Figure 1 ( A ) Chromatin immunoprecipitation ( ChIP ) analysis of receptor binding to the ER8 element ( region 1 ) of the nrf2 promoter . Immunoprecipitation with nonspecific IgG and PCR amplification of an adjacent region ( 2 ) served as controls . ChIP assays were also carried out to analyse binding to ER8s in the il10 , ddit3 and tyrobp genes . RAR , retinoic acid receptor ; VDR , vitamin D receptor . ( B ) Reverse transcription - PCR analysis of 1 , 25 ‐ dihydroxyvitamin D3 ( 1 , 25D3 ) ‐ or retinoic acid ( RA ) ‐ regulated expression of ddit3 , il10 , and tyrobp in U937 , Calu ‐ 3 and MCF ‐ 7 cells .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Chromatin immunoprecipitation (ChIP) analysis of receptor binding to the ER8 element (region 1) of the nrf2 promoter. Immunoprecipitation with nonspecific IgG and PCR amplification of an adjacent region (2) served as controls. ChIP assays were also carried out to analyse binding to ER8s in the il10, ddit3 and tyrobp genes. RAR, retinoic acid receptor; VDR, vitamin D receptor.(B) Reverse transcription-PCR analysis of 1,25‐dihydroxyvitamin D3 (1,25D3)‐ or retinoic acid (RA)‐regulated expression of ddit3, il10, and tyrobp in U937, Calu‐3 and MCF‐7 cells.


Figure 2 ( A ) Electrophoretic mobility shift assay ( EMSA ) of binding of vitamin D receptors ( VDRs ) / retinoid X receptors ( RXRs ) to thep19ink4dER8 , with binding to the mouseosteopontin ( mop ) DR3vitamin D responsive element as a control , along with a comparison of binding to ER6 and ER8 motifs ( right ‐ hand panel ) . ( B ) EMSA of retinoic acid receptor ( RAR ) / RXR binding to the p19ink4d ER8 , and the rarβ DR5 retinoic acid response element , as a control . ( C ) Chromatin immunoprecipitation ( ChIP ) analysis of binding of VDRs and RARs to the p19ink4d ER8 ( region 1 ) and RNA polymerase II ( POLII ) binding to the transcription start site . ( D ) ReChIP analysis confirms the presence of VDR / RXRs and RAR / RXRs but not of VDR / RARs on the p19ink4d ER8 . ( E ) Cloning of p19ink4d promoter containing or lacking ER8 upstream of a luciferase reporter . 3 ( 1 , 25D3 ) ‐ ( F ) and retinoic acid ( RA ) ‐ regulated ( G ) luciferase expression is dependent on the ER8 element in the p19ink4d promoter . Luciferase expression driven by a thymidine kinase ( tk ) control promoter is shown .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Electrophoretic mobility shift assay (EMSA) of binding of vitamin D receptors (VDRs)/retinoid X receptors (RXRs) to thep19ink4dER8, with binding to the mouseosteopontin (mop) DR3vitamin D responsive element as a control, along with a comparison of binding to ER6 and ER8 motifs (right‐hand panel).(B) EMSA of retinoic acid receptor (RAR)/RXR binding to the p19ink4d ER8, and the rarβ DR5 retinoic acid response element, as a control.(C) Chromatin immunoprecipitation (ChIP) analysis of binding of VDRs and RARs to the p19ink4d ER8 (region 1) and RNA polymerase II (POLII) binding to the transcription start site.(D) ReChIP analysis confirms the presence of VDR/RXRs and RAR/RXRs but not of VDR/RARs on the p19ink4d ER8.(E) Cloning of p19ink4d promoter containing or lacking ER8 upstream of a luciferase reporter. 3 (1,25D3)‐ (F) and retinoic acid (RA)‐regulated (G) luciferase expression is dependent on the ER8 element in the p19ink4d promoter. Luciferase expression driven by a thymidine kinase (tk) control promoter is shown.


Figure 33 and retinoic acid induce p19ink4d expression . Expression of p19ink4d messenger RNA by reverse transcription - PCR ( A ) or protein by western blotting ( B ) was carried out on extracts of cells treated with 3 ( 1 , 25D3 ) or retinoic acid ( RA ) , as indicated . ( C ) Treatment with 1 , 25D3 or RA transiently enhances co ‐ immunoprecipitation ( IP ) of p19INK4D with cyclin ‐ dependent kinase CDK4 in U937 cells . Similar results were obtained in SCC25 cells ( not shown ) . A control western blot ( W ) of CDK4 levels in ligand ‐ treated cells is shown below . ( D ) Treatment with 1 , 25D3 enhances co ‐ immunoprecipitation of p27KIP1 with CDK4 in U937 cells . Extracts of U937 cells were probed for p27KIP1 and CDK4 , and immunoprecipitated with an anti ‐ CDK antibody and probed for p27KIP1 co ‐ immunoprecipitation .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 33 and retinoic acid induce p19ink4d expression. Expression of p19ink4d messenger RNA by reverse transcription-PCR (A) or protein by western blotting (B) was carried out on extracts of cells treated with 3 (1,25D3) or retinoic acid (RA), as indicated.(C) Treatment with 1,25D3 or RA transiently enhances co‐immunoprecipitation (IP) of p19INK4D with cyclin‐dependent kinase CDK4 in U937 cells. Similar results were obtained in SCC25 cells (not shown). A control western blot (W) of CDK4 levels in ligand‐treated cells is shown below.(D) Treatment with 1,25D3 enhances co‐immunoprecipitation of p27KIP1 with CDK4 in U937 cells. Extracts of U937 cells were probed for p27KIP1 and CDK4, and immunoprecipitated with an anti‐CDK antibody and probed for p27KIP1 co‐immunoprecipitation.


Figure 4 ( A ) Short interfering RNAs ( siRNA ) knockdown of p19ink4d messenger RNA and protein in SCC25 cells . SiRNAs were directed against p19ink4d , scrambled p19ink4d ( Scr . ) or cyclophilin ( cyc . ) controls . ( B ) Combined knockdown of p27KIP1 and p19INK4D in SCC25 cells analysed by western blotting for p27KIP1 ( top ) and p19INK4D ( bottom ) . ( C , D ) Fluorescence ‐ activated cell sorting analysis of cell ‐ cycle distribution of control SCC25 cells , or cells transfected with scrambled ( Scr ) , p19ink4d ‐ or p27kip1 ‐ directed siRNAs , individually or together ( Dbl ) . ( C ) Cells were treated with vehicle or 1 , 25 ‐ dihydroxyvitamin D3 ( 1 , 25D3 ) , as indicated . ( D ) Cells were treated with DMSO vehicle , retinoic acid ( RA ) or 1 , 25D3 , as indicated . ( E ) Loss of p19 enhances lysosomal β ‐ galactosidase activity in SCC25 cells . ( Left ) Histogram of numbers of cells staining for β ‐ galactosidase activity in the absence ( − ) or presence of 1 , 25D3 in control cells ( − ) or in cells transfected with scrambled ( Scr ) or p19 siRNAs . ( Right ) Bright field images of untransfected cells ( − / − ) or p19INK4D ‐ depleted cells treated with 1 , 25D3 ( p19 / D3 ) . ( F ) Loss of p19INK4D induces lysosomal fusion . Analysis of lysosomal fusion ( autophagosomes ) in SCC25 cells treated as in ( E ) , stained for incorporation of lysotracker red . Nuclei were counterstained with Hoechst ( blue ) .

O O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Short interfering RNAs (siRNA) knockdown of p19ink4d messenger RNA and protein in SCC25 cells. SiRNAs were directed against p19ink4d, scrambled p19ink4d (Scr.) or cyclophilin (cyc.) controls.(B) Combined knockdown of p27KIP1 and p19INK4D in SCC25 cells analysed by western blotting for p27KIP1 (top) and p19INK4D (bottom).(C,D) Fluorescence‐activated cell sorting analysis of cell‐cycle distribution of control SCC25 cells, or cells transfected with scrambled (Scr), p19ink4d‐ or p27kip1‐directed siRNAs, individually or together (Dbl). (C) Cells were treated with vehicle or 1,25‐dihydroxyvitamin D3 (1,25D3), as indicated. (D) Cells were treated with DMSO vehicle, retinoic acid (RA) or 1,25D3, as indicated.(E) Loss of p19 enhances lysosomal β‐galactosidase activity in SCC25 cells. (Left) Histogram of numbers of cells staining for β‐galactosidase activity in the absence (−) or presence of 1,25D3 in control cells (−) or in cells transfected with scrambled (Scr) or p19 siRNAs. (Right) Bright field images of untransfected cells (−/−) or p19INK4D‐depleted cells treated with 1,25D3 (p19/D3).(F) Loss of p19INK4D induces lysosomal fusion. Analysis of lysosomal fusion (autophagosomes) in SCC25 cells treated as in (E), stained for incorporation of lysotracker red. Nuclei were counterstained with Hoechst (blue).


Figure 1A Immunofluorescence staining of endothelium ( CD31 , in red ) , pericytes ( NG2 , in green ) , smooth muscle cells ( α - SMA , in cyan ) , and nuclei ( DAPI , in blue ) on frozen sections of limbmuscles injected with myoblast clones expressing different VEGF levels ( V Low , V Med , and V High , respectively ) and treated with VEGF - Trap or saline after 10 and 17 days . Analysis was performed after 4 days of treatment ( 2 and 3 weeks , respectively ) . All normal vessels induced by low and medium VEGF doses displayed a similar coverage by normal pericytes , whereas aberrant structures induced by high VEGF were covered with α - SMA - positive smooth muscle cells . Asterisks indicate muscle fibers , which were mostly sectioned in longitudinal orientation , although some cross sections are visible depending on the orientation of muscle bundles . Scale bar = 25 μm . B , C Quantification of vessel length density ( VLD ) on the same samples showed that stabilization started already by 10 days for vessels induced by low VEGF , but not until 17 days for those induced by medium VEGF , despite similar pericyte coverage , while aberrant structures induced by high VEGF always regressed . Data represent the mean ± SEM of individual images ( n ) acquired from three muscles / group ; 2 weeks ( B ) : V Low saline , n = 9 ; V Med saline , n = 13 ; V High saline , n = 8 ; V Low TRAP , n = 28 ; V Med TRAP , n = 22 ; V High TRAP , n = 30 ; 3 weeks ( C ) : V Low saline , n = 9 ; V Med saline , n = 11 ; V High saline , n = 19 ; V Low TRAP , n = 43 ; V Med TRAP , n = 14 ; V High TRAP , n = 19 ; * P < 0 . 05 and * * * P < 0 . 001 ( all versus Ctrl ) by one - way ANOVA with Bonferroni multiple comparisons test ; 2 weeks ( B ) : V Low TRAP versus Ctrl P < 0 . 0001 ; 3 weeks ( C ) : V Low TRAP versus Ctrl P < 0 . 0001 ; V Med TRAP versus Ctrl P = 0 . 0158 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Immunofluorescence staining of endothelium (CD31, in red), pericytes (NG2, in green), smooth muscle cells (α-SMA, in cyan), and nuclei (DAPI, in blue) on frozen sections of limbmuscles injected with myoblast clones expressing different VEGF levels (V Low, V Med, and V High, respectively) and treated with VEGF-Trap or saline after 10 and 17 days. Analysis was performed after 4 days of treatment (2 and 3 weeks, respectively). All normal vessels induced by low and medium VEGF doses displayed a similar coverage by normal pericytes, whereas aberrant structures induced by high VEGF were covered with α-SMA-positive smooth muscle cells. Asterisks indicate muscle fibers, which were mostly sectioned in longitudinal orientation, although some cross sections are visible depending on the orientation of muscle bundles. Scale bar = 25 μm.B, C Quantification of vessel length density (VLD) on the same samples showed that stabilization started already by 10 days for vessels induced by low VEGF, but not until 17 days for those induced by medium VEGF, despite similar pericyte coverage, while aberrant structures induced by high VEGF always regressed. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group; 2 weeks (B): V Low saline, n = 9; V Med saline, n = 13; V High saline, n = 8; V Low TRAP,n = 28; V Med TRAP,n = 22; V High TRAP,n = 30; 3 weeks (C): V Low saline, n = 9; V Med saline, n = 11; V High saline, n = 19; V Low TRAP,n = 43; V Med TRAP,n = 14; V High TRAP,n = 19; *P < 0.05 and ***P < 0.001 (all versus Ctrl) by one-way ANOVA with Bonferroni multiple comparisons test; 2 weeks (B): V Low TRAP versus Ctrl P < 0.0001; 3 weeks (C): V Low TRAP versus Ctrl P < 0.0001; V Med TRAP versus Ctrl P = 0.0158.


Figure 2Muscles were harvested 7 days after implantation of V Low , V Med , and V High clones or control cells ( Ctrl ) . Gene expression of Pdgfb , Ang1 , Ang2 , Tgfb1 , and Sema3a was quantified by qRT - PCR and expressed as fold - change versus control muscles . Data represent individual values , with mean ± SEM ( n = 3 or 4 , as indicated ) ; * P < 0 . 05 and * * P < 0 . 01 by one - way ANOVA with Bonferroni multiple comparisons test , after data normalization by logarithmic transformation . TGF - β1 : V Low versus V Med P = 0 . 0047 ; V Low versus V High P = 0 . 0379 ; Sema3A : V Low versus V High P = 0 . 0042 ; V Med versus V High P = 0 . 0179 . Muscles were harvested 7 days after implantation of V Low , V Med , and V High clones or control cells ( Ctrl ) . Immunohistochemistry for Sema3A on frozen muscle sections confirmed a decreasing protein expression in the areas implanted with myoblasts producing increasing VEGF doses ( n = 4 ) . The right panels represent higher - magnification views of the left panels , which encompass the majority of the implantation sites . Scale bar = 50 μm . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Muscles were harvested 7 days after implantation of V Low, V Med, and V High clones or control cells (Ctrl).Gene expression of Pdgfb,Ang1,Ang2,Tgfb1, and Sema3a was quantified by qRT-PCR and expressed as fold-change versus control muscles. Data represent individual values, with mean ± SEM (n = 3 or 4, as indicated); *P < 0.05 and **P < 0.01 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation. TGF-β1: V Low versus V Med P = 0.0047; V Low versus V High P = 0.0379; Sema3A: V Low versus V High P = 0.0042; V Med versus V High P = 0.0179.Muscles were harvested 7 days after implantation of V Low, V Med, and V High clones or control cells (Ctrl). Immunohistochemistry for Sema3A on frozen muscle sections confirmed a decreasing protein expression in the areas implanted with myoblasts producing increasing VEGF doses (n = 4). The right panels represent higher-magnification views of the left panels, which encompass the majority of the implantation sites. Scale bar = 50 μm.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.


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

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Figure 3Immunofluorescence 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.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.


Figure 4Mice , 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Mice, 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.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;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.


Figure 5Relative 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Relative 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.


Figure 6A - 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A-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;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.


Figure 7A - 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A-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.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.


Figure 1 ( B ) H & amp ; amp ; E staining of representative atherosclerotic lesions in aortic root ( upper panel - 4x magnification ) and brachiocephalic arteries ( lower panel - 10x magnification ) at 14 week harvest . Lesion width of roots highlighted with black bars . ( C ) Quantification of root lesion size ( left panel , * p = 0 . 002 ) , and of brachiocephalic lesion size ( right panel , * p = 0 . 001 ) , both quantified as percent of total lumen area . Data in ( C ) are averaged lesion percentages from 4 - 6μm serial sections from each animal , n = 9 , 13 , 15 . All of the data represent the mean + SEM , * p < 0 . 05 versus LNA - control tissue . Level of significance was determined using one - way ANOVA with Bonferroni ' s post test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) H&amp;E staining of representative atherosclerotic lesions in aortic root (upper panel-4x magnification) and brachiocephalic arteries (lower panel-10x magnification) at 14 week harvest. Lesion width of roots highlighted with black bars. (C) Quantification of root lesion size (left panel, *p=0.002), and of brachiocephalic lesion size (right panel, *p=0.001), both quantified as percent of total lumen area. Data in (C) are averaged lesion percentages from 4-6μm serial sections from each animal, n=9, 13, 15. All of the data represent the mean + SEM, *p<0.05 versus LNA-control tissue. Level of significance was determined using one-way ANOVA with Bonferroni's post test.


Figure 2 ( A ) Movat pentachrome staining of aortic root ( upper panel - 4x magnification ) and brachiocephalic artery fibrous caps ( lower panel - 10x magnification ) at 14 week harvest , caps denoted with arrowheads and black bars ( roots only ) . ( B ) Quantification of aortic root ( left panel , * p = 0 . 005 , # p < 0 . 001 ) and brachiocephalic artery ( right panel * p = 0 . 011 , # p < 0 . 024 ) fibrous caps , graphed as percent of total plaque area . ( C ) Representative immunofluorescence microscopy ( IF ) staining of smooth muscle actin ( red ) , denoted by white arrowheads , in aortic rootlesions shown at 10x magnification . IF images in ( C ) are representative of data quantified in ( D ) calculated from 3 - 6μm serial sections from animals in independent cohorts . Level of significance was determined using Kruskal - Wallace one - way ANOVA on ranks , * p = 0 . 002 ( D ) . ( E ) Movat pentachrome staining of aortic root ( upper panel - 4x magnification , * p < 0 . 001 ) and brachiocephalic artery necrotic areas ( lower panel - 10x magnification ) at 14 week harvest , representative necrotic zones denoted by highlighted areas . ( F ) Quantification of aortic root ( left panel , * p < 0 . 001 ) and brachiocephalic artery ( right panel , # p = 0 . 044 ) necrotic areas , graphed as percent of total plaque area .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Movat pentachrome staining of aortic root (upper panel-4x magnification) and brachiocephalic artery fibrous caps (lower panel-10x magnification) at 14 week harvest, caps denoted with arrowheads and black bars (roots only). (B) Quantification of aortic root (left panel, *p=0.005, #p<0.001) and brachiocephalic artery (right panel*p=0.011, #p<0.024) fibrous caps, graphed as percent of total plaque area.(C) Representative immunofluorescence microscopy (IF) staining of smooth muscle actin (red), denoted by white arrowheads, in aortic rootlesions shown at 10x magnification. IF images in (C) are representative of data quantified in (D) calculated from 3-6μm serial sections from animals in independent cohorts. Level of significance was determined using Kruskal-Wallace one-way ANOVA on ranks, *p=0.002 (D).(E) Movat pentachrome staining of aortic root (upper panel-4x magnification, *p<0.001) and brachiocephalic artery necrotic areas (lower panel-10x magnification) at 14 week harvest, representative necrotic zones denoted by highlighted areas. (F) Quantification of aortic root (left panel, *p<0.001) and brachiocephalic artery (right panel, #p=0.044) necrotic areas, graphed as percent of total plaque area.


Figure 3ApoE - / - common carotid arteries ( CCA ) were dissected into plaque and adjacent non - plaque areas , then RNA isolated . ( A ) Graphed qPCR quantification of collagen 1A1 ( left panel ) and collagen 3A1 ( right panel ) mRNA expression in plaque ( P ) and non - plaque ( NP ) areas of ApoE - / - carotid arteries . Three assays were carried out , and the data represent mRNA expression normalized to NP , and the mean + SEM , n = 4 , 3 , 4 of mice / group . * represents p < 0 . 001 compared with LNA - miR - 29 non - plaque region . Picrosirius red staining of collagen in BCs ( 10x magnification ) ( B ) ; brightfield ( upper panel ) and polarized light ( lower panel ) imaging shown . Collagen quantification in BCs ( C , * p = 0 . 016 ) from polarized light images expressed as a percentage of plaque area . Picrosirius red staining of collagen in aortic roots ( 4x magnification ) ( D ) ; brightfield ( upper panel ) and polarized light ( lower panel ) imaging shown . Collagen quantification in roots ( E , * p = 0 . 003 , # p = 0 . 005 ) from polarized light images expressed as a percentage of plaque area .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3ApoE-/- common carotid arteries (CCA) were dissected into plaque and adjacent non-plaque areas, then RNA isolated. (A) Graphed qPCR quantification of collagen 1A1 (left panel) and collagen 3A1 (right panel) mRNA expression in plaque (P) and non-plaque (NP) areas of ApoE-/- carotid arteries. Three assays were carried out, and the data represent mRNA expression normalized to NP, and the mean + SEM, n=4, 3, 4 of mice/group. * represents p<0.001 compared with LNA-miR-29 non-plaque region.Picrosirius red staining of collagen in BCs (10x magnification) (B); brightfield (upper panel) and polarized light (lower panel) imaging shown. Collagen quantification in BCs(C, *p=0.016) from polarized light images expressed as a percentage of plaque area.Picrosirius red staining of collagen in aortic roots (4x magnification) (D); brightfield (upper panel) and polarized light (lower panel) imaging shown. Collagen quantification in roots (E, *p=0.003, #p=0.005) from polarized light images expressed as a percentage of plaque area.


Figure 4Proteome studies analyze the frequency distribution of protein changes after the transfection of primary murine aortic VSMCs with pre - miR - 29b , anti - miR - 29b , pre - miR - 30c , or anti - miR - 30c . A log2 fold change of − 1 or 1 was used as cutoff . Log2 ( fold change ) is calculated using the normalized spectral counts and averaged for the 3 biological replicates . ( A ) The log ( fold change ) for differentially secreted proteins following overexpression or inhibition of pre - miR - 29b was analyzed , and significantly changed miR - 29 confirmed target transcripts are shown . FDR indicates false discovery rate . The complete list of significant proteins is in ( Appendix Table S1A - B ) . ( B ) Thirty microliters of conditioned medium from VSMCs transfected with pre - miR - 29b and anti - miR - 29b were separated by SDS - PAGE before validation of COL3A1 by immunoblotting . ( C ) Densitometry analysis of COL3A1 signal from blot in ( B ) ( pre - miR , left * p < 0 . 001 , anti - miR , right * p = 0 . 002 ) . All data in ( C ) represent the mean + SEM , * p < 0 . 05 versus other groups in anti - and pre - groups . Level of significance was determined using one - way ANOVA with Bonferroni ' s post test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Proteome studies analyze the frequency distribution of protein changes after the transfection of primary murine aortic VSMCs with pre-miR-29b, anti-miR-29b, pre-miR-30c, or anti-miR-30c. A log2 fold change of −1 or 1 was used as cutoff. Log2 (fold change) is calculated using the normalized spectral counts and averaged for the 3 biological replicates. (A) The log (fold change) for differentially secreted proteins following overexpression or inhibition of pre-miR-29b was analyzed, and significantly changed miR-29 confirmed target transcripts are shown. FDR indicates false discovery rate. The complete list of significant proteins is in (Appendix Table S1A-B).(B) Thirty microliters of conditioned medium from VSMCs transfected with pre-miR-29b and anti-miR-29b were separated by SDS-PAGE before validation of COL3A1 by immunoblotting. (C) Densitometry analysis of COL3A1 signal from blot in (B) (pre-miR, left *p<0.001, anti-miR, right *p=0.002) . All data in (C) represent the mean + SEM, *p<0.05 versus other groups in anti- and pre- groups. Level of significance was determined using one-way ANOVA with Bonferroni's post test.


Figure 1C . Quantification of Cre activity by dual luciferase assays ( mean ± SD , n = 3 ) . Data information Npu : Nostoc punctiforme , Ssp : Synechocystis sp . , CDS : coding sequence . D . Quantification of tTA activity by dual luciferase assays ( mean ± SD , n = 3 ( N and C ) or n = 7 ( N / C ) ) . Data information : Npu : Nostoc punctiforme CDS : coding sequence .

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Figure 1C. Quantification of Cre activity by dual luciferase assays (mean±SD, n=3). Data information Npu: Nostoc punctiforme, Ssp: Synechocystis sp., CDS: coding sequence.D. Quantification of tTA activity by dual luciferase assays (mean±SD, n=3 (N and C) or n=7 (N/C)). Data information: Npu: Nostoc punctiforme CDS: coding sequence.


Figure 2A - B . Scheme of engineered Spc and Ccsp loci after integration of split - Cre and split - tTA effectors ( red mark = stop codon ) and comparison of endogenous SPC or CCSP expression and YFP or mCherry reporter expression in lung sections . Blue : DAPI . Scale bar : 100 µm . C - D . β - galactosidase and H & E staining of lung sections from BASC tracer and BASC viewer animals . Scale bars : 500 µm ( overview ) and 20 µm ( boxed magnification ) . E . Localization of β - gal + cells in adult BASC viewer animals . BADJ - associated and " alveolar " β - gal + cells are expressed as percentage of all lineage - labeled cells ( means ± SD n = 3 ) . F . Number of β - gal + cells in adult BASC tracer and BASC viewer animals , normalized to the number of terminal bronchioles ( TBs ) ( means ± SD n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-B. Scheme of engineered Spc and Ccsp loci after integration of split-Cre and split-tTA effectors (red mark = stop codon) and comparison of endogenous SPC or CCSP expression and YFP or mCherry reporter expression in lung sections. Blue: DAPI. Scale bar: 100 µm.C-D. β-galactosidase and H&E staining of lung sections from BASC tracer and BASC viewer animals. Scale bars: 500 µm (overview) and 20 µm (boxed magnification).E. Localization of β-gal+ cells in adult BASC viewer animals. BADJ-associated and "alveolar" β-gal+ cells are expressed as percentage of all lineage-labeled cells (means±SD n=3).F. Number of β-gal+ cells in adult BASC tracer and BASC viewer animals, normalized to the number of terminal bronchioles (TBs) (means±SD n=3).


Figure 3A . Sequential microscopic imaging of epifluorescence and β - galactosidase activity in lung sections of adult BASC viewer animals . Arrow highlights labeled BASC at BADJ ( magnified in box ) . Blue : DAPI . Scale bars : 100 µm ( overview ) and 20 µm ( boxed magnification ) . B . β - galactosidase staining of AT2 cells ( YFP + ) , Club cells ( mCherry + ) and BASCs ( mCherry + / YFP + ) isolated from BASC viewer mice by fluorescence - activated cell sorting ( FACS ) . Scale bar : 50 µm . C . Doxycycline - dependent reversible labeling of BASCs in BASC viewer mice . Scale bar : 200 µm .

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Figure 3A. Sequential microscopic imaging of epifluorescence and β-galactosidase activity in lung sections of adult BASC viewer animals. Arrow highlights labeled BASC at BADJ (magnified in box). Blue: DAPI. Scale bars: 100 µm (overview) and 20 µm (boxed magnification).B. β-galactosidase staining of AT2 cells (YFP+), Club cells (mCherry+) and BASCs (mCherry+/YFP+) isolated from BASC viewer mice by fluorescence-activated cell sorting (FACS). Scale bar: 50 µm.C. Doxycycline-dependent reversible labeling of BASCs in BASC viewer mice. Scale bar: 200 µm.


Figure 4A . Heatmaps for Spearman correlation . C . MA and Volcano plots , and Top50 DEGs ( FDR < 0 . 05 , Log2FC > ± 0 . 585 , Means > 5 ) for visualization of pairwise differential gene expression analysis of BASCs and Club cells . D . MA and Volcano plots , and Top50 DEGs ( FDR < 0 . 05 , Log2FC > ± 0 . 585 , Means > 5 ) for visualization of pairwise differential gene expression analysis of BASCs and AT2 cells . E . Heatmap of RNA - seq z - scores computed for the Top50 DEGs in BASC , AT2 , and Club cells ( FDR < 0 . 05 , Log2FC > ± 0 . 585 , Means > 5 ) .

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Figure 4A. Heatmaps for Spearman correlation.C. MA and Volcano plots, and Top50 DEGs (FDR<0.05, Log2FC>±0.585, Means>5) for visualization of pairwise differential gene expression analysis of BASCs and Club cells. D. MA and Volcano plots, and Top50 DEGs (FDR<0.05, Log2FC>±0.585, Means>5) for visualization of pairwise differential gene expression analysis of BASCs and AT2 cells. E. Heatmap of RNA-seq z-scores computed for the Top50 DEGs in BASC, AT2, and Club cells (FDR<0.05, Log2FC>±0.585, Means>5).


Figure 5B . Quantification of β - gal + cells in FACS - purified AT2 and Club cell fractions isolated from 3 months old BASC v - race mice ( mean ± SD , n = 3 ) and example of β - galactosidase label distribution in lung sections . Scale bar : 100 µm . C . BASCs emerge at early postnatal stages and contribute to epithelial turnover throughout life . β - galactosidase staining of lung sections derived from BASC v - race animals collected at indicated time points . Arrow highlights β - gal + cell at P6 . Scale bar : 200 µm .

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Figure 5B. Quantification of β-gal+ cells in FACS-purified AT2 and Club cell fractions isolated from 3 months old BASC v-race mice (mean±SD, n=3) and example of β-galactosidase label distribution in lung sections. Scale bar: 100 µm.C. BASCs emerge at early postnatal stages and contribute to epithelial turnover throughout life. β-galactosidase staining of lung sections derived from BASC v-race animals collected at indicated time points. Arrow highlights β-gal+ cell at P6. Scale bar: 200 µm.


Figure 6A . β - galactosidase staining of cleared whole lung preparations isolated from control and injured BASC v - race animals 21 days ( bleomycin , naphthalene ) or 35 days ( influenza ) following injury . Scale bar : 5 mm ( whole lungs ) and 1 mm ( magnification ) . B . Sequential microscopic imaging of epifluorescence and β - galactosidase activity in lung sections of control and injured BASC v - race mice . Clusters of BASC - derived AT2 ( YFP + ) and Club cells ( mCherry + ) are highlighted . Blue : DAPI . Scale bar : 200 µm . C . Contribution of BASCs to different cellular compartments . Bronchiolar and alveolar accumulations of lineage - traced BASC derivatives are expressed as percentage of all clusters containing ≥ 10 β - gal + cells . Data depicted as mean ± SD , n = 4 ( bleomycin , naphthalene ) or n = 5 ( influenza ) .

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Figure 6A. β-galactosidase staining of cleared whole lung preparations isolated from control and injured BASC v-race animals 21 days (bleomycin, naphthalene) or 35 days (influenza) following injury. Scale bar: 5 mm (whole lungs) and 1 mm (magnification).B. Sequential microscopic imaging of epifluorescence and β-galactosidase activity in lung sections of control and injured BASC v-race mice. Clusters of BASC-derived AT2 (YFP+) and Club cells (mCherry+) are highlighted. Blue: DAPI. Scale bar: 200 µm.C. Contribution of BASCs to different cellular compartments. Bronchiolar and alveolar accumulations of lineage-traced BASC derivatives are expressed as percentage of all clusters containing ≥ 10 β-gal+ cells. Data depicted as mean±SD, n=4 (bleomycin, naphthalene) or n=5 (influenza).


Figure 7A . β - galactosidase and H & E staining of lung sections from control ( non - infected ) and injured BASC viewer mice 7 days post infection ( 7 dpi ) with influenza virus . Scale bar : 200 µm . B . Abundance of β - gal + cells in control ( non - infected ) and injured ( 7 dpi ) BASC viewer animals . Data depicted as mean ± SD , n = 4 , * p = 0 . 0286 ( Mann - Whitney test ) , TB - terminal bronchiole . C . Club cell depletion / recovery after naphthalene treatment . Microscopic imaging of epifluorescence in lung sections from control and injured BASC viewer animals isolated 3 , 7 and 21 days post naphthalene ( dpn ) . Blue : DAPI . Scale bar : 200 µm . D . β - galactosidase staining of lung sections from control and injured BASC viewer animals 3 , 7 and 21 days post naphthalene ( dpn ) . Scale bar : 200 µm . E . Abundance of β - gal + cells in control and injured BASC viewer animals . The percentage of terminal bronchioles containing 0 , 1 , 2 , 3 , 4 , ≥ 5 β - gal + cells is shown . Data depicted as mean ± SD , n = 3 .

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Figure 7A. β-galactosidase and H&E staining of lung sections from control (non-infected) and injured BASC viewer mice 7 days post infection (7 dpi) with influenza virus. Scale bar: 200 µm. B. Abundance of β-gal+ cells in control (non-infected) and injured (7 dpi) BASC viewer animals. Data depicted as mean±SD, n = 4, *p = 0.0286 (Mann-Whitney test), TB - terminal bronchiole. C. Club cell depletion/recovery after naphthalene treatment. Microscopic imaging of epifluorescence in lung sections from control and injured BASC viewer animals isolated 3, 7 and 21 days post naphthalene (dpn). Blue: DAPI. Scale bar: 200 µm.D. β-galactosidase staining of lung sections from control and injured BASC viewer animals 3, 7 and 21 days post naphthalene (dpn). Scale bar: 200 µm.E. Abundance of β-gal+ cells in control and injured BASC viewer animals. The percentage of terminal bronchioles containing 0, 1, 2, 3, 4, ≥ 5 β-gal+ cells is shown. Data depicted as mean±SD, n=3.


Figure 8A . Quantification of lineage - traced Club cells in distal TBs and β - galactosidase staining of cleared accessory lobe preparation ( scale bar : 250 µm ) isolated from injured BASC v - race mice at 21 dpn ( mean ± SD , n = 4 ) . Bottom panel : example of sequential microscopic imaging to quantify β - gal + mCherry + cells in distal TBs . Arrows denote last airway bifurcation before bronchioalveolar duct junction ( BADJ ) . Scale bar : 200 µm . B . β - galactosidase staining of FACS - purified Club cells from control and injured BASC v - race animals . Scale bar : 50 µm . The percentage of lineage - traced Club cells is shown ( mean ± SD , n = 4 ( oil ctrl . ) or n = 5 ( 21 dpn ) , * p = 0 . 0159 ( Mann - Whitney test ) . C . Combined β - galactosidase ( blue ) and acetylated tubulin staining ( cilia , brown color ) of lung sections from BASC v - race animals at 21 dpn . Red : DAPI ( pseudocolor ) . Examples of lineage - traced ciliated cells ( arrowheads ) and Club cells ( asterisks ) are highlighted . Scale bar : 50 µm . D . β - galactosidase staining of cleared accessory lobe preparations ( scale bar : 5 mm ) and quantification of β - gal + bronchioles in lung sections from adult BASC v - race ( ctrl . ) and BASC - ablated mice ( DTA ) . Data depicted as mean ± SD , n = 3 . E . β - galactosidase staining of cleared accessory lobe preparations ( scale bar : 5 mm ) and fluorescence microscopy of lung sections from uninjured and naphthalene - exposed BASC v - race ( ctrl . ) and BASC - ablated ( DTA ) mice 12 days post naphthalene ( dpn ) . Dotted lines depict bronchiolar boundaries . Scale bar : 100 µm . F . Quantification of Club cell recovery in injured BASC v - race ( ctrl . ) and BASC - ablated ( DTA ) animals , expressed as percentage of bronchiolar circumference re - populated by mCherry + cells at 12 dpn . Data depicted as mean ± SD , n = 5 , * * p = 0 . 0079 ( Mann - Whitney test ) . DTA - diphtheria toxin fragment A .

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Figure 8A. Quantification of lineage-traced Club cells in distal TBs and β-galactosidase staining of cleared accessory lobe preparation (scale bar: 250 µm) isolated from injured BASC v-race mice at 21 dpn (mean±SD, n=4). Bottom panel: example of sequential microscopic imaging to quantify β-gal+ mCherry+ cells in distal TBs. Arrows denote last airway bifurcation before bronchioalveolar duct junction (BADJ). Scale bar: 200 µm.B. β-galactosidase staining of FACS-purified Club cells from control and injured BASC v-race animals. Scale bar: 50 µm. The percentage of lineage-traced Club cells is shown (mean±SD, n=4 (oil ctrl.) or n=5 (21 dpn), *p = 0.0159 (Mann-Whitney test).C. Combined β-galactosidase (blue) and acetylated tubulin staining (cilia, brown color) of lung sections from BASC v-race animals at 21 dpn. Red: DAPI (pseudocolor). Examples of lineage-traced ciliated cells (arrowheads) and Club cells (asterisks) are highlighted. Scale bar: 50 µm.D. β-galactosidase staining of cleared accessory lobe preparations (scale bar: 5 mm) and quantification of β-gal+ bronchioles in lung sections from adult BASC v-race (ctrl.) and BASC-ablated mice (DTA). Data depicted as mean±SD, n=3. E. β-galactosidase staining of cleared accessory lobe preparations (scale bar: 5 mm) and fluorescence microscopy of lung sections from uninjured and naphthalene-exposed BASC v-race (ctrl.) and BASC-ablated (DTA) mice 12 days post naphthalene (dpn). Dotted lines depict bronchiolar boundaries. Scale bar: 100 µm. F. Quantification of Club cell recovery in injured BASC v-race (ctrl.) and BASC-ablated (DTA) animals, expressed as percentage of bronchiolar circumference re-populated by mCherry+ cells at 12 dpn. Data depicted as mean±SD, n=5, **p = 0.0079 (Mann-Whitney test). DTA - diphtheria toxin fragment A.


Figure 1 ( B ) Immunoblot analysis of membrane fractions of HEK293 Flp - In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals increased levels of membrane - bound TREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor . An isotype antibody was used as a negative control . Calnexin served as a loading control . Levels of membrane - bound TREM2 were quantified by MSD ELISA . Data represent the mean ± SEM ( n = 3 ) . One - way ANOVA , Tukey ' s post hoc test ; p ( DMSO vs GM ) = 0 . 0011 ; p ( DMSO vs isotype ) = 0 . 992 ; p ( isotype vs 4D9 ) = 0 . 0005 ; n . s . , not significant . ( C ) Immunoblot analysis of conditioned media from HEK293 Flp - In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals decreased levels of sTREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor . An isotype antibody was used as a negative control . sAPPα served as a loading control . Note that heavy and light chains of the antibodies used for treatment are also detected and annotated . Levels of sTREM2 were quantified by MSD ELISA . Data represent the mean ± SEM ( n = 3 ) . One - way ANOVA , Tukey ' s post hoc test ; p ( DMSO vs GM ) < 0 . 0001 ; p ( DMSO vs isotype ) = 0 . 6372 ; p ( isotype vs 4D9 ) < 0 . 0001 ; n . s . , not significant . ( D ) 4D9 antibody selectively detects TREM2 on the cell surface of HEK293 Flp - In cells stably overexpressing mouse TREM2 and mouse DAP12 . An anti - HA antibody was used as a positive control while empty vector transfected HEK293 Flp - In cells were used as a negative control . Scale bar = 10µm . ( F ) Sequence comparison of mouse TREM2 and human TREM2 shows substantial sequence conservation around the 4D9 epitope ( upper panel ) . Immunoblot analysis demonstrates that antibody 4D9 is highly specific for mouse TREM2 and does not detect human TREM2 or mouse TREM1 ( lower panel ) . ( G ) 4D9 binding to mouse TREM2 ECD is competed off by a stalk region peptide . A competition ELISA demonstrates that a dose titration of stalk peptide reduces binding of 4D9 to TREM2 ECD with an EC50 of 1 . 3uM . Data represent the mean ± SEM ( n = 3 ) . ( H ) Surface plasmon resonance binding kinetics of increasing concentrations of 4D9 antibody to mouse TREM2 ectodomain evaluated by BIAcore , Kon = 5 . 9 x 105 M - 1s - 1 , Koff = 4 . 0 x 10 - 5 s - 1 , KD = 68 pM . 4D9 binding to human TREM2 or mouse TREM1 was undetectable .

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Figure 1(B) Immunoblot analysis of membrane fractions of HEK293 Flp-In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals increased levels of membrane-bound TREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor. An isotype antibody was used as a negative control. Calnexin served as a loading control. Levels of membrane-bound TREM2 were quantified by MSD ELISA. Data represent the mean ± SEM (n=3). One-way ANOVA, Tukey's post hoc test; p (DMSO vs GM) = 0.0011; p (DMSO vs isotype) = 0.992; p (isotype vs 4D9) = 0.0005; n.s., not significant.(C) Immunoblot analysis of conditioned media from HEK293 Flp-In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals decreased levels of sTREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor. An isotype antibody was used as a negative control. sAPPα served as a loading control. Note that heavy and light chains of the antibodies used for treatment are also detected and annotated. Levels of sTREM2 were quantified by MSD ELISA. Data represent the mean ± SEM (n=3). One-way ANOVA, Tukey's post hoc test; p (DMSO vs GM) < 0.0001; p (DMSO vs isotype) = 0.6372; p (isotype vs 4D9) < 0.0001; n.s., not significant.(D) 4D9 antibody selectively detects TREM2 on the cell surface of HEK293 Flp-In cells stably overexpressing mouse TREM2 and mouse DAP12. An anti-HA antibody was used as a positive control while empty vector transfected HEK293 Flp-In cells were used as a negative control. Scale bar= 10µm.(F) Sequence comparison of mouse TREM2 and human TREM2 shows substantial sequence conservation around the 4D9 epitope (upper panel). Immunoblot analysis demonstrates that antibody 4D9 is highly specific for mouse TREM2 and does not detect human TREM2 or mouse TREM1 (lower panel).(G) 4D9 binding to mouse TREM2 ECD is competed off by a stalk region peptide. A competition ELISA demonstrates that a dose titration of stalk peptide reduces binding of 4D9 to TREM2 ECD with an EC50 of 1.3uM. Data represent the mean ± SEM (n=3).(H) Surface plasmon resonance binding kinetics of increasing concentrations of 4D9 antibody to mouse TREM2 ectodomain evaluated by BIAcore, Kon = 5.9 x 105 M-1s-1, Koff = 4.0 x 10-5 s-1, KD = 68 pM. 4D9 binding to human TREM2 or mouse TREM1 was undetectable.


Figure 2 ( A ) Flow cytometry dose response curve for cell binding of 4D9 mAb ( EC50 = 0 . 29nM ) , 4D9 Fab ( EC50 = 0 . 17nM ) , and isotype to HEK cells stably overexpressing mouse TREM2 . Data represent the mean ± SEM ( n = 2 ) . ( B ) In vitro ADAM17 sheddase activity is blocked by 4D9 - effectorless mAb and 4D9 Fab fragment but not an isotype control . Fluorescence polarization of FAM conjugated TREM2 stalk peptide was detected in the presence or absence of ADAM17 and 4D9 mAb , 4D9 Fab and isotype control . Data represent the mean ± SEM ( n = 6 ) . One - way ANOVA , Tukey ' s post hoc test ; p ( 4D9 Fab vs 4D9 mAb ) = 0 . 8855 ; p ( 4D9 Fab vs uncleaved ) < 0 . 0001 ; p ( 4D9 mAb vs uncleaved ) < 0 . 0001 ; n . s . , not significant . ( C ) ELISA - mediated quantification of sTREM2 in conditioned media from ( D ) AlphaLISA - mediated quantification of pSYK levels in HEK293 Flp - In cells stably overexpressing mouse TREM2 and mouse DAP12 treated with a dose titration of 4D9 mAb , 4D9 Fab or isotype for 5 minutes . Data represent the mean ± SEM ( n = 3 ) . ( E ) AlphaLISA - mediated quantification of pSYK levels in HEK293 Flp - In cells stably overexpressing mouse TREM2 and mouse DAP12 or empty vector treated with 1mg / ml POPC / POPS liposomes and 20µg / ml 4D9 mAb , 4D9 Fab or isotype for 5 minutes . pSYK levels were also determined for cells treated with liposomes only . Data represent the mean ± SEM ( n = 3 ) . Two - way ANOVA , Tukey ' s post hoc test ( cell line effect : F1 , 16 = 365 . 7 , p = < 0 . 0001 ; treatment effect : F3 , 16 = 39 . 35 , p < 0 . 0001 ; cell line x treatment effect : F3 , 16 = 38 . 75 , p < 0 . 0001 ) ; p ( no Ab vs isotype ) = 0 . 6218 ; p ( no Ab vs 4D9 mAb ) < 0 . 0001 ; p ( no Ab vs 4D9 Fab ) = 0 . 7301 ; p ( isotype vs 4D9 mAb ) < 0 . 0001 ; p ( isotype vs 4D9 Fab ) > 0 . 9999 ; p ( 4D9 mAb vs 4D9 Fab ) < 0 . 0001 ; n . s . , not significant .

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Figure 2(A) Flow cytometry dose response curve for cell binding of 4D9 mAb (EC50= 0.29nM), 4D9 Fab (EC50=0.17nM), and isotype to HEK cells stably overexpressing mouse TREM2. Data represent the mean ± SEM (n=2).(B) In vitro ADAM17 sheddase activity is blocked by 4D9-effectorless mAb and 4D9 Fab fragment but not an isotype control. Fluorescence polarization of FAM conjugated TREM2 stalk peptide was detected in the presence or absence of ADAM17 and 4D9 mAb, 4D9 Fab and isotype control. Data represent the mean ± SEM (n=6). One-way ANOVA, Tukey's post hoc test; p (4D9 Fab vs 4D9 mAb) = 0.8855; p (4D9 Fab vs uncleaved) < 0.0001; p (4D9 mAb vs uncleaved) < 0.0001; n.s., not significant.(C) ELISA-mediated quantification of sTREM2 in conditioned media from (D) AlphaLISA-mediated quantification of pSYK levels in HEK293 Flp-In cells stably overexpressing mouse TREM2 and mouse DAP12 treated with a dose titration of 4D9 mAb, 4D9 Fab or isotype for 5 minutes. Data represent the mean ± SEM (n=3).(E) AlphaLISA-mediated quantification of pSYK levels in HEK293 Flp-In cells stably overexpressing mouse TREM2 and mouse DAP12 or empty vector treated with 1mg/ml POPC/POPS liposomes and 20µg/ml 4D9 mAb, 4D9 Fab or isotype for 5 minutes. pSYK levels were also determined for cells treated with liposomes only. Data represent the mean ± SEM (n=3). Two-way ANOVA, Tukey's post hoc test (cell line effect: F1,16=365.7, p=<0.0001; treatment effect: F3,16=39.35, p<0.0001; cell line x treatment effect: F3,16=38.75, p<0.0001); p (no Ab vs isotype) = 0.6218; p (no Ab vs 4D9 mAb) < 0.0001; p (no Ab vs 4D9 Fab) = 0.7301; p (isotype vs 4D9 mAb) < 0.0001; p (isotype vs 4D9 Fab) > 0.9999; p (4D9 mAb vs 4D9 Fab) < 0.0001; n.s., not significant.


Figure 3 ( A ) Flow cytometry detection of cell surface binding of 4D9 ( blue ) and isotype ( green ) to wild type and Trem2 - / - mouse bone marrow derived macrophages . Representative histograms are shown as MFI ( mean fluorescent intensity ) . Graphs represent the median signal intensity ± SEM ( n = 3 ) . Student ' s t - test ( two - tailed ) ; Trem2 + / + : p = 0 . 0005 ; Trem2 - / - : p = 0 . 4435 ; n . s . , not significant . ( B ) Immunoblot analysis of membrane fractions of bone marrow derived macrophages ( BMDM ) upon treatment with 4D9 antibody reveals increased levels of membrane - bound TREM2 relative to an isotype antibody . BMDM from TREM2 knockout mice were included to show the specificity of the anti - TREM2 antibody . Calnexin served as a loading control . Levels of membrane - bound TREM2 were quantified by MSD ELISA . TREM2 antibody clone 5F4 was used for detection . Data represent the mean ± SEM ( n = 5 - 6 ) . Unpaired t - test ( two - tailed ) with Welch ' s correction ; p < 0 . 0001 . No gender - specific differences could be observed . ( C ) Immunoblot analysis of sTREM2 in conditioned media from BMDM upon treatment with 4D9 and isotype antibodies . BMDM from TREM2 knockout mice were included to show the specificity of the anti - TREM2 antibody . Soluble APP served as a loading control . Note that heavy and light chains of the antibodies used for treatment are also detected and annotated as follows : Ab hc = antibody heavy chain ; Ab lc = antibody light chain . Levels of sTREM2 were quantified by MSD ELISA . TREM2 antibody clone 5F4 was used for detection . Data represent the mean ± SEM ( n = 6 ) . Unpaired t - test ( two - tailed ) with Welch ' s correction ; p < 0 . 0001 . No gender - specific differences could be observed . ( D ) Wildtype , Trem2 + / - , and Trem2 - / - BMDMs were plated in low concentration of M - CSF on 4D9 or isotype coated wells for 5 days and cellular ATP levels were measured by luminescence detection to indicate cell viability . 4D9 and isotype data for each genotype represent the mean ± SEM ( n = 2 and n = 1 , respectively ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Flow cytometry detection of cell surface binding of 4D9 (blue) and isotype (green) to wild type and Trem2-/- mouse bone marrow derived macrophages. Representative histograms are shown as MFI (mean fluorescent intensity). Graphs represent the median signal intensity ± SEM (n=3). Student's t-test (two-tailed); Trem2+/+: p = 0.0005; Trem2-/-: p = 0.4435; n.s., not significant.(B) Immunoblot analysis of membrane fractions of bone marrow derived macrophages (BMDM) upon treatment with 4D9 antibody reveals increased levels of membrane-bound TREM2 relative to an isotype antibody. BMDM from TREM2 knockout mice were included to show the specificity of the anti-TREM2 antibody. Calnexin served as a loading control. Levels of membrane-bound TREM2 were quantified by MSD ELISA. TREM2 antibody clone 5F4 was used for detection. Data represent the mean ± SEM (n=5-6). Unpaired t-test (two-tailed) with Welch's correction; p<0.0001. No gender-specific differences could be observed.(C) Immunoblot analysis of sTREM2 in conditioned media from BMDM upon treatment with 4D9 and isotype antibodies. BMDM from TREM2 knockout mice were included to show the specificity of the anti-TREM2 antibody. Soluble APP served as a loading control. Note that heavy and light chains of the antibodies used for treatment are also detected and annotated as follows: Ab hc= antibody heavy chain; Ab lc= antibody light chain. Levels of sTREM2 were quantified by MSD ELISA. TREM2 antibody clone 5F4 was used for detection. Data represent the mean ± SEM (n=6). Unpaired t-test (two-tailed) with Welch's correction; p<0.0001. No gender-specific differences could be observed.(D) Wildtype, Trem2+/-, and Trem2-/- BMDMs were plated in low concentration of M-CSF on 4D9 or isotype coated wells for 5 days and cellular ATP levels were measured by luminescence detection to indicate cell viability. 4D9 and isotype data for each genotype represent the mean ± SEM (n=2 and n=1, respectively).


Figure 4 ( A ) Flow cytometry detection of cell surface 4D9 ( red ) binding and isotype control ( blue ) on primary wild type and Trem2 - / - mouse microglia . Data are shown as MFI ( mean fluorescent intensity ) . ( B ) Uptake assay for fluorescently labelled myelin , Aβ ( 1 - 42 ) and inactivated E . coli particles . The top row shows that myelin and Aβ ( pseudocoloured in white ) accumulate within the plasma membrane of primary microglia ( labeled with dylight - 649 isolectin and pseudocoloured in red ) . Overnight pre - treatment with antibody 4D9 significantly increased the percentage of substrate positive cells ( middle row ) . Pre - treatment with the isotope control did not affect the uptake rate ( bottom row ) . No changes in the uptake of E . coli particles were observed . Substrate uptake in the absence of antibody is shown in the top row . Hoechst 33342 was used to counter stain the nuclei ( in cyan ) and to assess cell density . Scale bar = 20µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Flow cytometry detection of cell surface 4D9 (red) binding and isotype control (blue) on primary wild type and Trem2-/- mouse microglia. Data are shown as MFI (mean fluorescent intensity).(B) Uptake assay for fluorescently labelled myelin, Aβ(1-42) and inactivated E.coli particles. The top row shows that myelin and Aβ (pseudocoloured in white) accumulate within the plasma membrane of primary microglia (labeled with dylight-649 isolectin and pseudocoloured in red). Overnight pre-treatment with antibody 4D9 significantly increased the percentage of substrate positive cells (middle row). Pre-treatment with the isotope control did not affect the uptake rate (bottom row). No changes in the uptake of E.coli particles were observed. Substrate uptake in the absence of antibody is shown in the top row. Hoechst 33342 was used to counter stain the nuclei (in cyan) and to assess cell density. Scale bar= 20µm.


Figure 5 ( A ) 4D9 demonstrates standard IgG pharmacokinetics in vivo . Peripheral clearance rates of 4D9 on a human IgG effectorless backbone compared to an isotype control were determined in wild type mice by hIgG ELISA detection of plasma antibody concentrations 1 and 24 hours , 4 and 7 days after 10 mg / kg intravenous injection of antibody . ( B ) Brain antibody concentration was measured 24hrs post intravenous dosing of isotype hIgG at 100mg / kg and 4D9 - hIgG dosed intravenously at 100 , 50 and 10mg / kg . Detection of hIgG levels by ELISA demonstrated dose dependent brain concentrations of 4D9 in the single digit nM range . Animals were perfused to minimize IgG contribution from the plasma . Data represent the mean ± SEM ( n = 5 ) . ( D ) Target engagement timecourse demonstrated near 100 % 4D9 - bound sTREM2 in CSF of wildtype mice at 24 hrs post dose . Over a 10 day timecourse with timepoints at day 1 , 2 , 4 , 7 and study termination at day 10 , the bound / total sTREM2 reduces gradually to reach ~ 50 % by day 10 . Animals were dosed intravenously with 50mg / kg of isotype and 4D9 antibodies . Data represent the mean ± SEM ( n = 5 ) . ( E ) Target engagement dose response demonstrated saturated bound sTREM2 at 50 and 10mg / kg with > 50 % bound sTREM2 at 1mg / kg of antibody . 4D9 - bound sTREM2 was undetectable at 0 . 1mg / kg and lower . 4D9 was IV - dosed at 50 , 10 , 1 , 0 . 1 , 0 . 01 , 0 . 001 , 0 . 0001 mg / kg , and isotype dosed at 50mg / kg . CSF bound : total sTREM2 in wildtype mice was measured at 24hrs . Data represent the mean ± SEM ( n = 5 ) . ( F ) 4D9 antibody demonstrates dose dependent increase of total brain TREM2 levels . Quantification of total TREM2 in brain lysates from wildtype mice dosed with 4D9 or isotype control was performed by a MSD - platform based ELISA . Data represent the mean ± SEM ( n = 5 ) and are shown as pg TREM2 per ug of total protein . One - way ANOVA , Dunnett ' s post hoc test , p ( isotype vs 4D9 ( 100mg / kg ) ) < 0 . 0001 ; p ( isotype vs 4D9 ( 50mg / kg ) ) = 0 . 0007 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) 4D9 demonstrates standard IgG pharmacokinetics in vivo. Peripheral clearance rates of 4D9 on a human IgG effectorless backbone compared to an isotype control were determined in wild type mice by hIgG ELISA detection of plasma antibody concentrations 1 and 24 hours, 4 and 7 days after 10 mg/kg intravenous injection of antibody.(B) Brain antibody concentration was measured 24hrs post intravenous dosing of isotype hIgG at 100mg/kg and 4D9-hIgG dosed intravenously at 100, 50 and 10mg/kg. Detection of hIgG levels by ELISA demonstrated dose dependent brain concentrations of 4D9 in the single digit nM range. Animals were perfused to minimize IgG contribution from the plasma. Data represent the mean ± SEM (n=5).(D) Target engagement timecourse demonstrated near 100% 4D9-bound sTREM2 in CSF of wildtype mice at 24 hrs post dose. Over a 10 day timecourse with timepoints at day 1, 2, 4, 7 and study termination at day 10, the bound/total sTREM2 reduces gradually to reach ~50% by day 10. Animals were dosed intravenously with 50mg/kg of isotype and 4D9 antibodies. Data represent the mean ± SEM (n=5).(E) Target engagement dose response demonstrated saturated bound sTREM2 at 50 and 10mg/kg with >50% bound sTREM2 at 1mg/kg of antibody. 4D9-bound sTREM2 was undetectable at 0.1mg/kg and lower. 4D9 was IV-dosed at 50, 10, 1, 0.1, 0.01, 0.001, 0.0001 mg/kg, and isotype dosed at 50mg/kg. CSF bound: total sTREM2 in wildtype mice was measured at 24hrs. Data represent the mean ± SEM (n=5).(F) 4D9 antibody demonstrates dose dependent increase of total brain TREM2 levels. Quantification of total TREM2 in brain lysates from wildtype mice dosed with 4D9 or isotype control was performed by a MSD-platform based ELISA. Data represent the mean ± SEM (n=5) and are shown as pg TREM2 per ug of total protein. One-way ANOVA, Dunnett's post hoc test, p (isotype vs 4D9 (100mg/kg)) < 0.0001; p (isotype vs 4D9 (50mg/kg)) = 0.0007.