanted with control cells (Ctrl) or myoblasts expressing low (V Low) or medium (V Med) VEGF levels, were treated with the anti-NRP1A antibody blocking Sema3A/NRP1 binding (AbαNRP1A) or with control IgG2a. Analyses were performed after 1 week.A, B Quantification of recruited NEM in sites of new angiogenesis by increasing VEGF doses, expressed as number of CD11b+ cells/mm2 of area (A) or /cm of vessel length (B). Data represent the mean ± SEM of individual images (n) acquired from three muscles/group. (A) Ctrl IgG2a, n = 11; Ctrl AbαNRP1A, n = 17; V Low IgG2a, n = 24; V Low AbαNRP1A, n = 28; V Med IgG2a, n = 25; V Med AbαNRP1A, n = 34; *P < 0.05, **P < 0.01 and ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: V Low IgG2a versus Ctrl IgG2a P = 0.0068; V Low IgG2a versus V Low AbαNRP1A P = 0.0068; V Med IgG2a versus V Med AbαNRP1A P = 0.0460. (B) V Low IgG2a versus V Low AbαNRP1A P < 0.0001; V Low IgG2a versus V Med IgG2a P = 0.0193; V Med IgG2a versus V Med AbαNRP1A P = 0.0046.


Mice , implanted with control cells ( Ctrl ) or myoblasts expressing low ( V Low ) or medium ( V Med ) VEGF levels , were treated with the anti - NRP1A antibody blocking Sema3A / NRP1 binding ( AbαNRP1A ) or with control IgG2a . Analyses were performed after 1 week . C Quantification of vessel length density generated by increasing VEGF doses after the different treatments . Data represent the mean ± SEM of individual images ( n ) acquired from three muscles / group : Ctrl IgG2a , n = 6 ; Ctrl AbαNRP1A , n = 6 ; V Low IgG2a , n = 19 ; V Low AbαNRP1A , n = 20 ; V Med IgG2a , n = 20 ; V Med AbαNRP1A , n = 25 ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 by Kruskal - Wallis analysis with Dunn ' s multiple comparisons test : V Low IgG2a versus Ctrl IgG2a P < 0 . 0001 ; V Low AbαNRP1A versus Ctrl IgG2a P < 0 . 0001 ; V Low IgG2a versus V Med IgG2a P = 0 . 0014 ; V Low AbαNRP1A versus V Med AbαNRP1A P = 0 . 0179 .

B-ORGANISM O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O B-TISSUE 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-TISSUE O O O O B-GENEPROD 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 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 O O O O O O O O O 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 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 O O O O O O O O O O O

Mice, implanted with control cells (Ctrl) or myoblasts expressing low (V Low) or medium (V Med) VEGF levels, were treated with the anti-NRP1A antibody blocking Sema3A/NRP1 binding (AbαNRP1A) or with control IgG2a. Analyses were performed after 1 week.C Quantification of vessel length density generated by increasing VEGF doses after the different treatments. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: Ctrl IgG2a, n = 6; Ctrl AbαNRP1A, n = 6; V Low IgG2a, n = 19; V Low AbαNRP1A, n = 20; V Med IgG2a, n = 20; V Med AbαNRP1A, n = 25; *P < 0.05, **P < 0.01 and ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: V Low IgG2a versus Ctrl IgG2a P < 0.0001; V Low AbαNRP1A versus Ctrl IgG2a P < 0.0001; V Low IgG2a versus V Med IgG2a P = 0.0014; V Low AbαNRP1A versus V Med AbαNRP1A P = 0.0179.


Mice , implanted with control cells ( Ctrl ) or myoblasts expressing low ( V Low ) or medium ( V Med ) VEGF levels , were treated with the anti - NRP1A antibody blocking Sema3A / NRP1 binding ( AbαNRP1A ) or with control IgG2a . Analyses were performed after 1 week . D Tgfb1 gene expression in total skeletal muscles after the different treatments , expressed as fold - change versus the Ctrl IgG2a group . Data represent individual values , with mean ± SEM ( n = 3 or 4 , as indicated ) ; * P < 0 . 05 and * * P < 0 . 01 by one - way ANOVA with Bonferroni multiple comparisons test , after data normalization by logarithmic transformation ; V Low IgG2a versus Ctrl IgG2a P = 0 . 0083 ; V Low IgG2a versus V Low AbαNRP1A P = 0 . 0315 .

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

Mice, implanted with control cells (Ctrl) or myoblasts expressing low (V Low) or medium (V Med) VEGF levels, were treated with the anti-NRP1A antibody blocking Sema3A/NRP1 binding (AbαNRP1A) or with control IgG2a. Analyses were performed after 1 week.D Tgfb1 gene expression in total skeletal muscles after the different treatments, expressed as fold-change versus the Ctrl IgG2a group. Data represent individual values, with mean ± SEM (n = 3 or 4, as indicated); *P < 0.05 and **P < 0.01 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation; V Low IgG2a versus Ctrl IgG2a P = 0.0083; V Low IgG2a versus V Low AbαNRP1A P = 0.0315.


Mice , implanted with control cells ( Ctrl ) or myoblasts expressing low ( V Low ) or medium ( V Med ) VEGF levels , were treated with the anti - NRP1A antibody blocking Sema3A / NRP1 binding ( AbαNRP1A ) or with control IgG2a . Analyses were performed after 1 week . E Immunohistochemistry for Sema3A protein on frozen muscle sections with H & amp ; amp ; E counterstaining . The intensity of Sema3A stain was quantified as reciprocal intensity on a scale out of 250 . Scale bar = 100 μm . Data represent the mean ± SEM of individual images ( n ) acquired from three muscles / group : No stain , n = 3 ; Ctrl IgG2a , n = 23 ; Ctrl AbαNRP1A , n = 12 ; V Low IgG2a , n = 53 ; V Low AbαNRP1A , n = 38 ; * P < 0 . 05 and * * * P < 0 . 001 by Kruskal - Wallis analysis with Dunn ' s multiple comparisons test : V Low IgG2a versus No stain P = 0 . 0007 ; V Low IgG2a versus Ctrl IgG2a P < 0 . 0001 ; V Low AbαNRP1A versus Ctrl IgG2a P = 0 . 0142 ; V Low IgG2a versus V Low AbαNRP1A P = 0 . 0001 .

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

Mice, implanted with control cells (Ctrl) or myoblasts expressing low (V Low) or medium (V Med) VEGF levels, were treated with the anti-NRP1A antibody blocking Sema3A/NRP1 binding (AbαNRP1A) or with control IgG2a. Analyses were performed after 1 week.E Immunohistochemistry for Sema3A protein on frozen muscle sections with H&amp;E counterstaining. The intensity of Sema3A stain was quantified as reciprocal intensity on a scale out of 250. Scale bar = 100 μm. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: No stain, n = 3; Ctrl IgG2a, n = 23; Ctrl AbαNRP1A, n = 12; V Low IgG2a, n = 53; V Low AbαNRP1A, n = 38; *P < 0.05 and ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: V Low IgG2a versus No stain P = 0.0007; V Low IgG2a versus Ctrl IgG2a P < 0.0001; V Low AbαNRP1A versus Ctrl IgG2a P = 0.0142; V Low IgG2a versus V Low AbαNRP1A P = 0.0001.


Relative gene expression of Id1 , specifically induced by activated SMAD1 / 5 , and Pai1 , specifically induced by activated SMAD2 / 3 , in total muscle tissues 1 week after implantation with V Low - , V Med - and V High - expressing myoblast clones or control cells ( Ctrl ) , expressed as fold - change versus the Ctrl group . Data represent individual values , with mean ± SEM ( n = 3 or 4 , as indicated ) ; * * P < 0 . 01 and * * * P < 0 . 001 by one - way ANOVA with Bonferroni multiple comparisons test , after data normalization by logarithmic transformation ; V Low versus Ctrl P = 0 . 0002 ; V Low versus V Med P = 0 . 005 ; V Low versus V High P = 0 . 0016 .

O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD 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 B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative gene expression of Id1, specifically induced by activated SMAD1/5, and Pai1, specifically induced by activated SMAD2/3, in total muscle tissues 1 week after implantation with V Low-, V Med- and V High-expressing myoblast clones or control cells (Ctrl), expressed as fold-change versus the Ctrl group. Data represent individual values, with mean ± SEM (n = 3 or 4, as indicated); **P < 0.01 and ***P < 0.001 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation; V Low versus Ctrl P = 0.0002; V Low versus V Med P = 0.005; V Low versus V High P = 0.0016.


Immunofluorescence staining of endothelium ( CD31 , in red ) , nuclei ( DAPI , in blue ) , and phosphorylated SMAD2 / 3 ( in cyan ) on cryosections of similarly injected muscles confirmed that endothelial SMAD2 / 3 activation was inhibited by increasing VEGF doses . Scale bar = 10 μm ; n = 3 .

B-EXP_ASSAY O O B-TISSUE O B-GENEPROD O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O 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 O

Immunofluorescence staining of endothelium (CD31, in red), nuclei (DAPI, in blue), and phosphorylated SMAD2/3 (in cyan) on cryosections of similarly injected muscles confirmed that endothelial SMAD2/3 activation was inhibited by increasing VEGF doses. Scale bar = 10 μm; n = 3.


A - C Endothelial Sema3A expression is directly inhibited by VEGF and stimulated by TGF - β1 in vitro . Mouse aortic endothelial cells were stimulated with increasing doses of recombinant VEGF164 or TGF - β1 for 24 h . The expression of Sema3a and Tgfb1 was quantified by qRT - PCR and expressed as fold - change versus the non - stimulated cells . VEGF dose dependently inhibited Sema3a expression ( A ) , but did not affect that of Tgfb1 ( B ) , while TGF - β1 upregulated Sema3a expression in a dose - dependent fashion ( C ) . Data represent individual values , with mean ± SEM ( n = 4 ) ; * * P < 0 . 01 and * * * P < 0 . 001 by one - way ANOVA with Bonferroni multiple comparisons test , after data normalization by logarithmic transformation ; Sema3A expression upon VEGF stimulation : 40 versus 0 P = 0 . 0041 ; TGF - β1 upon VEGF stimulation : no significant differences ; Sema3A expression upon TGF - β1 stimulation : 1 versus 0 P < 0 . 0001 ; 2 . 5 versus 0 P < 0 . 0001 ; 40 versus 0 P < 0 . 0001 .

O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD 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 B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD 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 O O O

A-C Endothelial Sema3A expression is directly inhibited by VEGF and stimulated by TGF-β1 in vitro. Mouse aortic endothelial cells were stimulated with increasing doses of recombinant VEGF164 or TGF-β1 for 24 h. The expression of Sema3a and Tgfb1 was quantified by qRT-PCR and expressed as fold-change versus the non-stimulated cells. VEGF dose dependently inhibited Sema3a expression (A), but did not affect that of Tgfb1 (B), while TGF-β1 upregulated Sema3a expression in a dose-dependent fashion (C). Data represent individual values, with mean ± SEM (n = 4); **P < 0.01 and ***P < 0.001 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation; Sema3A expression upon VEGF stimulation: 40 versus 0 P = 0.0041; TGF-β1 upon VEGF stimulation: no significant differences; Sema3A expression upon TGF-β1 stimulation: 1 versus 0 P < 0.0001; 2.5 versus 0 P < 0.0001; 40 versus 0 P < 0.0001.


D , E Inhibition of TGF - β1 abolishes Sema3A production and NEM recruitment in vivo . Mice , implanted with control cells ( Ctrl ) or myoblasts expressing low ( V Low ) VEGF levels , were treated with a TGF - β1 blocking antibody . Analyses were preformed after 1 week . ( D ) Immunohistochemistry for Sema3A protein on frozen muscle sections with H & amp ; amp ; E counterstaining . The intensity of Sema3A stain was quantified as reciprocal intensity on a scale out of 250 . Scale bar = 50 μm . Data represent the mean ± SEM of individual images ( n ) acquired from three muscles / group : No stain , n = 10 ; Ctrl IgG1 , n = 17 ; Ctrl AbαTGF - β1 , n = 16 ; V Low IgG1 , n = 23 ; V Low AbαTGF - β1 , n = 28 . * * P < 0 . 01 and * * * P < 0 . 001 by Kruskal - Wallis analysis with Dunn ' s multiple comparisons test : V Low IgG1 versus No stain P = 0 . 0069 ; V Low IgG1 versus Ctrl IgG1 P < 0 . 0001 ; V Low AbαTGF - β1 versus V Low IgG1 P = 0 . 0002 .

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

D, E Inhibition of TGF-β1 abolishes Sema3A production and NEM recruitment in vivo. Mice, implanted with control cells (Ctrl) or myoblasts expressing low (V Low) VEGF levels, were treated with a TGF-β1 blocking antibody. Analyses were preformed after 1 week. (D) Immunohistochemistry for Sema3A protein on frozen muscle sections with H&amp;E counterstaining. The intensity of Sema3A stain was quantified as reciprocal intensity on a scale out of 250. Scale bar = 50 μm. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: No stain, n = 10; Ctrl IgG1, n = 17; Ctrl AbαTGF-β1, n = 16; V Low IgG1, n = 23; V Low AbαTGF-β1, n = 28. **P < 0.01 and ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: V Low IgG1 versus No stain P = 0.0069; V Low IgG1 versus Ctrl IgG1 P < 0.0001; V Low AbαTGF-β1 versus V Low IgG1 P = 0.0002.


D , E Inhibition of TGF - β1 abolishes Sema3A production and NEM recruitment in vivo . Mice , implanted with control cells ( Ctrl ) or myoblasts expressing low ( V Low ) VEGF levels , were treated with a TGF - β1 blocking antibody . Analyses were preformed after 1 week . ( E ) Quantification of recruited NEM ( number of CD11b + cells / mm2 of area ) . Scale bar = 25 μm . Data represent the mean ± SEM of individual images ( n ) acquired from three muscles / group : Ctrl IgG1 , n = 20 ; Ctrl AbαTGF - β1 , n = 19 ; V Low IgG1 , n = 20 ; V Low AbαTGF - β1 , n = 27 ; * * * P < 0 . 001 by Kruskal - Wallis analysis with Dunn ' s multiple comparisons test : V Low IgG1 versus Ctrl IgG1 P = 0 . 0005 ; V Low AbαTGF - β1 versus V Low IgG1 P < 0 . 0001 .

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

D, E Inhibition of TGF-β1 abolishes Sema3A production and NEM recruitment in vivo. Mice, implanted with control cells (Ctrl) or myoblasts expressing low (V Low) VEGF levels, were treated with a TGF-β1 blocking antibody. Analyses were preformed after 1 week. (E) Quantification of recruited NEM (number of CD11b+ cells/mm2 of area). Scale bar = 25 μm. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: Ctrl IgG1, n = 20; Ctrl AbαTGF-β1, n = 19; V Low IgG1, n = 20; V Low AbαTGF-β1, n = 27; ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: V Low IgG1 versus Ctrl IgG1 P = 0.0005; V Low AbαTGF-β1 versus V Low IgG1 P < 0.0001.


A - C Mice , implanted with myoblasts expressing low ( V Low ) or medium ( V Med ) VEGF levels , were treated on days 4 and 6 after cell implantation with intramuscular injections of Sema3A - Fc ( 0 . 1 or 10 mg / kg of average muscle tissue weight ) , or with control Fc protein . ( A ) Quantification of the number of NEM / cm of vessel length in sites of new angiogenesis after 1 week . Data represent the mean ± SEM of individual images ( n ) acquired from 3 to 5 muscles / group : V Low Fc , n = 21 ; V Low 0 . 1 mg / kg , n = 22 ; V Low 10 mg / kg , n = 28 ; V Med Fc , n = 20 ; V Med 0 . 1 mg / kg , n = 30 ; V Med 10 mg / kg , n = 25 ; * * P < 0 . 01 and * * * P < 0 . 001 by Kruskal - Wallis analysis with Dunn ' s multiple comparisons test : V Low Fc versus V Low 10 mg / kg P = 0 . 0027 ; V Med Fc versus V Med 0 . 1 mg / kg P = 0 . 001 ; V Med Fc versus V Med 10 mg / kg P < 0 . 0001 . ( B ) Quantification of vessel length density ( VLD ) in treated muscles after 1 week . Data represent the mean ± SEM of individual images ( n ) acquired from 3 to 4 muscles / group : V Low Fc , n = 8 ; V Low 0 . 1 mg / kg , n = 20 ; V Low 10 mg / kg , n = 18 ; V Med Fc , n = 8 ; V Med 0 . 1 mg / kg , n = 23 ; and V Med 10 mg / kg , n = 27 . Data were subjected to Kruskal - Wallis analysis with Dunn ' s multiple comparisons test , and no significant differences were detected . ( C ) Vascular stabilization rate was determined 2 weeks after cell implantation by measuring vessel length density after abrogation of VEGF signaling by VEGF - Trap . Treatment with 10 mg / kg Sema3A - Fc did not affect the stabilization of angiogenesis induced by low VEGF , but significantly increased the resistant fraction of vessels induced by medium VEGF . Data represent the mean ± SEM of individual images ( n ) acquired from 3 to 4 muscles / group : V Low Fc , n = 32 ; V Low Sema3A , n = 14 ; V Med Fc , n = 28 ; V Med Sema3A , n = 33 ; * P < 0 . 05 by one - way ANOVA with Bonferroni multiple comparisons test : V Med Fc versus V Med Sema3A P = 0 . 0458 .

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

A-C Mice, implanted with myoblasts expressing low (V Low) or medium (V Med) VEGF levels, were treated on days 4 and 6 after cell implantation with intramuscular injections of Sema3A-Fc (0.1 or 10 mg/kg of average muscle tissue weight), or with control Fc protein. (A) Quantification of the number of NEM/cm of vessel length in sites of new angiogenesis after 1 week. Data represent the mean ± SEM of individual images (n) acquired from 3 to 5 muscles/group: V Low Fc, n = 21; V Low 0.1 mg/kg, n = 22; V Low 10 mg/kg, n = 28; V Med Fc, n = 20; V Med 0.1 mg/kg, n = 30; V Med 10 mg/kg, n = 25; **P < 0.01 and ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: V Low Fc versus V Low 10 mg/kg P = 0.0027; V Med Fc versus V Med 0.1 mg/kg P = 0.001; V Med Fc versus V Med 10 mg/kg P < 0.0001. (B) Quantification of vessel length density (VLD) in treated muscles after 1 week. Data represent the mean ± SEM of individual images (n) acquired from 3 to 4 muscles/group: V Low Fc, n = 8; V Low 0.1 mg/kg, n = 20; V Low 10 mg/kg, n = 18; V Med Fc, n = 8; V Med 0.1 mg/kg, n = 23; and V Med 10 mg/kg, n = 27. Data were subjected to Kruskal-Wallis analysis with Dunn's multiple comparisons test, and no significant differences were detected. (C) Vascular stabilization rate was determined 2 weeks after cell implantation by measuring vessel length density after abrogation of VEGF signaling by VEGF-Trap. Treatment with 10 mg/kg Sema3A-Fc did not affect the stabilization of angiogenesis induced by low VEGF, but significantly increased the resistant fraction of vessels induced by medium VEGF. Data represent the mean ± SEM of individual images (n) acquired from 3 to 4 muscles/group: V Low Fc, n = 32; V Low Sema3A, n = 14; V Med Fc, n = 28; V Med Sema3A, n = 33; *P < 0.05 by one-way ANOVA with Bonferroni multiple comparisons test: V Med Fc versus V Med Sema3A P = 0.0458.


D - F Mice received intramuscular injections of adenovirus expressing VEGF ( AdVIC ) or CD8 control ( AdCD8 ) and were treated on days 4 and 6 after vector delivery with intramuscular injections of Sema3A - Fc ( 10 mg / kg of average muscle tissue weight ) or control Fc protein . ( D ) Immunofluorescence staining of endothelium ( CD31 , in red ) , pericytes ( NG2 , in green ) , smooth muscle cells ( α - SMA , in cyan ) , and nuclei ( DAPI , in blue ) , showing vessel density and morphology 1 week after injection of adenoviral vectors alone ( no treatment ) or after 3 weeks and with Fc or Sema3A - Fc treatment . Scale bar = 50 μm . ( E ) Quantification of vessel length density ( VLD ) on the same samples shows that treatment with Sema3A - Fc accelerated stabilization of vessels induced by transient and uncontrolled VEGF expression . Data represent the mean ± SEM of individual images ( n ) acquired from 3 to 4 muscles / group : Uninjected muscles , n = 72 ; AdCD8 1 week , n = 68 ; AdVIC 1 week , n = 53 ; AdVIC + Fc 3 weeks , n = 78 ; AdVIC + Sema3AFc 3 weeks , n = 91 ; * * P < 0 . 01 and * * * P < 0 . 001 by Kruskal - Wallis analysis with Dunn ' s multiple comparisons test : AdCD8 1 week versus AdVIC + Fc 3 weeks P = 0 . 0037 ; all other comparisons indicated P < 0 . 0001 .

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

D-F Mice received intramuscular injections of adenovirus expressing VEGF (AdVIC) or CD8 control (AdCD8) and were treated on days 4 and 6 after vector delivery with intramuscular injections of Sema3A-Fc (10 mg/kg of average muscle tissue weight) or control Fc protein. (D) Immunofluorescence staining of endothelium (CD31, in red), pericytes (NG2, in green), smooth muscle cells (α-SMA, in cyan), and nuclei (DAPI, in blue), showing vessel density and morphology 1 week after injection of adenoviral vectors alone (no treatment) or after 3 weeks and with Fc or Sema3A-Fc treatment. Scale bar = 50 μm. (E) Quantification of vessel length density (VLD) on the same samples shows that treatment with Sema3A-Fc accelerated stabilization of vessels induced by transient and uncontrolled VEGF expression. Data represent the mean ± SEM of individual images (n) acquired from 3 to 4 muscles/group: Uninjected muscles, n = 72; AdCD8 1 week, n = 68; AdVIC 1 week, n = 53; AdVIC+Fc 3 weeks, n = 78; AdVIC+Sema3AFc 3 weeks, n = 91; **P < 0.01 and ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: AdCD8 1 week versus AdVIC+Fc 3 weeks P = 0.0037; all other comparisons indicated P < 0.0001.


D - F Mice received intramuscular injections of adenovirus expressing VEGF ( AdVIC ) or CD8 control ( AdCD8 ) and were treated on days 4 and 6 after vector delivery with intramuscular injections of Sema3A - Fc ( 10 mg / kg of average muscle tissue weight ) or control Fc protein . ( F ) Immunofluorescence staining of endothelial cells ( CD31 , in red ) and NEM ( CD11b , in green ) on frozen sections of muscles 3 weeks after injection of adenoviral vectors and with Fc or Sema3A - Fc treatment . White arrows indicate NEM . Scale bar = 50 μm .

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

D-F Mice received intramuscular injections of adenovirus expressing VEGF (AdVIC) or CD8 control (AdCD8) and were treated on days 4 and 6 after vector delivery with intramuscular injections of Sema3A-Fc (10 mg/kg of average muscle tissue weight) or control Fc protein. (F) Immunofluorescence staining of endothelial cells (CD31, in red) and NEM (CD11b, in green) on frozen sections of muscles 3 weeks after injection of adenoviral vectors and with Fc or Sema3A-Fc treatment. White arrows indicate NEM. Scale bar = 50 μm.


A , Representative images of controls from the siRNA screen for HU - induced RPA accumulation . GFP - RPA1 and RFP - PCNA reporter cells were transfected with siRNAs and 48 hrs later treated with HU ( 3 mM ) for 2 hrs prior to pre - extraction , fixation and imaging .

O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD 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 O O O O O O O

A, Representative images of controls from the siRNA screen for HU-induced RPA accumulation. GFP-RPA1 and RFP-PCNA reporter cells were transfected with siRNAs and 48 hrs later treated with HU (3 mM) for 2 hrs prior to pre-extraction, fixation and imaging.