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 .

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

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 ) .

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

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 .

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

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 ) .

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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 .

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

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 ) .

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

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 .

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

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 .

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

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 ) .

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

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 ) .

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

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

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

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 .

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

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 .

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

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 .

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

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

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

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

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

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

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

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

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

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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 .

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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 .

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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.


B , Gene ranking based on siRNA scores from two independent screens . 219 genes were targeted by three individual siRNAs and ranked based on P - values derived from RSA and Fisher ' s analysis . Selected hits are highlighted .

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B, Gene ranking based on siRNA scores from two independent screens. 219 genes were targeted by three individual siRNAs and ranked based on P-values derived from RSA and Fisher's analysis. Selected hits are highlighted.


C , Validation of selected genes by single cell analysis of GFP - RPA1 intensity in pre - extracted cells . Only S - phase cells positive for RFP - PCNA were analysed . a , b , c denote independent siRNAs ; Ctrl , Control . Median with interquartile range of relative intensity per cell is shown , n > 4000 . Mann - Whitney : * * * p < 10 - 4 , n . s . non - significant . One representative experiment out of two biological replicas is shown .

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C, Validation of selected genes by single cell analysis of GFP-RPA1 intensity in pre-extracted cells. Only S-phase cells positive for RFP-PCNA were analysed. a, b, c denote independent siRNAs; Ctrl, Control. Median with interquartile range of relative intensity per cell is shown, n > 4000. Mann-Whitney: *** p < 10-4, n.s. non-significant. One representative experiment out of two biological replicas is shown.


D , Analysis of DNA synthesis rate . Cells were pulsed 15 min with EdU and EdU intensity was analysed in S - phase cells positive for RFP - PCNA . EdU incorporation is shown relative to siCtrl .

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D, Analysis of DNA synthesis rate. Cells were pulsed 15 min with EdU and EdU intensity was analysed in S-phase cells positive for RFP-PCNA. EdU incorporation is shown relative to siCtrl.


A , DNA replication measured by EdU incorporation . RPF - PCNA reporter cells were transfected with indicated siRNAs and pulsed with EdU for 20 min . S - phase cells positive for RFP - PCNA were analysed . n > 150 . Error bars , SD ; n = 3 biological replicas .

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A, DNA replication measured by EdU incorporation. RPF-PCNA reporter cells were transfected with indicated siRNAs and pulsed with EdU for 20 min. S-phase cells positive for RFP-PCNA were analysed. n > 150. Error bars, SD; n= 3 biological replicas.


B , Complementation analysis of DNA synthesis . Stable cell lines expressing BRPF3 resistant to the BRPF3 / a siRNA and control ( lacZ - V5 ) were siRNA transfected and analysed for EdU incorporation as in A . Error - bars , SD ; n = 6 biological replicas . Two - tailed t test , * * p < 10 - 2 .

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B, Complementation analysis of DNA synthesis. Stable cell lines expressing BRPF3 resistant to the BRPF3/a siRNA and control (lacZ-V5) were siRNA transfected and analysed for EdU incorporation as in A. Error-bars, SD; n = 6 biological replicas. Two-tailed t test, ** p<10-2.


C , Comparison of native BRPF1 and BRPF3 complexes purified from K562 cells . ( left ) mass - spec analysis ( spectral counts / total peptides identified ) and ( right ) verification of associated protein by western blot .

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C, Comparison of native BRPF1 and BRPF3 complexes purified from K562 cells. (left) mass-spec analysis (spectral counts/total peptides identified) and (right) verification of associated protein by western blot.


D , Immunoprecipitation analysis of BRPF3 deletion mutants . One representative experiment out of two is shown .

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D, Immunoprecipitation analysis of BRPF3 deletion mutants. One representative experiment out of two is shown.


E , Histone acetyltransferase assay ( HAT ) with purified BRPF1 / 3 complexes on free histones and mononucleosomes .

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E, Histone acetyltransferase assay (HAT) with purified BRPF1/3 complexes on free histones and mononucleosomes.


F , HAT assay with purified BRPF1 / 3 complexes on H3 peptides unmodified or acetylated ( ac ) / methylated ( me ) at K14 and K27 , respectively .

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F, HAT assay with purified BRPF1/3 complexes on H3 peptides unmodified or acetylated (ac)/methylated (me) at K14 and K27, respectively.


G , Analysis of histone acetylation in BRPF3 depleted cells . ( left ) Western blot of siRNA treated U - 2 - OS cells . TSA treatment ( 1 hr ) was included as a positive control . 2x , double amount of extract loaded in 1x . ( right ) H3K14 acetylation levels quantified relative to total H3 . Error bars , SD ; n = 3 biological replicates .

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G, Analysis of histone acetylation in BRPF3 depleted cells. (left) Western blot of siRNA treated U-2-OS cells. TSA treatment (1 hr) was included as a positive control. 2x, double amount of extract loaded in 1x. (right) H3K14 acetylation levels quantified relative to total H3. Error bars, SD; n = 3 biological replicates.


H , Complementation analysis of H3K14ac . Total cell extracts of siRNA treated lacZ - V5 and BRPF3 - V5 ( siBRPF3 / a resistant ) expressing cells were analysed by western blotting . One representative experiment out of four biological replicas is shown .

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H, Complementation analysis of H3K14ac. Total cell extracts of siRNA treated lacZ-V5 and BRPF3-V5 (siBRPF3/a resistant) expressing cells were analysed by western blotting. One representative experiment out of four biological replicas is shown.


A - B , Single - molecule analysis of DNA replication by DNA combing of siRNA transfected cells pulse - labelled with IdU ( 10 min ) and CIdU ( 20 min ) . ( A ) Distribution of inter - track distances . Bars represent the median . n > 100 . Mann - Whitney : * * * P < 10 - 3 . One representative experiment out of two biological replicas is shown . ( B ) Size distribution of CldU track length . Bars represent the median . n > 300 . Mann - Whitney : * * * P < 10 - 3 . One representative experiment out of two biological replicas is shown . C , Distribution of intertrack distances measured by IdU / CIdU pulse labelling and DNA fiber analysis . Bars represent the median . n > 110 . Mann - Whitney : * * * * P < 10 - 4 . One representative experiment out of two biological replicas is shown .

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A-B, Single-molecule analysis of DNA replication by DNA combing of siRNA transfected cells pulse-labelled with IdU (10 min) and CIdU (20 min). (A) Distribution of inter-track distances. Bars represent the median. n > 100. Mann-Whitney: *** P < 10-3. One representative experiment out of two biological replicas is shown. (B) Size distribution of CldU track length. Bars represent the median. n > 300. Mann-Whitney: *** P < 10-3. One representative experiment out of two biological replicas is shown. C, Distribution of intertrack distances measured by IdU/CIdU pulse labelling and DNA fiber analysis. Bars represent the median. n > 110. Mann-Whitney: **** P < 10-4. One representative experiment out of two biological replicas is shown.


D , Scatter plot of chromatin - bound GFP - RPA1 intensity per cell . GFP - RPA1 and RFP - PCNA reporter cells were transfected with indicated siRNAs and treated 2 hrs with UCN - 01 ( 300 nM ) prior to pre - extraction and fixation . GFP - RPA1 levels were measured in PCNA positive cells . Lines represent medians . n > 150 . t - tests : * * * * P < 10 - 4 , * P < 0 . 05 ; a . u . , arbitrary unit .

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D, Scatter plot of chromatin-bound GFP-RPA1 intensity per cell. GFP-RPA1 and RFP-PCNA reporter cells were transfected with indicated siRNAs and treated 2 hrs with UCN-01 (300 nM) prior to pre-extraction and fixation. GFP-RPA1 levels were measured in PCNA positive cells. Lines represent medians. n > 150. t-tests: **** P < 10-4, * P < 0.05; a.u., arbitrary unit.


E , Analysis of replication factor loading . Soluble proteins from siRNA transfected cells treated as in D where removed by CSK - T extraction and chromatin pellets analysed by western blotting . One representative experiment out of three independent biological replicas is shown . F , Quantification of chromatin - bound MCM2 ( left ) and CDC45 ( right ) from the three independent experiments described in ( E ) . MCM2 and CDC45 levels were normalized to histone H4 levels and shown relative to untreated control . t - tests : * * * P < 10 - 3 , * * P < 0 . 01 , * P < 0 . 05 , n . s . non - significant .

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E, Analysis of replication factor loading. Soluble proteins from siRNA transfected cells treated as in D where removed by CSK-T extraction and chromatin pellets analysed by western blotting. One representative experiment out of three independent biological replicas is shown. F, Quantification of chromatin-bound MCM2 (left) and CDC45 (right) from the three independent experiments described in (E). MCM2 and CDC45 levels were normalized to histone H4 levels and shown relative to untreated control. t-tests: *** P < 10-3, ** P < 0.01, * P < 0.05, n.s. non-significant.


G , Analysis of chromatin - bound proteins in HBO1 depleted cells as in E . One representative experiment out of two biological replicas is shown .

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G, Analysis of chromatin-bound proteins in HBO1 depleted cells as in E. One representative experiment out of two biological replicas is shown.


A , ChIP - seq analysis of BRPF3 binding on the human genome . BRPF3 enrichment signal obtained by ChIP - seq with asynchronous ( AS ) , synchronized ( S ) and released into HU ( S + HU ) RKO cells , on the MCM4 ( left ) and Lamin B2 ( right ) replication origins . Signals were compared to the published enrichment signals for ORC1 in HeLa cells ( ( Dellino et al , 2013 ) , GSE37583 ) , HBO1 in RKO cells ( Avvakumov et al . , 2012 , GSE33221 ) , and H3K14ac in IMR90 cells ( GSM521881 ) . The negative control IgG signal shown is from the S + HUchromatin .

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A, ChIP-seq analysis of BRPF3 binding on the human genome. BRPF3 enrichment signal obtained by ChIP-seq with asynchronous (AS), synchronized (S) and released into HU (S+HU) RKO cells, on the MCM4 (left) and Lamin B2 (right) replication origins. Signals were compared to the published enrichment signals for ORC1 in HeLa cells ((Dellino et al, 2013), GSE37583), HBO1 in RKO cells (Avvakumov et al., 2012, GSE33221), and H3K14ac in IMR90 cells (GSM521881). The negative control IgG signal shown is from the S+HUchromatin.


B , Venn diagrams illustrating the overlap of genome binding sites between ( top ) BRPF3 , HBO1 and ORC1 , and ( bottom ) BRPF3 , BRPF1 / 2 and ORC1 in human cells .

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B, Venn diagrams illustrating the overlap of genome binding sites between (top) BRPF3, HBO1 and ORC1, and (bottom) BRPF3, BRPF1/2 and ORC1 in human cells.


C , Heatmaps of BRPF3 , HBO1 and H3K14ac ChIP - seq signal on ± 5kb surrounding the TSS of genes . Genes were sorted by BRPF3 abundance from high to low . A . U . arbitrary unit .

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C, Heatmaps of BRPF3, HBO1 and H3K14ac ChIP-seq signal on ± 5kb surrounding the TSS of genes. Genes were sorted by BRPF3 abundance from high to low. A.U. arbitrary unit.


D , Average profiles of BRPF3 and H3K14ac ChIP - seq signal on ± 2 . 5kb around the centre of ( top ) BRPF3 and ( bottom ) ORC1 genome binding sites .

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D, Average profiles of BRPF3 and H3K14ac ChIP-seq signal on ± 2.5kb around the centre of (top) BRPF3 and (bottom) ORC1 genome binding sites.


E , ChIP - qPCR analysis of H3K14ac on well - characterized replication origins . H3K14ac enrichments measured relative to IgG control are shown relative to control siRNA treated cells . Error bars , range ; n = 2 biological replicas .

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E, ChIP-qPCR analysis of H3K14ac on well-characterized replication origins. H3K14ac enrichments measured relative to IgG control are shown relative to control siRNA treated cells. Error bars, range; n= 2 biological replicas.


F , Expression levels of DNA replication factors were not altered by the lack of BRPF3 . Each point represents the expression levels of a gene in control and BRPF3 depleted cells ( GSE65065 ) . Replication factors were classified as described previously ( Alabert et al , 2014 ) . Total genes ( grey ) , DNA replication factors ( colors ) and BRPF3 ( black ) expression levels are shown . Continuous black lines mark 1 . 5 fold change in expression levels .

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F, Expression levels of DNA replication factors were not altered by the lack of BRPF3. Each point represents the expression levels of a gene in control and BRPF3 depleted cells (GSE65065). Replication factors were classified as described previously (Alabert et al, 2014). Total genes (grey), DNA replication factors (colors) and BRPF3 (black) expression levels are shown. Continuous black lines mark 1.5 fold change in expression levels.


A , Western blot of DNA damage signalling in BRPF3 depleted cells . siRNA transfected U - 2 - OS cells were treated with HU for 2 h or 24 h . One representative experiment out of two biological experiments is shown .

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A, Western blot of DNA damage signalling in BRPF3 depleted cells. siRNA transfected U-2-OS cells were treated with HU for 2 h or 24 h. One representative experiment out of two biological experiments is shown.


B - C , Recovery from replication stress . siRNA treated cells were treated for 24 hrs with HU ( 0 h ) , and released into normal medium for the indicated times ( see also Fig . S6A , B ) . ( B ) DNA replication rate measured by EdU incorporation . Cells were pulsed with EdU for 15 min and gated as shown in Figure S6A to analyse EdU positive cells . n > 1000 . Mann - Whitney : * * * * P < 10 - 4 . ( C ) Cell cycle progression measured by FACS analysis of DNA content . One representative experiment out of two independent biological replicas is shown .

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B-C, Recovery from replication stress. siRNA treated cells were treated for 24 hrs with HU (0 h), and released into normal medium for the indicated times (see also Fig. S6A, B). (B) DNA replication rate measured by EdU incorporation. Cells were pulsed with EdU for 15 min and gated as shown in Figure S6A to analyse EdU positive cells. n > 1000. Mann-Whitney: **** P < 10-4. (C) Cell cycle progression measured by FACS analysis of DNA content. One representative experiment out of two independent biological replicas is shown.


A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated . ( A ) Cells were incubated for 2 hours in the presence or absence of 50 nM of lysosomal inhibitor chloroquine ( CQ ) . Cell lysates were evaluated by immunoblotting .

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A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated. (A) Cells were incubated for 2 hours in the presence or absence of 50 nM of lysosomal inhibitor chloroquine (CQ). Cell lysates were evaluated by immunoblotting.


A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated . ( B ) Cells were infected , for 1 and 4 hours , with K . pneumonia at MOI of 10 followed by 1 hour of gentamycin treatment . Cell lysates were plated in LB agar for 18 hours and CFU were determined . Data information : the triangles and dots represent the individual test ( three technical replicates per individual test ) on control and Atg7 knock down cells . The bars represent the mean CFU of each group ( three biological replicates each group ) , error bars represent standard deviation . Statistical significance between assays were calculated using two - tailed student ' s t test with Welch ' s correction . NS : not significant ; * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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

A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated. (B) Cells were infected, for 1 and 4 hours, with K. pneumonia at MOI of 10 followed by 1 hour of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined. Data information: the triangles and dots represent the individual test (three technical replicates per individual test) on control and Atg7 knock down cells. The bars represent the mean CFU of each group (three biological replicates each group), error bars represent standard deviation. Statistical significance between assays were calculated using two-tailed student's t test with Welch's correction. NS: not significant; *p ≤ 0.05; **p ≤ 0.01.


A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated . ( D - E ) Cells were infected , for 1 - 4 hours , with WT ( D ) or ΔpscD P . aeruginosa ( E ) at MOI of 10 . All infections were followed by 1 hour of gentamycin treatment . Cell lysates were plated in LB agar for 18 hours and CFU were determined . Data information : , the triangles and dots represent the individual test ( three technical replicates per individual test ) on control and Atg7 knock down cells . The bars represent the mean CFU of each group ( three biological replicates each group ) , error bars represent standard deviation . Statistical significance between assays were calculated using two - tailed student ' s t test with Welch ' s correction . NS : not significant ; * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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

A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated. (D-E) Cells were infected, for 1-4 hours, with WT (D) or ΔpscD P. aeruginosa (E) at MOI of 10. All infections were followed by 1 hour of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined. Data information: , the triangles and dots represent the individual test (three technical replicates per individual test) on control and Atg7 knock down cells. The bars represent the mean CFU of each group (three biological replicates each group), error bars represent standard deviation. Statistical significance between assays were calculated using two-tailed student's t test with Welch's correction. NS: not significant; *p ≤ 0.05; **p ≤ 0.01.


( A ) A549 cells were infected with WT P . aeruginosa for various time points and cell lysates were evaluated by immunoblotting .

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(A) A549 cells were infected with WT P. aeruginosa for various time points and cell lysates were evaluated by immunoblotting.


( B ) A549 cells infected with WT or ΔpscD P . aeruginosa for 4 hours or left uninfected ( NI ) in the presence of lysosomal inhibitor CQ ( 50 nM ) . The cell lysates were evaluated by immunoblotting .

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(B) A549 cells infected with WT or ΔpscD P. aeruginosa for 4 hours or left uninfected (NI) in the presence of lysosomal inhibitor CQ (50 nM). The cell lysates were evaluated by immunoblotting.


( C ) A549 cells stably expressing GFP - LC3 were left non - infected or infected , for 4 hours , with WT or ΔpscD P . aeruginosa , and evaluated by fluorescence microscopy . Cell nuclei were counterstained with DAPI ( blue ) and autophagosome were visualized as green puncta . Graph represents quantitative analysis of GFP - LC3 positive autophagosome per cell , scale bars : 10 µm . Each bar represents mean value of three independent experiments , error bars represent standard deviation . Statistical significance between assays was determined using One - way ANOVA followed by Dunn ' s Multiple Comparison Test . * * p ≤ 0 . 01 .

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(C) A549 cells stably expressing GFP-LC3 were left non-infected or infected, for 4 hours, with WT or ΔpscD P. aeruginosa, and evaluated by fluorescence microscopy. Cell nuclei were counterstained with DAPI (blue) and autophagosome were visualized as green puncta. Graph represents quantitative analysis of GFP-LC3 positive autophagosome per cell, scale bars: 10 µm. Each bar represents mean value of three independent experiments, error bars represent standard deviation. Statistical significance between assays was determined using One-way ANOVA followed by Dunn's Multiple Comparison Test. **p ≤ 0.01.


( D - E ) A549 cells were treated for 16 hours with rapamycin 0 . 8 μg / ml or DMSO , then infected with either WT ( D ) or ΔpscD P . aeruginosa and followed by 1 hour of gentamycin treatment ( E ) . The circles and squares represent the individual test ( three technical replicates per individual test ) with DMSO and rapamycin treatment . The bars represent the means of CFU from three independent experiments , error bars represent standard deviation . The significance of differences between different drug treatment was determined using two - tailed student ' s t test with Welch ' s correction . NS : not significant ; * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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

(D-E) A549 cells were treated for 16 hours with rapamycin 0.8 μg/ml or DMSO, then infected with either WT (D) or ΔpscD P. aeruginosa and followed by 1 hour of gentamycin treatment (E). The circles and squares represent the individual test (three technical replicates per individual test) with DMSO and rapamycin treatment. The bars represent the means of CFU from three independent experiments, error bars represent standard deviation. The significance of differences between different drug treatment was determined using two-tailed student's t test with Welch's correction. NS: not significant; *p ≤ 0.05; **p ≤ 0.01.


A549 cells were infected with one of several P . aeruginosa mutants . Cell lysates were evaluated by immunoblotting .

A549 cells were infected with one of several P. aeruginosa mutants. Cell lysates were evaluated by immunoblotting.


( B ) primary human bronchial epithelial cells were infected with one of several P . aeruginosa mutants . Cell lysates were evaluated by immunoblotting .

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(B) primary human bronchial epithelial cells were infected with one of several P. aeruginosa mutants. Cell lysates were evaluated by immunoblotting.


( C ) A549 cells , stably expressing GFP - LC3 , were infected with WT or P . aeruginosa mutants and evaluated by fluorescence microscopy , scale bars : 10 µm . GFP - LC3 puncta representing the autophagosomes was quantified and presented in the graph . Each bar represents mean value from three independent experiments and error bars represent standard deviation . Statistical significance between assays was determined using One - way ANOVA followed by Dunn ' s Multiple Comparison Test . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 . P . aeruginosa mutant strains legend : ΔSTY ( mutant deficient for the three cytotoxins ) , WT ( contains all cytotoxin ) , ΔS ( deficient for ExoS but still express ExoT and ExoY ) , ΔTY ( express ExoS but deficient for ExoT and ExoY ) .

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

(C) A549 cells, stably expressing GFP-LC3, were infected with WT or P. aeruginosa mutants and evaluated by fluorescence microscopy, scale bars: 10 µm. GFP-LC3 puncta representing the autophagosomes was quantified and presented in the graph. Each bar represents mean value from three independent experiments and error bars represent standard deviation. Statistical significance between assays was determined using One-way ANOVA followed by Dunn's Multiple Comparison Test. *p ≤ 0.05; **p ≤ 0.01. P. aeruginosa mutant strains legend: ΔSTY (mutant deficient for the three cytotoxins), WT (contains all cytotoxin), ΔS (deficient for ExoS but still express ExoT and ExoY), ΔTY (express ExoS but deficient for ExoT and ExoY).


( D , E ) A549 cells , stably expressing GFP - LC3 , were infected for 4 hours with WT or with one of P . aeruginosa mutants ( ΔSTY , ΔS and ΔTY ) and evaluated by fluorescence microscopy ( D ) . P . aeruginosa were visualized using specific antibody ( red ) and the colocalized bacteria signals with GFP - LC3 puncta ( green ) were merged and presented as yellow . Scale bars : 10 µm . ( E ) Quantitative analysis of co - localization of internalized P . aeruginosa with GFP - LC3 positive puncta . The colocalization of these two signals was quantified with 10 independent visual fields of more than 100 cells . Data represent mean ± SD of three independent experiments . Statistical significance between assays was determined using One - way ANOVA followed by Dunn ' s Multiple Comparison Test . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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

(D,E) A549 cells, stably expressing GFP-LC3, were infected for 4 hours with WT or with one of P. aeruginosa mutants (ΔSTY, ΔS and ΔTY) and evaluated by fluorescence microscopy (D). P. aeruginosa were visualized using specific antibody (red) and the colocalized bacteria signals with GFP-LC3 puncta (green) were merged and presented as yellow. Scale bars: 10 µm. (E) Quantitative analysis of co-localization of internalized P. aeruginosa with GFP-LC3 positive puncta. The colocalization of these two signals was quantified with 10 independent visual fields of more than 100 cells. Data represent mean ± SD of three independent experiments. Statistical significance between assays was determined using One-way ANOVA followed by Dunn's Multiple Comparison Test. *p ≤ 0.05; **p ≤ 0.01.


( F ) Representative transmission electron microscopy images of A549 cells infected with P . aeruginosa containing ExoS ( ΔTY ) or deficient for the three T3SS effector proteins ( ΔSTY ) for 4 hours . ΔSTY P . aeruginosa ( white star ) was engulfed by multiple membrane autophagosomes ( black arrow ) . ΔTY P . aeruginosa ( white star ) were found in cytoplasm with single membrane surrounded , scale bars : 0 . 5 µm .

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(F) Representative transmission electron microscopy images of A549 cells infected with P. aeruginosa containing ExoS (ΔTY) or deficient for the three T3SS effector proteins (ΔSTY) for 4 hours. ΔSTY P. aeruginosa (white star) was engulfed by multiple membrane autophagosomes (black arrow). ΔTY P. aeruginosa (white star) were found in cytoplasm with single membrane surrounded, scale bars: 0.5 µm.


( G ) A549 cells , stably expressing lentiviral vectors containing random shRNA or Atg7 shRNA , were infected with WT or P . aeruginosa mutants ( ΔSTY , ΔTY and ΔS ) at MOI of 10 . All infections were followed by 1 hours of gentamycin treatment . Cell lysates were plated in LB agar for 18 hours and CFU were determined . Data information : The triangles and dots represent the individual test ( three technical replicates per individual test ) on control and Atg7 knock down cells from three biological replicates . The bars represent the means of CFU of each group ( three biological replicates each group ) , error bars represent standard deviation . The significance of differences between different assay was determined using two - tailed student ' s t test with Welch ' s correction , NS : not significant ; * * p ≤ 0 . 01 .

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

(G) A549 cells, stably expressing lentiviral vectors containing random shRNA or Atg7 shRNA, were infected with WT or P. aeruginosa mutants (ΔSTY, ΔTY and ΔS) at MOI of 10. All infections were followed by 1 hours of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined. Data information: The triangles and dots represent the individual test (three technical replicates per individual test) on control and Atg7 knock down cells from three biological replicates. The bars represent the means of CFU of each group (three biological replicates each group), error bars represent standard deviation. The significance of differences between different assay was determined using two-tailed student's t test with Welch's correction, NS: not significant; **p ≤ 0.01.


( A ) Primary airway epithelial cells were isolated from tracheas of Atg7 + / + and Atg7 - / - mouse and were evaluated by immunoblotting .

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(A) Primary airway epithelial cells were isolated from tracheas of Atg7+/+ and Atg7-/- mouse and were evaluated by immunoblotting.


( B ) Sections of tracheas from Atg7 + / + and Atg7 - / - mouse were evaluated by immunofluorescence microscopy following staining of nuclear DNA with DAPI ( Blue ) , ciliated epithelial cell marker β - tubulin IV ( green ) and Atg7 ( red ) . Scale bars : 20 µm .

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(B) Sections of tracheas from Atg7+/+ and Atg7-/- mouse were evaluated by immunofluorescence microscopy following staining of nuclear DNA with DAPI (Blue), ciliated epithelial cell marker β-tubulin IV (green) and Atg7 (red). Scale bars: 20 µm.


( C , D ) Atg7 + / + and Atg7 - / - mice were intranasally injected with 3 x107 CFU of WT , ΔS , ΔTY or ΔSTY P . aeruginosa . After 24 hours , lungs ( C ) , spleens and livers ( D ) were harvested , homogenized and diluted for CFU counting . Each data point represents bacterial count ( Log10 CFU / g ) from individual mouse . The horizontal lines represent the mean value from each group ( 6 mice each group ) , error bars represent standard deviation . The significance of differences between different drug treatment was determined using One - way ANOVA and Dunn ' s multiple comparisons test . , NS : not significant ; * p ≤ 0 .

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(C,D) Atg7+/+ and Atg7-/- mice were intranasally injected with 3 x107 CFU of WT, ΔS, ΔTY or ΔSTY P. aeruginosa. After 24 hours, lungs (C), spleens and livers (D) were harvested, homogenized and diluted for CFU counting. Each data point represents bacterial count (Log10 CFU/g) from individual mouse. The horizontal lines represent the mean value from each group (6 mice each group), error bars represent standard deviation. The significance of differences between different drug treatment was determined using One-way ANOVA and Dunn's multiple comparisons test., NS: not significant; *p ≤ 0.05


( E , F ) Atg7 + / + and Atg7 - / - mice were intranasally injected with PBS with or without 1 x109 CFU of ΔTY ( E ) ΔSTY ( F ) . Mice survival was monitored for 10 days post infection . Kaplan - Meyer survival curves were made based on the observation of 12 - 18 mice / group from two independent experiments . The P - values were calculated using the log - rank test ( ΔTYAtg7 + / + vs ΔTYAtg7 - / - , P = 0 . 2186 ; ΔSTYAtg7 + / + vs ΔSTYAtg7 - / - , P = 0 . 0145 ) .

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(E,F) Atg7+/+ and Atg7-/- mice were intranasally injected with PBS with or without 1 x109 CFU of ΔTY (E) ΔSTY (F). Mice survival was monitored for 10 days post infection. Kaplan-Meyer survival curves were made based on the observation of 12-18 mice/group from two independent experiments. The P-values were calculated using the log-rank test (ΔTYAtg7+/+ vs ΔTYAtg7-/-, P = 0.2186; ΔSTYAtg7+/+ vs ΔSTYAtg7-/-, P=0.0145).


( A A549 cells were infected for 4 hours with ExoS - deficient P . aeruginosa ( ΔS ) , ExoS containing P . aeruginosa ( SWT ) , P . aeruginosa containing ExoS with loss - of - function mutations in the GTPase activating domain ( SG - A + ) , in the ADP - ribosylation domain ( SG + A - ) , or in both domains ( SG - A - ) . Cell lysates were evaluated by immunoblotting .

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(A A549 cells were infected for 4 hours with ExoS-deficient P. aeruginosa (ΔS), ExoS containing P. aeruginosa (SWT), P. aeruginosa containing ExoS with loss-of-function mutations in the GTPase activating domain (SG-A+), in the ADP-ribosylation domain (SG+A-), or in both domains (SG-A-). Cell lysates were evaluated by immunoblotting.


( B ) A549 cells were transfected , for 24hours , with a plasmid expressing empty vector ( EV ) , WT GFP - ExoS , a GFP - ExoS mutant for ADPRT domain ( SG + A - ) or a GFP - ExoS mutant deficient in both GAP and ADPRT domains ( SG - A - ) . Cell lysates were evaluated by immunoblotting .

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(B) A549 cells were transfected, for 24hours, with a plasmid expressing empty vector (EV), WT GFP-ExoS, a GFP-ExoS mutant for ADPRT domain (SG+A-) or a GFP-ExoS mutant deficient in both GAP and ADPRT domains (SG-A-). Cell lysates were evaluated by immunoblotting.


( C ) A549 cells were treated for 24 hours with 100 µM of the ADPRT activity inhibitor MIBG and then left uninfected ( NI ) or infected with P . aeruginosa containing ExoS with ADPRT activity only ( SG - A + ) . Cell lysates were evaluated by immunoblotting .

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(C) A549 cells were treated for 24 hours with 100 µM of the ADPRT activity inhibitor MIBG and then left uninfected (NI) or infected with P. aeruginosa containing ExoS with ADPRT activity only (SG-A+). Cell lysates were evaluated by immunoblotting.


A549 cells were infected for 4 hours with WT or P . aeruginosa mutants ( ΔpscD Cell lysates were evaluated by immunoblotting .

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A549 cells were infected for 4 hours with WT or P. aeruginosa mutants (ΔpscD Cell lysates were evaluated by immunoblotting.


A549 cells were infected for 4 hours with WT or P . aeruginosa mutants STY , ΔS and ΔTY ) . Cell lysates were evaluated by immunoblotting .

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A549 cells were infected for 4 hours with WT or P. aeruginosa mutants STY, ΔS and ΔTY). Cell lysates were evaluated by immunoblotting.


F ) A549 cells were infected for 4 hours with ExoS - deficient P . aeruginosa ( ΔS ) , ExoS containing P . aeruginosa ( SWT ) , P . aeruginosa containing ExoS with loss - of - function mutations in the GTPase activating domain ( SG - A + ) , in the ADP - ribosylation domain ( SG + A - ) , or in both domains ( SG - A - ) . Cell lysates were evaluated by immunoblotting .

F) A549 cells were infected for 4 hours with ExoS-deficient P. aeruginosa (ΔS), ExoS containing P. aeruginosa (SWT), P. aeruginosa containing ExoS with loss-of-function mutations in the GTPase activating domain (SG-A+), in the ADP-ribosylation domain (SG+A-), or in both domains (SG-A-). Cell lysates were evaluated by immunoblotting.


( A ) A549 cells were transfected with empty vector ( VE ) and vector contains Ras - G12V for 24 hours , followed by a 4 - hours infection with P . aeruginosa ΔSTY or ΔTY . Cell lysates were evaluated by immunoblotting .

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(A) A549 cells were transfected with empty vector (VE) and vector contains Ras-G12V for 24 hours, followed by a 4-hours infection with P. aeruginosa ΔSTY or ΔTY. Cell lysates were evaluated by immunoblotting.


( B ) A549 cells were transfected , for 24 hours , with empty vector ( VE ) and vectors contain Ras - G12V or Ras - G12V & R41K followed by a 4 - hours infection with P . aeruginosa ΔSTY or ΔTY . Cell lysates were evaluated by immunoblotting .

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(B) A549 cells were transfected, for 24 hours, with empty vector (VE) and vectors contain Ras-G12V or Ras-G12V&R41K followed by a 4-hours infection with P. aeruginosa ΔSTY or ΔTY. Cell lysates were evaluated by immunoblotting.


( A ) A549 cells were transfected , for 48 hours , with a mCherry - DFCP1 plasmid and then infected , for 4 hours , with P . aeruginosa ( WT , ΔSTY , ΔS , ΔTY , SG - A - , SG - A + or SG + A - ) and the mCherry puncta number per cell was analyzed by fluorescence microscopy . Representative immunofluorescence images were showed , and scale bars represent 10 µm . ( B ) Quantitative analysis of mCherry - DFCP1 puncta by visually count more than 100 cells for each sample . Each bar represents mean value of from three independent experiments and error bars represent standard deviation . The significance of differences between assays was determined using ANOVA with Dunnett multiple - comparison posttest , NS : not significant ; * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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

(A) A549 cells were transfected, for 48 hours, with a mCherry-DFCP1 plasmid and then infected, for 4 hours, with P. aeruginosa (WT, ΔSTY, ΔS, ΔTY, SG-A-, SG-A+ or SG+A-) and the mCherry puncta number per cell was analyzed by fluorescence microscopy. Representative immunofluorescence images were showed, and scale bars represent 10 µm. (B) Quantitative analysis of mCherry-DFCP1 puncta by visually count more than 100 cells for each sample. Each bar represents mean value of from three independent experiments and error bars represent standard deviation. The significance of differences between assays was determined using ANOVA with Dunnett multiple-comparison posttest, NS: not significant; *p ≤ 0.05; **p ≤ 0.01.


( A A549 cells , transfected for 48 hours with vector encoding Flag - Atg14L , were infected with P . aeruginosa ( WT , ΔSTY , ΔS , ΔTY Flag - Atg14L - Vps34 complex was immunoprecipitated ( IP ) by Flag - antibody magnetic beads and evaluated by immunoblotting .

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

(A A549 cells, transfected for 48 hours with vector encoding Flag-Atg14L, were infected with P. aeruginosa (WT, ΔSTY, ΔS, ΔTY Flag-Atg14L-Vps34 complex was immunoprecipitated (IP) by Flag-antibody magnetic beads and evaluated by immunoblotting.