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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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


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

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


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

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


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

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

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


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 .

O O O O O O O O O O O O O O O O O O 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, 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 .

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

C, 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 .

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

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 .

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

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 .

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

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 .

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

C, 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 .

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 .

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 .

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

F, 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 .

O O 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

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 .

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

H, 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 .

O O O O O O O O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

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 .

O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

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

E, 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 .

O O O 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, 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 .

O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

O O O O O O O O O O O O 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, 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 .

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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 .

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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 .

(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 ) .

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(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 .

(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 .

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(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 .

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 .

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(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 .

(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.


( B , The IP complexes were subjected to in vitro kinase assay using phosphatidylinositol as substrate . The products radiolabeled PI3P were separated by TLC and detected by autoradiography . Data are mean ± SD of three independent experiments . The significance of differences between different assays was determined using One - way ANOVA analysis followed Dunn ' s multiple - comparison posttest , NS : not significant ; * p ≤ 0 . 05 .

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(B, The IP complexes were subjected to in vitro kinase assay using phosphatidylinositol as substrate. The products radiolabeled PI3P were separated by TLC and detected by autoradiography. Data are mean ± SD of three independent experiments. The significance of differences between different assays was determined using One-way ANOVA analysis followed Dunn's multiple-comparison posttest, NS: not significant; *p ≤ 0.05.


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

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


D ) The IP complexes were subjected to in vitro kinase assay using phosphatidylinositol as substrate . The products radiolabeled PI3P were separated by TLC and detected by autoradiography . Data are mean ± SD of three independent experiments . The significance of differences between different assays was determined using One - way ANOVA analysis followed Dunn ' s multiple - comparison posttest , NS : not significant ; * p ≤ 0 . 05 .

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D) The IP complexes were subjected to in vitro kinase assay using phosphatidylinositol as substrate. The products radiolabeled PI3P were separated by TLC and detected by autoradiography. Data are mean ± SD of three independent experiments. The significance of differences between different assays was determined using One-way ANOVA analysis followed Dunn's multiple-comparison posttest, NS: not significant; *p ≤ 0.05.


( A ) UBQLN4 associates with polyubiquitinated proteasomal substrates . Flag - tagged UBQLN4 protein was affinity - purified from extracts of HeLa cells that were treated with ( + ) or without ( - ) 20 μM MG - 132 for 4 h before harvesting , and the Flag - precipitates ( IP : Flag ) were blotted with anti - polyubiquitin ( FK2 ) , anti - BAG6 , and anti - Flag antibodies . Mock indicates empty vector transfection . Note that UBQLN4 - BAG6 co - precipitation can be observed exclusively in the presence of polyubiquitinated substrates .

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(A) UBQLN4 associates with polyubiquitinated proteasomal substrates. Flag-tagged UBQLN4 protein was affinity-purified from extracts of HeLa cells that were treated with (+) or without (-) 20 μM MG-132 for 4 h before harvesting, and the Flag-precipitates (IP:Flag) were blotted with anti-polyubiquitin (FK2), anti-BAG6, and anti-Flag antibodies. Mock indicates empty vector transfection. Note that UBQLN4-BAG6 co-precipitation can be observed exclusively in the presence of polyubiquitinated substrates.


( B ) Polyubiquitinated proteins associated with UBQLN4 are sensitive to the translation inhibitor cycloheximide ( CHX ) . HeLa cells expressing Flag - tagged UBQLN4 were treated with 5 μM MG - 132 as well as CHX ( either 10 or 25 μg / mL ) for 4 h as indicated , and then subjected to immunoprecipitation with an anti - Flag antibody as in ( A ) .

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(B) Polyubiquitinated proteins associated with UBQLN4 are sensitive to the translation inhibitor cycloheximide (CHX). HeLa cells expressing Flag-tagged UBQLN4 were treated with 5 μM MG-132 as well as CHX (either 10 or 25 μg/mL) for 4 h as indicated, and then subjected to immunoprecipitation with an anti-Flag antibody as in (A).


( D ) HeLa cells were treated as in ( C ) , and Flag - precipitates were blotted as in ( B ) .

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(D) HeLa cells were treated as in (C), and Flag-precipitates were blotted as in (B).


( A ) UBQLN4 co - localizes with cytoplasmic aggregates after puromycin treatment . HeLa cells expressing Flag - tagged UBQLN4 were treated with 5 µg / mL puromycin for 2 h and then followed by 1 % Triton X - 100 extraction and 4 % paraformaldehyde fixation . Fixed cells were stained with anti - polyubiquitin FK2 ( shown in red to detect ubiquitin - positive cytoplasmic aggregates , ALIS ) and anti - Flag antibodies ( shown in green ) .

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(A) UBQLN4 co-localizes with cytoplasmic aggregates after puromycin treatment. HeLa cells expressing Flag-tagged UBQLN4 were treated with 5 µg/mL puromycin for 2 h and then followed by 1% Triton X-100 extraction and 4% paraformaldehyde fixation. Fixed cells were stained with anti-polyubiquitin FK2 (shown in red to detect ubiquitin-positive cytoplasmic aggregates, ALIS) and anti-Flag antibodies (shown in green).


( B , C ) Puromycin - treated cells with UBQLN4 siRNA contain increased number of ALIS . At 72 h after transfection of the two distinct siRNA duplexes for UBQLN4 ( UBQLN4 siRNA # 1 and # 2 ) or control siRNA ( 5 nM each ) , the cells were treated with 5 µg / mL puromycin for 2 h , and then subjected to immunostaining with an anti - polyubiquitin FK2 antibody ( shown in green , B ) as in ( A ) . The number of ALIS was determined . The quantified data in ( C ) represent mean ± SEM . n = 76 cells for control siRNA , n = 89 cells for UBQLN4 siRNA # 1 , and n = 81 cells for UBQLN4 siRNA # 2 . P ‐ values were calculated by Welch ' s t - test between the siRNA ‐ transfected and the respective control condition ( * P < 0 . 01 ) .

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(B, C) Puromycin-treated cells with UBQLN4 siRNA contain increased number of ALIS. At 72 h after transfection of the two distinct siRNA duplexes for UBQLN4 (UBQLN4 siRNA#1 and #2) or control siRNA (5 nM each), the cells were treated with 5 µg/mL puromycin for 2 h, and then subjected to immunostaining with an anti-polyubiquitin FK2 antibody (shown in green, B) as in (A). The number of ALIS was determined. The quantified data in (C) represent mean ± SEM. n = 76 cells for control siRNA, n = 89 cells for UBQLN4 siRNA#1, and n = 81 cells for UBQLN4 siRNA #2. P‐values were calculated by Welch's t-test between the siRNA‐transfected and the respective control condition (*P < 0.01).


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( D ) Depletion of UBQLN4 makes HeLa cells more sensitive to puromycin - induced cell death . After 48 h of UBQLN4 siRNA treatment , the cells were treated with 5 µg / mL puromycin for 15 . 5 h and then viability was measured . The data represent mean ± SD calculated from 3 independent experiments ( n = 3 ) . P ‐ values were calculated by Student ' s t - test compared with puromycin - treated control siRNA cells ( * P < 0 . 01 ) .

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(D) Depletion of UBQLN4 makes HeLa cells more sensitive to puromycin-induced cell death. After 48 h of UBQLN4 siRNA treatment, the cells were treated with 5 µg/mL puromycin for 15.5 h and then viability was measured. The data represent mean ± SD calculated from 3 independent experiments (n=3). P‐values were calculated by Student's t-test compared with puromycin-treated control siRNA cells (*P < 0.01).


( B ) IL - 2Rα ΔSS mislocalized to the cytosolic fraction . HeLa cells , expressing Flag - tagged IL - 2Rα WT or ΔSS , were fractionated to the cytosolic ( Cyt . ) and membrane ( Mem . ) fractions . Tubulin was used as a cytoplasmic marker , while calnexin was used for the ER membrane fraction .

(B) IL-2Rα ΔSS mislocalized to the cytosolic fraction. HeLa cells, expressing Flag-tagged IL-2Rα WT or ΔSS, were fractionated to the cytosolic (Cyt.) and membrane (Mem.) fractions. Tubulin was used as a cytoplasmic marker, while calnexin was used for the ER membrane fraction.


( C ) IL - 2Rα ΔSS was not glycosylated . Flag - tagged IL - 2Rα WT or ΔSS proteins was expressed in HeLa cells , and immunoprecipitated with an anti - Flag antibody . The precipitates were incubated with ( + ) or without ( - ) 10 unit of the deglycosylation enzyme PNGase F for 2 h , and subjected to western blot analysis with an anti - Flag antibody . Glycosylated and non - glycosylated signals are indicated .

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(C) IL-2Rα ΔSS was not glycosylated. Flag-tagged IL-2Rα WT or ΔSS proteins was expressed in HeLa cells, and immunoprecipitated with an anti-Flag antibody. The precipitates were incubated with (+) or without (-) 10 unit of the deglycosylation enzyme PNGase F for 2 h, and subjected to western blot analysis with an anti-Flag antibody. Glycosylated and non-glycosylated signals are indicated.


( D ) The glycosylated form of IL - 2Rα WT is a stable protein , while mislocalized IL - 2Rα ΔSS is degraded rapidly via its TMD . A series of IL - 2Ra deletion proteins were expressed in HeLa cells and then chased with 20 μg / mL CHX for the indicated periods . Actin was used as a loading control . The right panel is a quantified graph of the left blot signals represent mean ± SD calculated from 4 independent experiments ( n = 4 ) .

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(D) The glycosylated form of IL-2Rα WT is a stable protein, while mislocalized IL-2Rα ΔSS is degraded rapidly via its TMD. A series of IL-2Ra deletion proteins were expressed in HeLa cells and then chased with 20 μg/mL CHX for the indicated periods. Actin was used as a loading control. The right panel is a quantified graph of the left blot signals represent mean ± SD calculated from 4 independent experiments (n=4).


( E ) IL - 2Rα ΔSS is degraded in a proteasome - dependent manner . HeLa cells expressing Flag - tagged IL - 2Rα ΔSS were treated with 20 μg / mL CHX as well as 0 . 1 % DMSO ( DM . ) , 10 μM MG - 132 ( MG . ) , 2 μM Bortezomib ( Bor . ) , 10 μM Leupeptin ( Leu . ) , 0 . 1 μM Bafilomycin A1 ( Baf . ) , or 10 μg / ml Pepstatin A ( pep . A ) / E64d for the indicated periods . The right panel indicates that the quantified data of the left blot signals represent mean ± SD calculated from 3 independent experiments ( n = 3 ) .

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(E) IL-2Rα ΔSS is degraded in a proteasome-dependent manner. HeLa cells expressing Flag-tagged IL-2Rα ΔSS were treated with 20 μg/mL CHX as well as 0.1% DMSO (DM.), 10 μM MG-132 (MG.), 2 μM Bortezomib (Bor.), 10 μM Leupeptin (Leu.), 0.1 μM Bafilomycin A1 (Baf.), or 10 μg/ml Pepstatin A (pep. A)/E64d for the indicated periods. The right panel indicates that the quantified data of the left blot signals represent mean ± SD calculated from 3 independent experiments (n=3).


( F ) The Flag - tagged IL - 2Rα ΔSS mutant and T7 - tagged ubiquitin were co - expressed in HeLa cells and the cells were treated with ( + ) or without ( - ) 10 μM MG - 132 . After 4 h , Flag - precipitates were blotted with anti - T7 and anti - Flag antibodies .

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(F) The Flag-tagged IL-2Rα ΔSS mutant and T7-tagged ubiquitin were co-expressed in HeLa cells and the cells were treated with (+) or without (-) 10 μM MG-132. After 4 h, Flag-precipitates were blotted with anti-T7 and anti-Flag antibodies.


( G ) HeLa cells expressing Flag - tagged IL - 2RaWT , ΔSS , and ΔSSΔTM proteins were treated with ( + ) or without ( - ) 10 μM MG - 132 . At 4 h after MG - 132 treatment , the cells were lysed and analyzed by immunoblotting using the indicated antibodies . Note that a higher migrating form of non - glycosylated ( and thus defective ) IL - 2Ra WT ( indicated by the arrowhead ) is detected strongly in the presence of MG - 132 .

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(G) HeLa cells expressing Flag-tagged IL-2RaWT, ΔSS, and ΔSSΔTM proteins were treated with (+) or without (-) 10 μM MG-132. At 4 h after MG-132 treatment, the cells were lysed and analyzed by immunoblotting using the indicated antibodies. Note that a higher migrating form of non-glycosylated (and thus defective) IL-2Ra WT (indicated by the arrowhead) is detected strongly in the presence of MG-132.


( A , B ) IL - 2Rα ΔSS is stabilized in UBQLN4 - knockdown cells . HeLa cells were transfected with three distinct siRNA duplexes for UBQLN4 ( UBQLN4 siRNA # 1 ~ # 3 ) or control siRNA . At 48 h after siRNA transfection , Flag tagged - IL - 2Rα ΔSS was expressed in the cells . At 24 h after IL - 2RaSS transfection , the cells were chased with 20 μg / mL CHX and harvested at the indicated time after CHX addition . Anti - Flag signals in the control or UBQLN4 siRNA - treated cells ( siRNA # 1 ~ # 3 ) were quantified at the indicated time points . The data represent mean ± SEM calculated from 4 independent experiments ( n = 4 ) . The UBQLN4 signal is marked by an arrowhead , while the band just below it ( indicated by an asterisk ) is a non - specific band , since this signal was never affected by any of the UBQLN4 siRNAs . Actin was used as a loading control .

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(A, B) IL-2Rα ΔSS is stabilized in UBQLN4-knockdown cells. HeLa cells were transfected with three distinct siRNA duplexes for UBQLN4 (UBQLN4 siRNA#1~#3) or control siRNA. At 48 h after siRNA transfection, Flag tagged-IL-2Rα ΔSS was expressed in the cells. At 24 h after IL-2RaSS transfection, the cells were chased with 20 μg/mL CHX and harvested at the indicated time after CHX addition. Anti-Flag signals in the control or UBQLN4 siRNA-treated cells (siRNA#1~#3) were quantified at the indicated time points. The data represent mean ± SEM calculated from 4 independent experiments (n=4). The UBQLN4 signal is marked by an arrowhead, while the band just below it (indicated by an asterisk) is a non-specific band, since this signal was never affected by any of the UBQLN4 siRNAs. Actin was used as a loading control.


( C ) Polyubiquitin modification of IL - 2Rα ΔSS was not diminished , but rather strengthened , in UBQLN4 knockdown cells . At 48 h after transfection of HeLa cells with siRNA for UBQLN4 or BAG6 , Flag - tagged IL - 2Rα ΔSS and T7 - tagged ubiquitin were expressed with ( + ) or without ( - ) 10 mM MG - 132 . From these cells , Flag - tagged IL - 2Rα ΔSS was affinity - purified using Hot lysis analysis , and Flag - precipitates were blotted with an anti - T7 antibody to detect polyubiquitination of IL - 2Rα ΔSS .

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(C) Polyubiquitin modification of IL-2Rα ΔSS was not diminished, but rather strengthened, in UBQLN4 knockdown cells. At 48 h after transfection of HeLa cells with siRNA for UBQLN4 or BAG6, Flag-tagged IL-2Rα ΔSS and T7-tagged ubiquitin were expressed with (+) or without (-) 10 mM MG-132. From these cells, Flag-tagged IL-2Rα ΔSS was affinity-purified using Hot lysis analysis, and Flag-precipitates were blotted with an anti-T7 antibody to detect polyubiquitination of IL-2Rα ΔSS.


( A ) A series of Flag - tagged truncated mutants of IL - 2Ra substrates were expressed in HeLa cells with T7 - tagged UBQLN4 and treated with ( + ) or without ( - ) 10 µM MG - 132 for 4 h . Flag - IL - 2Ra substrates were immunoprecipitated and their co - precipitation with UBQLN4 was analyzed .

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(A) A series of Flag-tagged truncated mutants of IL-2Ra substrates were expressed in HeLa cells with T7-tagged UBQLN4 and treated with (+) or without (-) 10 µM MG-132 for 4 h. Flag-IL-2Ra substrates were immunoprecipitated and their co-precipitation with UBQLN4 was analyzed.


( B ) T7 - UBQLN4 was immunoprecipitated , and its interactions with a series of IL - 2Ra proteins were detected .

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(B) T7-UBQLN4 was immunoprecipitated, and its interactions with a series of IL-2Ra proteins were detected.


( B ) UBQLN4 dominantly co - precipitates IL - 2Rα ΔSS over UBQLN1 . Precipitates of Flag - tagged UBQLN1 or UBQLN4 from HeLa cell lysates were probed with IL - 2Rα ΔSS .

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(B) UBQLN4 dominantly co-precipitates IL-2Rα ΔSS over UBQLN1. Precipitates of Flag-tagged UBQLN1 or UBQLN4 from HeLa cell lysates were probed with IL-2Rα ΔSS.


( C ) The complete amino acid sequences of human UBQLN1 and UBQLN4 were analyzed using the disorder / order structure prediction program PONDR - FIT . The STI - II region of UBQLN4 is indicated by the red box , and that of UBQLN1 is boxed blue . Note that the disorder tendency of the UBL , STI - I , and UBA regions is nearly identical between UBQLN1 and UBQLN4 ( indicated by black and green boxes ) , while that of the STI - II region is quite distinct , and the STI - II of UBQLN4 shows a high probability of an ordered structure . The numbers denote the corresponding amino acid positions in these proteins .

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(C) The complete amino acid sequences of human UBQLN1 and UBQLN4 were analyzed using the disorder/order structure prediction program PONDR-FIT. The STI-II region of UBQLN4 is indicated by the red box, and that of UBQLN1 is boxed blue. Note that the disorder tendency of the UBL, STI-I, and UBA regions is nearly identical between UBQLN1 and UBQLN4 (indicated by black and green boxes), while that of the STI-II region is quite distinct, and the STI-II of UBQLN4 shows a high probability of an ordered structure. The numbers denote the corresponding amino acid positions in these proteins.


( E ) Substitution of the STI - II sequence of UBQLN4 with that of UBQLN1 ( designated as the 4 - STI - II - 1 mutant protein ) abolished its high affinity to the IL - 2Rα ΔSS client protein .

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(E) Substitution of the STI-II sequence of UBQLN4 with that of UBQLN1 (designated as the 4-STI-II-1 mutant protein) abolished its high affinity to the IL-2Rα ΔSS client protein.


( F ) Substitution of the STI - II sequence of UBQLN1 with that of UBQLN4 ( designated as the 1 - STI - II - 4 ) did not enable UBQLN1 to recognize IL - 2Rα ΔSS .

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(F) Substitution of the STI-II sequence of UBQLN1 with that of UBQLN4 (designated as the 1-STI-II-4) did not enable UBQLN1 to recognize IL-2Rα ΔSS.