Detection of nascent synthesis in mitochondrial fraction of ovary . C . Nascent synthesis of Tamas TFAM was decreased in mdi1 ovary , whereas mtSSB was not affected . Tfamgfp , Tamasgfp mtSSBgfp were expressed in wt or mdi1 background . Nascent were labeled by AHA incorporation then GFP - tagged was immunopurified with a GFP antibody nascent synthesis was detected by anti - biotin antibody .

O O O O O B-SUBCELLULAR O O B-TISSUE O O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD B-TISSUE O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O

Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. C. Nascent protein synthesis of Tamas and TFAM was decreased in mdi1 ovary, whereas mtSSB was not affected. Tfamgfp, Tamasgfp and mtSSBgfp were expressed in wt or mdi1 background. Nascent proteins were labeled by AHA incorporation and then the GFP-tagged protein was immunopurified with a GFP antibody and the nascent protein synthesis was detected by anti-biotin antibody.


Detection of nascent synthesis in mitochondrial fraction of ovary . D . Representative profile of 254 nm absorbance of wt , mdi1 mdi1 / TL ovary extracts .

O O O O O B-SUBCELLULAR O O B-TISSUE O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-TISSUE O O

Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. D. Representative profile of 254 nm absorbance of wt, mdi1 and mdi1/TL ovary extracts.


Detection of nascent synthesis in mitochondrial fraction of ovary . E . Polysome mRNA profiling for tamas , tfam , mtSSB , cox4 mRPL19 in wt , mdi1 mdi1 / TL ovary . of mRNA for each gene in non - polysomal fractions ( N . P , including ribosomal subunits monosome - associated ) polysomal fractions ( poly ) was calculated plotted . fractions of tamas , tfam , cox4 mRPL19 mRNAs in polysomal fractions were significantly decreased in mdi1 compared to wt , but were restored in mdi1 / TL flies . N = 4 for samples . P values of comparing wt to mdi1 : tamas , P = 0 . 0055 ; tfam , P = 0 . 001 ; cox4 , P = 0 . 0066 ; mRPL19 , P = 0 . 0097 . P values of comparing mdi1 / TL to mdi1 : tamas , P = 0 . 0206 ; tfam , P = 0 . 0346 ; cox4 , P = 0 . 0036 ; mRPL19 , P = 0 . 0016 .

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

Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. E. Polysome mRNA profiling for tamas, tfam, mtSSB, cox4 and mRPL19 in wt, mdi1 and mdi1/TL ovary. The percentage of mRNA for each gene in non-polysomal fractions (N.P, including ribosomal subunits and monosome-associated) and polysomal fractions (poly) was calculated and plotted. The fractions of tamas, tfam, cox4 and mRPL19 mRNAs in the polysomal fractions were significantly decreased in mdi1 compared to wt, but were restored in mdi1/TL flies. N=4 for all samples. P values of comparing wt to mdi1: tamas, P= 0.0055; tfam, P= 0.001; cox4, P=0.0066; mRPL19, P=0.0097. P values of comparing mdi1/TL to mdi1: tamas, P= 0.0206; tfam, P= 0.0346; cox4, P=0.0036; mRPL19, P=0.0016.


Detection of nascent synthesis in mitochondrial fraction of ovary . F . nascent synthesis of COX4 mRPL19 were decreased in mdi1 ovary were restored mdi1 / TL flies . were immunopurified with antibodies against endogenous .

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

Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. F. The nascent protein synthesis of COX4 and mRPL19 were decreased in mdi1 ovary and were restored mdi1/TL flies. The proteins were immunopurified with antibodies against the endogenous proteins.


A . Mitochondrial localization of a Tom20 - LarpGFP ( Tom20 - Larp ) fusion in an mdi1 egg chamber stained with ATP - S to reveal mitochondria . Bars , 20 m .

O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD B-TISSUE I-TISSUE O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O O

A. Mitochondrial localization of a Tom20-LarpGFP (Tom20-Larp) fusion protein in an mdi1 egg chamber stained with ATP-S to reveal mitochondria. Bars, 20 m.


B . Hatching rates mtDNA contents of eggs produced by flies with different genotypes relative to wt control ; each data point represents mean of 3 independent replicates . Bars , std . Expression of Tom20 - Larp in mdi1 ( mdi1 ; Tom20 - Larp ) significantly restored mtDNA level hatching rate . N = 3 x > 100 eggs / genotype for hatching rate . relative mtDNA level was average of 3 biological repeats . P values of comparing mdi1 ; Tom20 - Larp to mdi1 : for mtDNA level , P = 0 . 0209 ; for hatching rate , P = 3 . 4758E - 05 .

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

B. Hatching rates and mtDNA contents of eggs produced by flies with different genotypes relative to wt control; each data point represents the mean of 3 independent replicates. Bars, std. Expression of Tom20-Larp in mdi1 (mdi1;Tom20-Larp) significantly restored the mtDNA level and hatching rate. N= 3 x >100 eggs / genotype for hatching rate. The relative mtDNA level was the average of 3 biological repeats. P values of comparing mdi1;Tom20-Larp to mdi1: for mtDNA level, P=0.0209; for hatching rate, P=3.4758E-05.


C Representative D - type motor neurons in wild - type mxl - 1 mutant 24 h after surgery . In wild - type , a severed axon has regenerated a growth cone ( arrow ) . In mutants , proximal ends of axons failed to regenerate ( arrowhead ) . Scale bar = 10 μm .

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

C Representative D-type motor neurons in wild-type and mxl-1 mutant animals 24 h after laser surgery. In wild-type animals, a severed axon has regenerated a growth cone (arrow). In mutants, proximal ends of axons failed to regenerate (arrowhead). Scale bar = 10 μm.


D Percentages of axons that initiated regeneration 24 h after surgery . numbers ( n ) of examined are shown . Error bars indicate 95 % confidence intervals ( CI ) . Data information statistical significance was determined by Fisher ' s exact ; * P < 0 . 05 ; * * * P < 0 . 001 ; NS ; not significant .

O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Percentages of axons that initiated regeneration 24 h after laser surgery. The numbers (n) of animals examined are shown. Error bars indicate 95% confidence intervals (CI). Data information statistical significance was determined by Fisher's exact test; *P < 0.05; ***P <0.001; NS; not significant.


Percentages of axons that initiated regeneration 24 h after surgery . numbers ( n ) of examined are shown . Error bars indicate 95 % CI . Data information : In ( A ) , statistical significance was determined by Fisher ' s exact ; * * * P < 0 . 001 ; NS ; not significant .

O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Percentages of axons that initiated regeneration 24 h after laser surgery. The numbers (n) of animals examined are shown. Error bars indicate 95% CI. Data information: In (A), statistical significance was determined by Fisher's exact test; ***P <0.001; NS; not significant.


B Induction of Psvh - 2 : : nls : : venus expression in D - type motor neurons by surgery . Expression of in D - type motor neurons of wild - type , mxl - 1 , tdpt - 1 mxl - 1 ; tdpt - 1 3 h after surgery are shown . Yellow arrowheads arrows indicate axon bodies , respectively , of D - type neurons . arrows indicate sites of surgery . D neurons are visualized by CFP under control of unc - 25 promoter . bodies of D - type neurons are magnified shown within red boxes . Most of intestinal in these photos is from endogenous variable background autofluorescence . A schematic representation of D - type motor neurons is shown on bottom . Scale bars = 10 μm .

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

B Induction of Psvh-2::nls::venus expression in D-type motor neurons by laser surgery. Expression of fluorescent proteins in D-type motor neurons of wild-type, mxl-1, tdpt-1 and mxl-1; tdpt-1 animals 3 h after laser surgery are shown. Yellow arrowheads and white arrows indicate axon and cell bodies, respectively, of D-type neurons. White arrows indicate the sites of laser surgery. D neurons are visualized by CFP under control of the unc-25 promoter. Cell bodies of D-type neurons are magnified and shown within the red boxes. Most of the intestinal fluorescence in these photos is from endogenous and variable background autofluorescence. A schematic representation of D-type motor neurons is shown on the bottom. Scale bars = 10 μm.


Percentages of axons that initiated regeneration 24 h after surgery . numbers ( n ) of examined are shown . Error bars indicate 95 % CI . Data information : statistical significance was determined by Fisher ' s exact ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; NS ; not significant .

O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Percentages of axons that initiated regeneration 24 h after laser surgery. The numbers (n) of animals examined are shown. Error bars indicate 95% CI. Data information: statistical significance was determined by Fisher's exact test; *P < 0.05; **P < 0.01; ***P < 0.001; NS; not significant.


B Interaction of MXL - 1 with TDPT - 1 by yeast two - hybrid assays . reporter yeast L40u was co - transformed with expression vectors encoding LexA DBD - MXL - 1 GAL4 AD - TDPT - 1 as indicated . Yeasts carrying indicated plasmids were grown on a selective plate lacking histidine containing 10 mM 5 - aminotriazole for 4 days .

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

B Interaction of MXL-1 with TDPT-1 by yeast two-hybrid assays. The reporter yeast strain L40u was co-transformed with expression vectors encoding LexA DBD-MXL-1 and GAL4 AD-TDPT-1 as indicated. Yeasts carrying the indicated plasmids were grown on a selective plate lacking histidine and containing 10 mM 5-aminotriazole for 4 days.


B Interaction between ETS - 4 TDPT - 1 . COS - 7 were transfected with plasmids encoding T7 - TDPT - 1 , HA - ETS - 4 ( WT ) , HA - ETS - 4 ( S73E ) ( SE ) HA - ETS - 4 ( S73A ) ( SA ) , as indicated . Whole - extracts ( WCE ) immunoprecipitated complexes obtained with anti - T7 antibody ( IP : T7 ) were analyzed by immunoblotting ( IB ) .

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

B Interaction between ETS-4 and TDPT-1. COS-7 cells were transfected with plasmids encoding T7-TDPT-1, HA-ETS-4 (WT), HA-ETS-4(S73E) (SE) and HA-ETS-4(S73A) (SA), as indicated. Whole-cell extracts (WCE) and immunoprecipitated complexes obtained with anti-T7 antibody (IP: T7) were analyzed by immunoblotting (IB).


C In vitro SUMOylation of ETS - 4 . COS - 7 were transfected with plasmids encoding HA - ETS - 4 ( WT ) HA - ETS - 4 ( K32A ; K83A ) ( 2KA ) , as indicated . lysates were immunoprecipitated with anti - HA antibody . immunoprecipitates were subjected to in vitro SUMOylation assays . Reaction mixtures were analyzed by immunoblotting ( IB ) with anti - SUMO - 2 / 3 anti - HA antibodies . Arrows mark SUMOylated - ETS - 4 bands .

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

C In vitro SUMOylation of ETS-4. COS-7 cells were transfected with plasmids encoding HA-ETS-4 (WT) and HA-ETS-4(K32A; K83A) (2KA), as indicated. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates were subjected to in vitro SUMOylation assays. Reaction mixtures were analyzed by immunoblotting (IB) with anti-SUMO-2/3 and anti-HA antibodies. Arrows mark the SUMOylated-ETS-4 bands.


E SUMOylation of ETS - 4 in mammalian . HEK293 were transfected with plasmids encoding HA - ETS - 4 ( WT ) , HA - ETS - 4 ( K32A ; K83A ) ( 2KA ) , T7 - TDPT - 1 , His - SUMO - 1 , His - SUMO - 2 Myc - MXL - 1 , as indicated . SUMO - conjugated were isolated by cobalt affinity chromatography . ETS - 4 was detected by immunoblotting with anti - HA antibody . Whole - extracts ( WCE ) were analyzed by immunoblotting ( IB ) .

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

E SUMOylation of ETS-4 in mammalian cells. HEK293 cells were transfected with plasmids encoding HA-ETS-4 (WT), HA-ETS-4(K32A; K83A) (2KA), T7-TDPT-1, His-SUMO-1, His-SUMO-2 and Myc-MXL-1, as indicated. SUMO-conjugated proteins were isolated by cobalt affinity chromatography. ETS-4 was detected by immunoblotting with anti-HA antibody. Whole-cell extracts (WCE) were analyzed by immunoblotting (IB).


A Effect of phosphorylation of ETS - 4 on SUMOylation of ETS - 4 in mammalian . HEK293 were transfected with plasmids encoding HA - ETS - 4 ( WT ) , HA - ETS - 4 ( K32A ; K83A ) ( 2KA ) , HA - ETS - 4 ( S73A ) ( SA ) , HA - ETS - 4 ( S73E ) ( SE ) , T7 - TDPT - 1 His - SUMO - 2 , as indicated . SUMO - conjugated were isolated by cobalt affinity chromatography . ETS - 4 was detected by immunoblotting with anti - HA antibody . Whole - extracts ( WCE ) were analyzed by immunoblotting ( IB ) .

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

A Effect of phosphorylation of ETS-4 on SUMOylation of ETS-4 in mammalian cells. HEK293 cells were transfected with plasmids encoding HA-ETS-4 (WT), HA-ETS-4(K32A; K83A) (2KA), HA-ETS-4(S73A) (SA), HA-ETS-4(S73E) (SE), T7-TDPT-1 and His-SUMO-2, as indicated. SUMO-conjugated proteins were isolated by cobalt affinity chromatography. ETS-4 was detected by immunoblotting with anti-HA antibody. Whole-cell extracts (WCE) were analyzed by immunoblotting (IB).


B Effect of SUMOylation of ETS - 4 on PKA phosphorylation of ETS - 4 in vitro . COS - 7 were transfected with plasmids encoding HA - ETS - 4 ( WT ) , HA - ETS - 4 ( S73A ) ( SA ) HA - ETS - 4 ( K32A ; K83A ) ( 2KA ) , as indicated . lysates were immunoprecipitated with anti - HA antibody . immunoprecipitates were subjected to in vitro PKA kinase assays or in vitro SUMOylation then in vitro PKA kinase assays , using recombinant GST - fused active PKA , as indicated . Reaction mixtures were analyzed by immunoblotting ( IB ) with anti - phospho - PKA substrate , anti - SUMO - 2 / 3 anti - HA antibodies . Arrows mark SUMOylated - ETS - 4 bands . Asterisk indicates non - SUMOylated - phosphorylated - ETS - 4 bands .

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

B Effect of SUMOylation of ETS-4 on PKA phosphorylation of ETS-4 in vitro. COS-7 cells were transfected with plasmids encoding HA-ETS-4 (WT), HA-ETS-4(S73A) (SA) and HA-ETS-4(K32A; K83A) (2KA), as indicated. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates were subjected to in vitro PKA kinase assays or in vitro SUMOylation and then in vitro PKA kinase assays, using recombinant GST-fused active PKA, as indicated. Reaction mixtures were analyzed by immunoblotting (IB) with anti-phospho-PKA substrate, anti-SUMO-2/3 and anti-HA antibodies. Arrows mark the SUMOylated-ETS-4 bands. Asterisk indicates the non-SUMOylated- and phosphorylated-ETS-4 bands.


Percentages of axons that initiated regeneration 24 h after surgery are shown . numbers ( n ) of examined are shown . Error bars indicate 95 % CI . Data information statistical significance was determined by Fisher ' s exact ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; NS ; not significant .

O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Percentages of axons that initiated regeneration 24 h after laser surgery are shown. The numbers (n) of animals examined are shown. Error bars indicate 95% CI. Data information statistical significance was determined by Fisher's exact test; *P < 0.05; **P < 0.01; ***P < 0.001; NS; not significant.


Percentages of axons that initiated regeneration 24 h after surgery are shown . numbers ( n ) of examined are shown . Error bars indicate 95 % CI . Data information : statistical significance was determined by Fisher ' s exact ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; NS ; not significant .

O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Percentages of axons that initiated regeneration 24 h after laser surgery are shown. The numbers (n) of animals examined are shown. Error bars indicate 95% CI. Data information: statistical significance was determined by Fisher's exact test; *P < 0.05; **P < 0.01; ***P < 0.001; NS; not significant.


( B ) Autophagy ‐ negative phenotype of apg16Δ . autophagic ability of wild ‐ type ( TN125 ) , apg12Δ ( YNM107 ) apg16Δ ( YNM114 ) was measured by alkaline phosphatase before ( black bars ) after ( bars ) nitrogen starvation for 4 h . Error bars indicate standard deviation of three independent experiments .

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

(B) Autophagy‐negative phenotype of the apg16Δ strain. The autophagic ability of wild‐type (TN125), apg12Δ (YNM107) and apg16Δ cells (YNM114) was measured by the alkaline phosphatase assay before (black bars) and after (white bars) nitrogen starvation for 4 h. Error bars indicate the standard deviation of three independent experiments.


( C ) Loss of viability during starvation . Wild ‐ type ( KA311B , ○ ) , apg12Δ ( YNM101 , ● ) apg16Δ ( YNM124 , ▴ ) were cultured in nitrogen starvation medium , their viability was determined by phloxine B .

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

(C) Loss of viability during starvation. Wild‐type (KA311B, ○), apg12Δ (YNM101, ●) and apg16Δ cells (YNM124, ▴) were cultured in nitrogen starvation medium, and their viability was determined by phloxine B staining.


( D ) Defect in API maturation in apg16Δ . Transport of pro ‐ API to vacuole was examined by immunoblotting with anti ‐ API antiserum . positions of precursor mature API are indicated .

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(D) Defect in API maturation in apg16Δ cells. Transport of pro‐API to the vacuole was examined by immunoblotting with anti‐API antiserum. The positions of precursor and mature API are indicated.


( E ) Apg12p - Apg5p conjugation in apg16Δ . Total lysates were prepared by NaOH / 2 ‐ mercaptoethanol extraction method from apg12Δ ( YNM107 ) apg12Δapg16Δ ( YNM115 ) , both carrying pHA ‐ APG12 ( CEN ) . Western blot was performed using anti ‐ HA antibody .

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(E) The Apg12p-Apg5p conjugation in apg16Δ cells. Total lysates were prepared by the NaOH/2‐mercaptoethanol extraction method from apg12Δ (YNM107) and apg12Δapg16Δ cells (YNM115), both carrying pHA‐APG12 (CEN). Western blot analysis was performed using anti‐HA antibody.


Apg16p interacts with Apg12p - Apg5p conjugate but not with Apg12p monomer . apg12Δapg16Δ ( YNM115 ) were transformed with high copy ( 2μ ) plasmids encoding MycApg12p / or HAApg16p as indicated . Total lysates were immunoprecipitated with anti ‐ Myc ( lanes 1 - 4 ) or anti ‐ HA antibody ( lanes 5 - 8 ) detected by immunoblotting using anti ‐ Myc or anti ‐ HA antibody . positions of IgG heavy chain κ chain are indicated .

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Apg16p interacts with the Apg12p-Apg5p conjugate but not with the Apg12p monomer. The apg12Δapg16Δ cells (YNM115) were transformed with high copy (2μ) plasmids encoding MycApg12p and/or HAApg16p as indicated. Total lysates were immunoprecipitated with anti‐Myc (lanes 1-4) or anti‐HA antibody (lanes 5-8) and detected by immunoblotting using anti‐Myc or anti‐HA antibody. The positions of the IgG heavy chain and κ light chain are indicated.


Apg16p interacts preferentially with Apg12p ‐ modified Apg5p . Total lysates were prepared from apg16Δ ( YNM124 ) or apg12Δapg16Δ ( YNM115 ) harboring both pHA ‐ APG5 pMyc ‐ APG16 CEN plasmids . lysates were immunoprecipitated analyzed as described in Figure 3 .

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Apg16p interacts preferentially with Apg12p‐modified Apg5p. Total lysates were prepared from apg16Δ cells (YNM124) or apg12Δapg16Δ cells (YNM115) harboring both pHA‐APG5 and pMyc‐APG16 CEN plasmids. The lysates were immunoprecipitated and analyzed as described in Figure 3.


Apg16p interacts preferentially with Apg12p ‐ modified Apg5p . Total lysates were prepared from apg16Δcells ( YNM124 ) or apg12Δapg16Δ ( YNM115 ) harboring both pHA ‐ APG5 pMyc ‐ APG16 CEN plasmids . lysates were immunoprecipitated analyzed as described in Figure 3 .

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Apg16p interacts preferentially with Apg12p‐modified Apg5p. Total lysates were prepared from apg16Δcells (YNM124) or apg12Δapg16Δ cells (YNM115) harboring both pHA‐APG5 and pMyc‐APG16 CEN plasmids. The lysates were immunoprecipitated and analyzed as described in Figure 3.


( A ) PJ69 ‐ 4A were co ‐ transformed with each pGBD pGAD plasmid as indicated . Transformants were selected on Trp − Leu − plates , then two ‐ hybrid interaction ( + or − ) was assessed for growth on Ade − Trp − Leu − plates .

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(A) PJ69‐4A cells were co‐transformed with each pGBD and pGAD plasmid as indicated. Transformants were selected on Trp− Leu− plates, and then two‐hybrid interaction (+ or −) was assessed for growth on Ade− Trp− Leu− plates.


( B ) Self ‐ multimerization of Apg16p in vivo . Lysates of apg16Δ ( YNM124 ) transformed with pMyc ‐ APG16 ( 2μ ) / or pHA ‐ APG16 ( 2μ ) were immunoprecipitated with anti ‐ Myc antibody detected with anti ‐ HA antibody .

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(B) Self‐multimerization of Apg16p in vivo. Lysates of apg16Δ cells (YNM124) transformed with pMyc‐APG16 (2μ) and/or pHA‐APG16 (2μ) were immunoprecipitated with anti‐Myc antibody and detected with anti‐HA antibody.


Apg16p mediates Apg5p assembly . Immunoprecipitation was carried out as described in Figure 3 , using apg12Δ ( YNM107 ) apg12Δapg16Δ ( YNM115 ) carrying pMyc ‐ APG12 ( CEN ) pHA ‐ APG12 ( CEN ) ( A ) , or pMyc ‐ APG12 ( CEN ) pHA ‐ APG5 ( CEN ) ( B ) . Following SDS - PAGE , immunoprecipitates were subjected to immunoblot using anti ‐ HA or anti ‐ Myc antibody as indicated . A model of Apg12p - Apg5p - Apg16p is illustrated in each panel . Apg16p forms a homo ‐ oligomer through its C ‐ terminal coiled ‐ coil region . It is not clear whether Apg16p forms a dimer or a larger multimer . N ‐ terminal region of each Apg16p interacts with Apg5p ; most of these complexes are conjugated to Apg12p ( A ) but a small population are unmodified ( B ) . In absence of Apg16p , separate Apg5ps do not assemble .

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Apg16p mediates Apg5p assembly. Immunoprecipitation was carried out as described in Figure 3, using apg12Δ (YNM107) and apg12Δapg16Δ cells (YNM115) carrying pMyc‐APG12 (CEN) and pHA‐APG12 (CEN) (A), or pMyc‐APG12 (CEN) and pHA‐APG5 (CEN) (B). Following SDS-PAGE, immunoprecipitates were subjected to immunoblot analysis using anti‐HA or anti‐Myc antibody as indicated. A model of the Apg12p-Apg5p-Apg16p complex is illustrated in each panel. Apg16p forms a homo‐oligomer through its C‐terminal coiled‐coil region. It is not clear whether Apg16p forms a dimer or a larger multimer. The N‐terminal region of each Apg16p interacts with Apg5p; most of these complexes are conjugated to Apg12p (A) but a small population are unmodified (B). In the absence of Apg16p, separate Apg5ps do not assemble.


Apg16p mediates Apg5p assembly . Immunoprecipitation was carried out as described in Figure 3 , using apg12Δ ( YNM107 ) apg12Δapg16Δ ( YNM115 ) carrying pMyc ‐ APG12 ( CEN ) pHA ‐ APG5 ( CEN ) ( B ) . Following SDS - PAGE , immunoprecipitates were subjected to immunoblot using anti ‐ HA or anti ‐ Myc antibody as indicated . A model of Apg12p - Apg5p - Apg16p is illustrated in each panel . Apg16p forms a homo ‐ oligomer through its C ‐ terminal coiled ‐ coil region . It is not clear whether Apg16p forms a dimer or a larger multimer . N ‐ terminal region of each Apg16p interacts with Apg5p ; most of these complexes are conjugated to Apg12p ( A ) but a small population are unmodified ( B ) . In absence of Apg16p , separate Apg5ps do not assemble .

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Apg16p mediates Apg5p assembly. Immunoprecipitation was carried out as described in Figure 3, using apg12Δ (YNM107) and apg12Δapg16Δ cells (YNM115) carrying pMyc‐APG12 (CEN) and pHA‐APG5 (CEN) (B). Following SDS-PAGE, immunoprecipitates were subjected to immunoblot analysis using anti‐HA or anti‐Myc antibody as indicated. A model of the Apg12p-Apg5p-Apg16p complex is illustrated in each panel. Apg16p forms a homo‐oligomer through its C‐terminal coiled‐coil region. It is not clear whether Apg16p forms a dimer or a larger multimer. The N‐terminal region of each Apg16p interacts with Apg5p; most of these complexes are conjugated to Apg12p (A) but a small population are unmodified (B). In the absence of Apg16p, separate Apg5ps do not assemble.


Expression of Apg16p are dependent on Apg12p - Apg5p conjugate . Total lysates were prepared by NaOH / 2 ‐ mercaptoethanol extraction method from apg16Δ ( YNM124 ) , apg5Δapg16Δ ( YNM126 ) apg12Δapg16Δ ( YNM115 ) carrying pHA ‐ APG16 ( CEN ) . Western blot was performed using anti ‐ HA antibody .

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Expression levels of Apg16p are dependent on the Apg12p-Apg5p conjugate. Total lysates were prepared by the NaOH/2‐mercaptoethanol extraction method from apg16Δ (YNM124), apg5Δapg16Δ (YNM126) and apg12Δapg16Δ cells (YNM115) carrying pHA‐APG16 (CEN). Western blot analysis was performed using anti‐HA antibody.


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Apg16p exists in a pellet fraction in an Apg5p ‐ dependent manner . Spheroplasts were generated from harboring pHA ‐ APG5 ( A ) , pHA ‐ APG16 ( B ) or pHA ‐ APG16 ( 2μ ) with or without pHA ‐ APG5 ( 2μ ) ( C ) . They were lysed by Dounce homogenization . After unbroken spheroplasts were removed by low ‐ speed centrifugation , supernatant ( T ) was fractionated by 100 000 g centrifugation for 60 min to generate a supernatant ( S ) pellet ( P ) fraction . Each fraction was subjected to immunoblot using anti ‐ HA antibody .

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Apg16p exists in a pellet fraction in an Apg5p‐dependent manner. Spheroplasts were generated from cells harboring pHA‐APG5 (A), pHA‐APG16 (B) or pHA‐APG16 (2μ) with or without pHA‐APG5 (2μ) (C). They were lysed by Dounce homogenization. After unbroken spheroplasts were removed by low‐speed centrifugation, the supernatant (T) was fractionated by 100 000 g centrifugation for 60 min to generate a supernatant (S) and pellet (P) fraction. Each fraction was subjected to immunoblot analysis using anti‐HA antibody.


Pregnant dams were injected with 4 - OHT at days E10 . 5 E11 . 5 for at day E12 . 5 or E10 . 5 , E11 . 5 , E12 . 5 of development for later times of Developmental loss of VE - cadherin leads to severe edema . Fetuses of genotypes listed in top row were analyzed at developmental stages in first column . Numbers in brackets denote with signs of edema . Last column lists of viable fetuses in VE - cadherin deleted cohort Fetuses were explanted at day E14 . 5 . Arrowheads indicate prominent edema along back . are representative for 42 ( Cdh5lox / lox ) 25 ( Cdh5lox / lox ; Prox1 - CreERT2 ) analyzed

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Pregnant dams were injected with 4-OHT at days E10.5 and E11.5 for analysis at day E12.5 or E10.5, E11.5, and E12.5 of development for later times of analysis Developmental loss of VE-cadherin leads to severe edema. Fetuses of the genotypes listed in the top row were analyzed at the developmental stages in the first column. Numbers in brackets denote embryos with signs of edema. Last column lists the percentage of viable fetuses in the VE-cadherin deleted cohort Fetuses were explanted at day E14.5. Arrowheads indicate prominent edema along the back. The images are representative for 42 (Cdh5lox/lox) and 25 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals


Pregnant dams were injected with 4 - OHT at days E10 . 5 E11 . 5 for at day E12 . 5 or E10 . 5 , E11 . 5 , E12 . 5 of development for later times of Skin wholemount preparations stained for PROX1 , PECAM1 VEGFR - 3 . Maximum projections ( MIPs ) of tiled confocal stacks ( Cdh5lox / lox 2075µm x 2075µm , z = 50µm ; Cdh5lox / lox ; Prox1 - CreERT2 3765µm x 3765 µm , z = 61µm ) . Scale bars = 100 μm . data are representative for 6 ( Cdh5lox / lox ) 6 ( Cdh5lox / lox ; Prox1 - CreERT2 ) analyzed from 3

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Pregnant dams were injected with 4-OHT at days E10.5 and E11.5 for analysis at day E12.5 or E10.5, E11.5, and E12.5 of development for later times of analysis Skin wholemount preparations stained for PROX1, PECAM1 and VEGFR-3. Maximum intensity projections (MIPs) of tiled confocal stacks (Cdh5lox/lox 2075µm x 2075µm, z=50µm; Cdh5lox/lox; Prox1-CreERT2 3765µm x 3765 µm, z=61µm). Scale bars = 100 μm. The data are representative for 6 (Cdh5lox/lox) and 6 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals from 3 litters


Pregnant dams were injected with 4 - OHT at days E10 . 5 E11 . 5 for at day E12 . 5 or E10 . 5 , E11 . 5 , E12 . 5 of development for later times of Enumeration of PROX1 - expressing ( PROX1 + ) nuclei in three different VE - cadherin deleted wholemount preparations analogous to Fig . 1B . Nuclei were counted in a MIP corresponding to an of 1 . 5 mm2 normalized to number of nuclei in Cdh5lox / lox control preparations covered by lymphatic vessels was determined in samples evaluated in ( D ) is depicted as relative compared to Cdh5lox / lox controls . calculations measurements were obtained from three three confocal stacks per biological group . PROX1 - positive nuclei were counted using particle tool of

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Pregnant dams were injected with 4-OHT at days E10.5 and E11.5 for analysis at day E12.5 or E10.5, E11.5, and E12.5 of development for later times of analysis Enumeration of the PROX1-expressing (PROX1+) nuclei in three different VE-cadherin deleted wholemount preparations analogous to Fig. 1B. Nuclei were counted in a MIP corresponding to an area of 1.5 mm2 and normalized to the number of nuclei in Cdh5lox/lox control preparations The area covered by lymphatic vessels was determined in the samples evaluated in (D) and is depicted as relative area compared to Cdh5lox/lox controls. The calculations and measurements were obtained from three animals and three confocal stacks per biological group. PROX1-positive nuclei were counted using the particle analysis tool of Fiji


Deletion of VE - cadherin in lymphatic endothelial of newborn pups was induced by two optimal doses of 4 - OHT at days P2 P4 . For 6 later ear punches were subjected to wholemount immunostaining of dermal vasculature . A - H ( A , B , E , F ) Overview MIPs of ear skin wholemount preparations immunostained for indicated antigens . VEC , VE - cadherin ; PRX , PROX1 . ( C , D , G , H ) Magnified views of areas in red boxes in ( A , E ) . Areas delimited by stippled yellow boxes in ( C ) ( G ) are enlarged in insets ( yellow solid boxes ) . Yellow arrows indicate widened lumen , yellow arrowheads denote oak leaf - shaped LECs with discontinuous junctions . Scale bars in ( A E ) correspond to 100 μm in ( C G ) to 50 µm . data are representative for 6 ( Cdh5lox / lox ) 5 ( Cdh5lox / lox ; Prox1 - CreERT2 ) analyzed from 2 litters

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Deletion of VE-cadherin in lymphatic endothelial cells of newborn pups was induced by two optimal doses of 4-OHT at days P2 and P4. For analysis 6 weeks later ear punches were subjected to wholemount immunostaining of the dermal vasculature. A-H (A, B, E, F) Overview MIPs of ear skin wholemount preparations immunostained for the indicated antigens. VEC, VE-cadherin; PRX, PROX1. (C, D, G, H) Magnified views of the areas in the red boxes in (A, E). Areas delimited by the stippled yellow boxes in (C) and (G) are enlarged in the insets (yellow solid boxes). Yellow arrows indicate widened lumen, yellow arrowheads denote oak leaf-shaped LECs with discontinuous junctions. Scale bars in (A and E) correspond to 100 μm and in (C and G) to 50 µm. The data are representative for 6 (Cdh5lox/lox) and 5 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals from 2 litters


A - L VE - cadherin deletion was initiated either in newborn pups ( A , B , E , F ) by two applications of 4 - OHT at days P2 P4 or in mice at 11 ( C , G ) 42 ( D , H ) of ( three applications of Tamoxifen at 2 day intervals via oral gavage ) . Ear skins were prepared 6 ( A , B , E , F ) or 5 ( C , D , G , H ) after induction immunostained for indicated . Shown are MIPs of confocal tile scans ( approx . 750 µm x 750 µm ) covering 40 µm in depth . arrows in ( F ) highlight distorted partially fragmented lymphatic vessels arrowheads denote aberrantly pointed vessels ( F ) . ( I - L ) Lymphatic valves in dermis of ear were maintained for 5 despite deletion of VE - cadherin at 11 of ( yellow arrows in K , L ) . PEC1 , PECAM1 ; PRX1 , PROX1 ; VR3 , VEGFR - 3 . Scale bars correspond to 100 μm . data represent wholemount stainings from 6 ( A , E ) , 5 ( B , F ) , 6 ( C , G , K , L ) , 3 ( D , H ) 6 ( I , J ) analyzed

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A-L VE-cadherin deletion was initiated either in newborn pups (A, B, E, F) by two applications of 4-OHT at days P2 and P4 or in mice at 11 weeks (C, G) and 42 weeks (D, H) of age (three applications of Tamoxifen at 2 day intervals via oral gavage). Ear skins were prepared 6 weeks (A, B, E, F) or 5 weeks (C, D, G, H) after induction and immunostained for the indicated proteins. Shown are MIPs of confocal tile scans (approx. 750 µm x 750 µm) covering 40 µm in depth. White arrows in (F) highlight distorted and partially fragmented lymphatic vessels and white arrowheads denote aberrantly pointed vessels (F). (I-L) Lymphatic valves in the dermis of the ear were maintained for 5 weeks despite deletion of VE-cadherin at 11 weeks of age (yellow arrows in K, L). PEC1, PECAM1; PRX1, PROX1;VR3, VEGFR-3. Scale bars correspond to 100 μm. The data represent wholemount stainings from 6 (A, E), 5 (B, F), 6 (C, G, K, L), 3 (D, H) and 6 (I, J) analyzed animals


Fig . 4 : VE - cadherin is indispensable for maintenance of mesenteric lymphatic vessels at ages . A - F Deletion of VE - cadherin was induced in mice at various ( 4 ( A ) , neonate ( B ) , 6 month ( C , D , E ) or 1 year ( F ) ) by either two applications of 4 - OHT at days P2 P4 ( neonates ) or three applications of Tamoxifen at 2 day intervals via oral gavage ( other ages ) . mesentery was prepared as indicated 4 , 6 , 8 or 11 later wholemount preparations were immunostained for specified in color ( left ) . ( B ) Inset in top panel shows a magnification of outlined by dashed ( for further magnification see expanded view Fig . 1A B ) . Arrows indicate previous position of lymphatic valves ; arrowheads indicate PECAM1 + capillaries supplying intestinal fat tissue lining vessels ( magnified in expanded view Fig . 1C ) ; yellow arrowheads denote island of LECs that display intracellular VEGFR - 3 . ( C ) Arrows denote LECs that have converted from growth as tubes to sheets ; arrowheads , thinning of lymphatic vessels ; yellow arrowheads , aberrant sprouting of LECs . outlined by a box is depicted magnified in ( D ) provides details on LECs growing as a sheet of . ( E ) Formation of ectopic sprouts from valve of degenerating lymphatic vessels . ( F ) Fully deteriorated lymphatic vessel with emanating sprouts that initiate sheet - like growth . PEC1 , PECAM1 ; PRX1 , PROX1 ; VR3 , VEGFR - 3 . Scale bars = 100 μm . data represent n = 5 ( A ) , n = 5 ( B ) , n = 3 ( C - E ) n = 6 ( F ) .

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

Fig. 4: VE-cadherin is indispensable for the maintenance of mesenteric lymphatic vessels at all ages. A-F Deletion of VE-cadherin was induced in mice at various age (4 weeks (A), neonate (B), 6 month (C, D, E) or 1 year (F)) by either two applications of 4-OHT at days P2 and P4 (neonates) or three applications of Tamoxifen at 2 day intervals via oral gavage (all other ages). The mesentery was prepared as indicated 4, 6, 8 or 11 weeks later and the wholemount preparations were immunostained for the proteins specified in color (left). (B) Inset in top panel shows a magnification of the area outlined by the white dashed line (for further magnification see expanded view Fig. 1A and B). Arrows indicate the previous position of lymphatic valves; white arrowheads indicate PECAM1+ capillaries supplying the intestinal fat tissue lining the vessels (magnified in expanded view Fig. 1C); yellow arrowheads denote island of LECs that display intracellular VEGFR-3 staining. (C) Arrows denote LECs that have converted from growth as tubes to sheets; white arrowheads, thinning of lymphatic vessels; yellow arrowheads, aberrant sprouting of LECs. The area outlined by a white box is depicted magnified in (D) and provides details on LECs growing as a sheet of cells. (E) Formation of ectopic sprouts from the valve area of degenerating lymphatic vessels. (F) Fully deteriorated lymphatic vessel with emanating sprouts that initiate sheet-like growth. PEC1, PECAM1; PRX1, PROX1; VR3, VEGFR-3. Scale bars = 100 μm. The data represent n=5 (A), n=5 (B), n=3 (C-E) and n=6 (F) animals.


Fig . 5 : Deletion of VE - cadherin results in fragmentation distension of intestinal lacteals . A - E Deletion of VE - cadherin was induced in mice at various ages ( neonatal ( C ) , 16 ( D ) or 1 year ( E ) ) by either two applications of 4 - OHT at days P2 P4 ( neonates ) or three applications of Tamoxifen at 2 day intervals via oral gavage ( other ages ) . Control mice of same were subjected to an identical regimen ( A , B ) . Intestinal wholemount preparations of jejunum ileum were generated 5 - 8 after Tamoxifen induction immunostained for indicated , scale bars = 100 μm . data represent wholemount stainings from 3 ( D ) , 5 ( B , E ) 6 ( A , C ) analyzed for each genotype .

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Fig. 5: Deletion of VE-cadherin results in fragmentation and distension of the intestinal lacteals. A-E Deletion of VE-cadherin was induced in mice at various ages (neonatal (C), 16 weeks (D) or 1 year (E)) by either two applications of 4-OHT at days P2 and P4 (neonates) or three applications of Tamoxifen at 2 day intervals via oral gavage (all other ages).Control mice of the same age were subjected to an identical regimen (A, B). Intestinal wholemount preparations of the jejunum and ileum were generated 5 - 8 weeks after Tamoxifen induction and immunostained for the indicated proteins, scale bars = 100 μm. The data represent wholemount stainings from 3 (D), 5 (B,E) and 6 (A, C) animals analyzed for each genotype.


Deletion of VE - cadherin was induced in adult mice ( 13 ) EdU incorporation into PROX1 - positive nuclei was determined 6 after Tamoxifen administration . Shown are mesenteric wholemount stainings prepared from control ( A , B ; n = 3 ) or VE - cadherin - deleted mice ( C , D ; n = 3 ) . ( B ) ( D ) show a magnification of outlined by dashed in ( A ) ( C ) . Antigens shown in addition to EdU incorporation are depicted in colour on left side of first panel . PEC , PECAM1 ; PRX1 , PROX1 . Scale bars = 100 µm Enumeration of EdU - positive LECs . measurements were obtained from three nine confocal stacks per biological group . PROX1 - positive nuclei were counted using particle tool of Fiji . Co - localization of PROX1 EdU was determined manually in each confocal plane , using counting tool of Fiji . data represent mean ± SD . * * * * p ≤ 0 . 0001 . Two - unpaired Student ' s t

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

Deletion of VE-cadherin was induced in adult mice (13 weeks) and EdU incorporation into PROX1-positive cell nuclei was determined 6 weeks after Tamoxifen administration. Shown are mesenteric wholemount stainings prepared from control (A, B; n=3) or VE-cadherin-deleted mice (C, D; n=3). (B) and (D) show a magnification of the area outlined by the white dashed line in (A) and (C). Antigens shown in addition to the EdU incorporation are depicted in colour on the left side of the first panel. PEC, PECAM1; PRX1, PROX1. Scale bars = 100 µm Enumeration of EdU-positive LECs. The measurements were obtained from three animals and nine confocal stacks per biological group. PROX1-positive nuclei were counted using the particle analysis tool of Fiji. Co-localization of PROX1 and EdU was determined manually in each confocal plane, using the cell counting tool of Fiji. The data represent mean ± SD. **** p ≤ 0.0001. Two-tailed unpaired Student's t test


Aberrantly enhanced VEGFR - 3 phosphorylation in VE - cadherin - deficient mesenteric LECs of six old mice that had received 4 - OHT injections at P2 P4 . Shown are wholemount immunostainings for antigens shown on left in color . pVR3 , pVEGFR - 3 , PRX1 , PROX1 ; PEC1 , PECAM1 . ( G ) ( I ) show a magnification of outlined by dashed box in ( F ) ( H ) . Scale bars = 50 μm . data are representative for 6 ( Cdh5lox / lox ) 5 ( Cdh5lox / lox ; Prox1 - CreERT2 ) analyzed from 2

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Aberrantly enhanced VEGFR-3 phosphorylation in VE-cadherin-deficient mesenteric LECs of six weeks old mice that had received 4-OHT injections at P2 and P4. Shown are wholemount immunostainings for the antigens shown on the left in color. pVR3, pVEGFR-3, PRX1, PROX1; PEC1, PECAM1. (G) and (I) show a magnification of the area outlined by the white dashed box in (F) and (H). Scale bars = 50 μm. The data are representative for 6 (Cdh5lox/lox) and 5 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals from 2 litters


Dermal , mesenteric intestinal tissue pieces of 15 week old control ( Cdh5lox / lox ) mice were dissociated total was extracted immediately . For reverse transcription 2 . 0 µg of total was used for each tissue type . Expression of lymphangiogenic factors VEGF - C VEGF - D was determined by quantitative real - time ( qRT ) - PCR using specific TaqMan probes . Samples were measured in triplicate diagrams are representative for 3 independent experiments . Relative expression ( ΔΔCt ) of target genes was normalized to control transcript

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Dermal, mesenteric and intestinal tissue pieces of 15 week old control (Cdh5lox/lox) mice were dissociated and total RNA was extracted immediately. For reverse transcription 2.0 µg of total RNA was used for each tissue type. Expression of lymphangiogenic factors VEGF-C and VEGF-D was determined by quantitative real-time (qRT)-PCR using specific TaqMan probes. Samples were measured in triplicate and diagrams are representative for 3 independent experiments. Relative expression (ΔΔCt) of target genes was normalized to the control transcript UBC


Loss of VEGFR - 3 surface expression on mesenteric LECs growing in sheets was associated with concomitant upregulation of VEGFR - 2 , scale bars = 50 μm . data are representative for 4 analyzed from 2 litters . VR2 , VEGFR - 2 ; VR3 , VEGFR - 3 , PRX1 ,

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Loss of VEGFR-3 surface expression on mesenteric LECs growing in cell sheets was associated with concomitant upregulation of VEGFR-2, scale bars = 50 μm. The data are representative for 4 analyzed animals from 2 litters.VR2, VEGFR-2; VR3, VEGFR-3, PRX1, PROX1


Expression of transcriptional co - activators YAP TAZ was analyzed in mesenteric wholemount preparations 8 after induction of VE - Cadherin deletion at 1 year of by three doses of Tamoxifen . Respective antigens depicted are indicated on top of panels in colour . highly related molecules YAP TAZ were both recognized by antibody used are therefore collectively abbreviated Y / T . Scale bars = 100

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Expression of the transcriptional co-activators YAP and TAZ was analyzed in mesenteric wholemount preparations 8 weeks after induction of VE-Cadherin deletion at 1 year of age by three doses of Tamoxifen. Respective antigens depicted are indicated on the top of the panels in colour. The highly related molecules YAP and TAZ were both recognized by the antibody used and are therefore collectively abbreviated Y/T. Scale bars = 100 μm


Fig . 7 : Loss of VE - cadherin from lymphatic endothelial results in increased circumferential actin a reduced number of focal adhesions . A , B dermal LECs were isolated as described in appendix Fig . S8 A . PdLECs were used for up to four passages control VE - cadherin - deleted pdLECs had been cultured for same number of passages . were seeded at a density of 2 . 5 x 104 on gelatine - coated 15mm glass coverslips co - stained after 3 days of culture for actin cytoskeleton ( Phalloidin ) either Vinculin ( A ) or FAK ( B ) for visualization of focal adhesions . PROX1 in ( A ) identifies LECs , nuclei in ( B ) were counterstained with Hoechst . Scale bars correspond to 50 µm . ( C , D ) Bar diagrams show number of focal adhesions / , which were enumerated using Image J from Vinculin FAK stainings shown in ( A B ) . Data are mean values + S . E . M . derived from three lines 9 viewfields of 256 µm x 256 µm per biological group . * * * p ≤ 0 . 001 . Two - unpaired Student ' s t .

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

Fig. 7: Loss of VE-cadherin from lymphatic endothelial cells results in increased circumferential actin and a reduced number of focal adhesions. A, B Primary dermal LECs were isolated as described in appendix Fig. S8 A. PdLECs were used for up to four passages and control and VE-cadherin-deleted pdLECs had been cultured for the same number of passages. Cells were seeded at a density of 2.5 x 104 cells on gelatine-coated 15mm glass coverslips and co-stained after 3 days of culture for the actin cytoskeleton (Phalloidin) and either Vinculin (A) or FAK (B) for the visualization of focal adhesions. PROX1 staining in (A) identifies LECs, nuclei in (B) were counterstained with Hoechst. Scale bars correspond to 50 µm. (C, D) Bar diagrams show the number of focal adhesions/cell, which were enumerated using Image J from the Vinculin and FAK stainings shown in (A and B). Data are mean values + S.E.M. derived from three cell lines and 9 viewfields of 256 µm x 256 µm per biological group. *** p ≤ 0.001. Two-tailed unpaired Student's t test.


Fig . 8 : Enhanced collective migration in VE - cadherin - deficient lymphatic endothelial . ( A ) Collective migration of VE - cadherin - deficient control LECs was assessed in wound scratch assays . LECs were grown up to four passages seeded at a density of 5x104 on fibronectin - coated 8 well µ - chamber slides ( growth 0 . 22 cm2 ) ; scale bar 500 µm ( B ) of zone denuded from is depicted in curve diagram showing mean values ± SD of three experiments conducted in quadruplets . * p ≤ 0 . 01 . Two - unpaired Student ' s t . Expanded View Figures - Legends

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Fig. 8: Enhanced collective cell migration in VE-cadherin-deficient lymphatic endothelial cells. (A) Collective cell migration of VE-cadherin-deficient and control LECs was assessed in wound scratch assays. LECs were grown up to four passages and seeded at a density of 5x104 cells on fibronectin-coated 8 well µ-chamber slides (growth area 0.22 cm2); scale bar 500 µm (B) The area of the zone denuded from cells is depicted in the curve diagram showing mean values ± SD of three individual experiments conducted in quadruplets. * p ≤ 0.01. Two-tailed unpaired Student's t test. Expanded View Figures - Legends


A . Endogenous Cyclin B1 - YFP localisation during mitosis live in RPE1 ( still from Movie EV1 ) .

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A. Endogenous Cyclin B1-YFP localisation during mitosis live in RPE1 cells (still from Movie EV1).


Immunofluorescence ( B ) of relative Cyclin B1 MAD1 at unattached attached kinetochores in arrested in STLC .

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Immunofluorescence images (B) of relative Cyclin B1 and MAD1 levels at unattached and attached kinetochores in cells arrested in STLC.


C . Immunofluorescence quantifications ( C ) of relative Cyclin B1 MAD1 at unattached attached kinetochores in arrested in STLC . Each dot represents a kinetochore , data is from 40 kinetochore pairs ( 13 , max 5 kinetochore pairs / ) .

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C. Immunofluorescence quantifications (C) of relative Cyclin B1 and MAD1 levels at unattached and attached kinetochores in cells arrested in STLC. Each dot represents a kinetochore, data is from 40 kinetochore pairs (13 cells, max 5 kinetochore pairs/cell).


D , E . of relative kinetochore intensities of Cyclin B1 MAD1 in nocodazole - arrested ( noco ) treated with MPS1 inhibitors , AZ - 3146 ( 5μM ) or Reversine ( 500nM ) , either before ( D ) or after ( E ) mitotic entry .

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D,E. Quantification of relative kinetochore intensities of Cyclin B1 and MAD1 in nocodazole-arrested cells (noco) treated with the MPS1 inhibitors, AZ-3146 (5μM) or Reversine (500nM), either before (D) or after (E) mitotic entry.


F . Immunofluorescence of LacI - MAD1 Cyclin B1 in U2OS containing a LacO arrays on chromosome

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F. Immunofluorescence images of LacI-MAD1 and Cyclin B1 in U2OS cells containing a LacO arrays on chromosome 1


G . Live imaging of Cyclin B1 - mCherry ( CycB1 - mCh ) in LacO - U2OS transfected with LacI - MAD1 - FL ( full : aa1 - 718 ) or various LacI - MAD1 truncations ( acid numbers indicated ) .

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G. Live imaging of Cyclin B1-mCherry (CycB1-mCh) in LacO-U2OS cells transfected with LacI-MAD1-FL (full length: aa1-718) or various LacI-MAD1 truncations (amino acid numbers indicated).


H , I . Immunofluorescence ( H ) quantifications ( I ) of Cyclin B1 MAD1 kinetochore in control ( MAD1 - WT ) or MAD1β HeLa ( two independent clones : C13 or C24 ) treated with nocodazole .

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H,I. Immunofluorescence images (H) and quantifications (I) of Cyclin B1 and MAD1 kinetochore levels in control (MAD1-WT) or MAD1β HeLa cells (two independent clones: C13 or C24) treated with nocodazole.


J , K . Immunofluorescence ( J ) ( K ) of Cyclin B1 MAD1 kinetochore localisation in doxycycline - inducible MAD1α β knockouts treated with or without dox for 10 days then arrested in nocodazole . were selected that had full MAD1 knockout in doxycycline treatment ( this constituted approximately 30 % of ) .

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J,K. Immunofluorescence images (J) and quantification (K) of Cyclin B1 and MAD1 kinetochore localisation in doxycycline-inducible MAD1α and β knockouts treated with or without dox for 10 days and then arrested in nocodazole. Cells were selected that had full MAD1 knockout in the doxycycline treatment (this constituted approximately 30% of cells).


L . Relative kinetochore volumes occupied by Cyclin B1 MAD1 ( relative to CenpC ) in nocodazole - arrested MAD1α MAD1β ( calculated from experiments shown in ( H , I ) .

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

L. Relative kinetochore volumes occupied by Cyclin B1 and MAD1 (relative to CenpC) in nocodazole-arrested MAD1α and MAD1β cells (calculated from experiments shown in (H,I).


A . Elution profiles SDS - PAGE for SEC runs on indicated column of Cyclin B1 : CDK1 ( blue profile ) , MBP - MAD1 : MAD2 ( red ) , their combination ( green ) . Note that same Cyclin B1 : CDK1 elution profile SDS - PAGE is also displayed as reference in panels D F to improve clarity . For same reason , MBP - MAD1 : MAD2 elution profile SDS - PAGE is also displayed in panels 2A - C . B . Elution profiles SDS - PAGE for SEC runs on indicated column of CDK1 ( blue ) , MBP - MAD1 : MAD2 ( red ) , their combination ( green ) . C . Elution profiles SDS - PAGE for SEC runs on indicated column of Cyclin B1 ( blue ) , MBP - MAD1 : MAD2 ( red ) , their combination ( green ) . D . Elution profiles SDS - PAGE for SEC runs of Cyclin B1 : CDK1 ( blue ) , MBP - MAD1 ∆ 93 : MAD2 ( green ) , their combination ( red ) .

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

A. Elution profiles and SDS-PAGE for SEC runs on the indicated column of the Cyclin B1:CDK1 complex (blue profile), MBP-MAD1:MAD2 (red), and their combination (green). Note that the same Cyclin B1:CDK1 elution profile and SDS-PAGE is also displayed as reference in panels D and F to improve clarity. For the same reason, the MBP-MAD1:MAD2 elution profile and SDS-PAGE is also displayed in panels 2A-C. B. Elution profiles and SDS-PAGE for SEC runs on the indicated column of CDK1 (blue), MBP-MAD1:MAD2 (red), and their combination (green). C. Elution profiles and SDS-PAGE for SEC runs on the indicated column of Cyclin B1 (blue), MBP-MAD1:MAD2 (red), and their combination (green). D. Elution profiles and SDS-PAGE for SEC runs of the Cyclin B1:CDK1 complex (blue), MBP-MAD1∆93:MAD2 (green), and their combination (red).


F . Elution profiles SDS - PAGE for SEC runs of Cyclin B1 : CDK1 ( blue ) , MBP - MAD13EK : MAD2 ( E52K , E53K , E56K mutations ; red ) , their combination ( green ) . In A - D , potential binding partners were combined at a of 5 µM .

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

F. Elution profiles and SDS-PAGE for SEC runs of Cyclin B1:CDK1 (blue), MBP-MAD13EK:MAD2 (E52K, E53K, and E56K mutations; red), and their combination (green). In A-D, individual potential binding partners were combined at a concentration of 5 µM.


A . Western blot of indicated vsv - MAD1 - WT or 3EK HeLa clones treated with or without doxycycline for 10 days .

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A. Western blot analysis of indicated vsv-MAD1-WT or 3EK HeLa clones treated with or without doxycycline for 10 days.


B . Immunofluorescence showing MAD1 ZW10 kinetochore in nocodazole - arrested MAD1 - WT - C13 3EK - C14 just after envelope breakdown ( early prometaphase ) or later in mitosis when is condensed ( late prometaphase ) . Note , that early late prometaphase was defined on level of condensation .

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B. Immunofluorescence images showing MAD1 and ZW10 kinetochore levels in nocodazole-arrested MAD1-WT-C13 AND 3EK-C14 just after nuclear envelope breakdown (early prometaphase) or later in mitosis when the chromatin is condensed (late prometaphase). Note, that early and late prometaphase was defined based on the level of chromatin condensation.


C , D . Relative kinetochore volumes occupied by MAD1 ( C ) ZW10 ( D ) ( relative to CenpC ) in MAD1 - WT - 3EK in early late prometaphase .

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C,D. Relative kinetochore volumes occupied by MAD1 (C) and ZW10 (D) (relative to CenpC) in MAD1-WT and -3EK cells in early and late prometaphase.


E . of MAD1 kinetochore from indicated MAD1 - WT 3EK clones treated as in ( B ) .

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E. Quantification of MAD1 kinetochore intensity from indicated MAD1-WT and 3EK clones treated as in (B).


A - C . Immunofluorescence ( A ) quantifications ( B , C ) of relative MAD1 MAD2 kinetochore in indicated lines arrested in nocodazole treated with / without AZ - 3146 for 30 min to inhibit MPS1 . In kinetochore graphs , each dot represents a , horizontal lines indicate median vertical bars show 95 % confidence interval . Note , when these vertical bars do not overlap difference is considered statistically significant at a level of at least p < 0 . 05 ( see methods ) . kinetochore graphs display data that is relative to WT C11 controls in nocodazole , which are normalised to 1 . mean level of normalised controls is indicated by dotted lines . 30 from 3 experiments . Scale bars = 5µM . D . Duration of mitotic arrest in indicated lines arrested in nocodazole then treated with 1 . 25 µM AZ - 3146 . Graph shows cumulative mean ( ± ) of 3 experiments , 50 per condition per experiment .

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

A-C. Immunofluorescence images (A) and quantifications (B,C) of relative MAD1 and MAD2 kinetochore levels in indicated cells lines arrested in nocodazole and treated with/without AZ-3146 for 30 min to inhibit MPS1. In all kinetochore intensity graphs, each dot represents a cell, horizontal lines indicate the median and vertical bars show the 95% confidence interval. Note, when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p<0.05 (see methods). All kinetochore intensity graphs display data that is relative to WT C11 controls in nocodazole, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines. 30 cells from 3 experiments. Scale bars = 5µM. D. Duration of mitotic arrest in indicated cell lines arrested in nocodazole and then treated with 1.25 µM AZ-3146. Graph shows cumulative mean (±SEM) of 3 experiments, 50 cells per condition per experiment.


A . Immunofluorescence of MAD1 MAD1 - pT716 kinetochore in nocodazole - arrested RPE1 treated with or without AZ - 3146 . Scale bars = 5µM .

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

A. Immunofluorescence images of MAD1 and MAD1-pT716 kinetochore levels in nocodazole-arrested RPE1 cells treated with or without AZ-3146. Scale bars = 5µM.


B . Electron of rotary - shadowed MAD193 - 718 : MAD2 ( top row ) , MBP - MAD1 : MAD2 ( middle row ) , MBP - MAD1 : MAD2 in with GST - CDK1 : Cyclin B1 ( bottom row ) . Scale bar = 50 nm .

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

B. Electron micrographs of rotary-shadowed MAD193-718:MAD2 (top row), MBP-MAD1:MAD2 (middle row), and MBP-MAD1:MAD2 in complex with GST-CDK1:Cyclin B1 (bottom row). Scale bar = 50 nm.


C . Quantifications ( top ) corresponding immunofluorescence ( underneath ) of relative kinetochore MAD1 MAD1 - pT716 in nocodazole - arrested of MAD1 - WT - C14 MAD1 - 3EK - C12 treated with different doses of AZ - 3146 for 30 min . MG132 was included at time of AZ - 3146 addition to prevent mitotic exit . Each dot represents a , horizontal lines indicate median error bars show 95 % confidence interval . Note , when these vertical bars do not overlap difference is considered statistically significant at a level of at least p < 0 . 05 ( see methods ) . 30 from 3 experiments . Both kinetochore graphs display data that is relative to WT - C14 untreated controls , which are normalised to 1 . mean level of normalised controls is indicated by dotted lines .

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

C. Quantifications (top) and corresponding immunofluorescence images (underneath) of relative kinetochore MAD1 and MAD1-pT716 levels in nocodazole-arrested of MAD1-WT-C14 and MAD1-3EK-C12 cells treated with different doses of AZ-3146 for 30 min. MG132 was included at the time of AZ-3146 addition to prevent mitotic exit. Each dot represents a cell, horizontal lines indicate the median and error bars show the 95% confidence interval. Note, when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p<0.05 (see methods). 30 cells from 3 experiments. Both kinetochore intensity graphs display data that is relative to the WT-C14 untreated controls, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines.


D . Duration of mitotic arrest in MAD1 - WT C14 or MAD1 - 3EK C12 arrested in nocodazole then treated with indicated concentrations of AZ - 3146 . Note , 1 . 25 µM AZ - 3146 data is displayed in Figure 4D , but also included here to allow comparison with other drug doses . Graph shows cumulative mean ( ± ) of 3 experiments , 50 per condition per experiment .

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

D. Duration of mitotic arrest in MAD1-WT C14 or MAD1-3EK C12 cells arrested in nocodazole and then treated with indicated concentrations of AZ-3146. Note, the 1.25 µM AZ-3146 data is displayed in Figure 4D, but also included here to allow comparison with other drug doses. Graph shows cumulative mean (±SEM) of 3 experiments, 50 cells per condition per experiment.


shows dynamic expression of genes in SF3A / 3B ( E ) SR family ( F ) at each indicated differentiation stage . Both heatmaps were row normalized . heatmaps were scaled with Z - Score using log2 ( FPKM ) expression of indicated components . n = 3 technical replicates .

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

The heatmap shows the dynamic expression of genes in the SF3A/3B complex (E) and SR family (F) at each indicated differentiation stage. Both heatmaps were row normalized. The heatmaps were scaled with Z-Score using the log2(FPKM) expression of indicated components. n=3 technical replicates.


mRNA expression of splicing regulator SRSF2 splicing factor PCBP2 during hematopoietic differentiation were measured by RT - qPCR . ACTB gene was used as a control . Results given are mean ± standard deviation ( SD ) . P - values were determined by an unpaired two - Student ' s t - . n ≥ 3 biological replicates .

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

The mRNA expression of splicing regulator SRSF2 and splicing factor PCBP2 during hematopoietic differentiation were measured by RT-qPCR. The ACTB gene was used as a control. Results given are mean ± standard deviation (SD). P-values were determined by an unpaired two-tailed Student's t-test. n ≥ 3 biological replicates.


experimental schematic ( upper panel ) . APLNR + on day 2 were FACS - purified treated with various dosages of PLB from day 2 . 5 to 5 during inducing hematopoietic differentiation . On day 5 , generation of CD31 + CD34 + EPCs CD31 + CD34 + CD73 - HEPs were assessed . representative FACS plots showed frequency of APLNR + , CD31 + CD34 + EPCs CD31 + CD34 + CD73 - HEPs without or with PLB treatment . bar graphs show of CD31 + CD34 + EPCs CD31 + CD34 + CD73 - HEPs without or with PLB treatment .

O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O B-SMALL_MOLECULE O O

The experimental schematic (upper panel). APLNR+ cells on day 2 were FACS-purified and treated with various dosages of PLB from day 2.5 to 5 during inducing hematopoietic differentiation. On day 5, the generation of CD31+CD34+ EPCs and CD31+CD34+CD73- HEPs were assessed. The representative FACS plots showed the frequency of APLNR+ cells, CD31+CD34+ EPCs and CD31+CD34+CD73- HEPs without or with PLB treatment. The bar graphs show the percentage of CD31+CD34+ EPCs and CD31+CD34+CD73- HEPs without or with PLB treatment.


bar graph represents of CD31 + CD34 + from ( H ) . P - values were calculated by one - way ANOVA followed by Tukey ' s .

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The bar graph represents the percentage of CD31+CD34+ cells from (H). P-values were calculated by one-way ANOVA followed by Tukey's test.


bar graph representing of CD31 + CD34 + from ( K ) . P - values were determined by an unpaired two - Student ' s t - .

The bar graph representing the percentage of CD31+CD34+ cells from (K). P-values were determined by an unpaired two-tailed Student's t-test.


representative FACS plots show frequency of CD31 + CD34 + EPCs in GFP + population at day 5 with 1 . 25 nM PLB supplementation alone or in combination with NUMB _ S ectopic expression from day 2 . 5 . bar graph denotes of CD31 + CD34 + in GFP + as described in ( F ) . P - values were determined by an unpaired two - Student ' s t - .

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

The representative FACS plots show the frequency of CD31+CD34+ EPCs in the GFP+ population at day 5 with 1.25 nM PLB supplementation alone or in combination with NUMB_S ectopic expression from day 2.5. The bar graph denotes the percentage of CD31+CD34+ in GFP+ cells as described in (F). P-values were determined by an unpaired two-tailed Student's t-test.


On day 5 of hematopoietic differentiation , inclusion or exclusion of NUMB exon 9 were detected by RT - PCR with 1 . 25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2 . 5 . bar graph showing signaling of each specific band from RT - PCR electropherogram .

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

On day 5 of hematopoietic differentiation, the inclusion or exclusion of NUMB exon 9 were detected by RT-PCR with 1.25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2.5. The bar graph showing the signaling intensity of each specific band from the RT-PCR electropherogram.


top panel depicts location of specific primers targeting NUMB _ S NUMB _ L isoform . bottom bar graph showing expression of NUMB _ S NUMB _ L in on day 5 of differentiation with 1 . 25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2 . 5 with RT - qPCR .

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

The top panel depicts the location of specific primers targeting NUMB_S and NUMB_L isoform. The bottom bar graph showing the expression of NUMB_S and NUMB_L in cells on day 5 of differentiation with 1.25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2.5 with RT-qPCR.


plotting expression of NUMB _ S key components in NOTCH signaling in day 2 - APLNR + , day 5 - DMSO treated day 5 - PLB treated ( 2 . 5 nM ) , respectively . NOTCH components were divided into groups of upregulated , downregulated , unchanged by comparison between DMSO PLB treated . was scaled with Z - Score using log2 ( FPKM ) expression of indicated genes . n = 2 technical

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

The heatmap plotting the expression of NUMB_S and the key components in NOTCH signaling in day 2-APLNR+ cells, day 5-DMSO treated and day 5-PLB treated cells (2.5 nM), respectively. The NOTCH components were divided into groups of upregulated, downregulated, and unchanged by comparison between DMSO and PLB treated cells. The heatmap was scaled with Z-Score using the log2(FPKM) expression of indicated genes. n=2 technical replicates


representative FACS plots indicating generation of CD31 + CD34 + EPCs on day 5 of differentiation with or without HES1 overexpression . DOX was treated from day 2 . 5 to 5 to induce HES1 overexpression . Statistical of of CD31 + CD34 + EPCs of ( H ) .

O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O

The representative FACS plots indicating the generation of CD31+CD34+ EPCs on day 5 of differentiation with or without HES1 overexpression. DOX was treated from day 2.5 to 5 to induce HES1 overexpression. Statistical analysis of the percentage of CD31+CD34+ EPCs of (H).


B In vivo transduction profile of luciferase reporter vectors displaying variants of enriched library capsid motifs or unmodified wild type AAV2 control capsid . Four after i . v . injection of 5x1010 genomic particles / mouse containing a CMV - luciferase reporter gene , luminescence was measured in brain control organs . Data are shown as bars ( mean ) with plotted data points ( n = 3 / group , 8 - 12 ) .

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

B In vivo transduction profile of luciferase reporter vectors displaying variants of the enriched library capsid sequence motifs or unmodified wild type AAV2 control capsid. Four weeks after i.v. injection of 5x1010 genomic particles/mouse containing a CMV-luciferase reporter gene, luminescence was measured in the brain and control organs. Data are shown as bars (mean) with plotted individual data points (n = 3 animals/group, age 8-12 weeks).


A Luciferase reporter gene vectors displaying brain - targeted peptide NRGTEWD ( " BR1 " ) , unmodified wild type AAV2 capsid , or previously reported brain - targeting peptide DSPAHPS ( PPS ) . Vectors were intravenously injected into mice ( 5x1010 genomic particles / mouse ) . Panels show representative examples of n = 5 , 8 - 12 per group . were imaged in dorsal ( left panel ) , ventral ( second from left panel ) , lateral ( second from right right panel ) position , 14 days after vector injection . B Close - up imaging of AAV - BR1 - treated mice . Mice were imaged in dorsal lateral position in a different color scheme allowing detailed visualization of transduced brain in living , even as late as at day 264 after vector injection . C Virtual sections : sagittal , coronal , transaxial ( left panels ) three - dimensional reconstruction ( right panel ) of luminescence of a mouse injected with BR1 vector ( as in A ) . were obtained by measuring different wavelengths of emitted ( Living Image software ) , confirming brain as exclusive source of luminescence .

O B-GENEPROD O O O O B-TISSUE O O O O O O B-GENEPROD O O O O O O B-ORGANISM B-SUBCELLULAR O O O O B-TISSUE O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O

A Luciferase reporter gene vectors displaying the brain-targeted peptide NRGTEWD ("BR1"), unmodified wild type AAV2 capsid, or the previously reported brain-targeting peptide DSPAHPS (PPS). Vectors were intravenously injected into mice (5x1010 genomic particles/mouse). Panels show representative examples of n = 5 animals, age 8-12 weeks per group. Animals were imaged in dorsal (left panel), ventral (second from left panel), and lateral (second from right and right panel) position, 14 days after vector injection.B Close-up imaging of AAV-BR1-treated mice. Mice were imaged in dorsal and lateral position in a different color scheme allowing detailed visualization of the transduced brain in living animals, even as late as at day 264 after vector injection.C Virtual sections : sagittal, coronal, and transaxial (left panels) and three-dimensional reconstruction (right panel) of the luminescence images of a mouse injected with BR1 vector (as in A). Images were obtained by measuring different wavelengths of the emitted light (Living Image software), confirming the brain as exclusive source of luminescence.


AAV - BR1 vector harboring luciferase gene under control of CAG promoter was administered intravenously ( 5x1010 genomic particles / mouse , 8 ) . Long - term transgene expression was analyzed by in vivo bioluminescence imaging at 16 time points during a 665 - day period ( n = 2 ) . Original of analyzed ( above ) of luminescence in brain as region of interest = ROI ( bottom ) .

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AAV-BR1 vector harboring the luciferase gene under control of the CAG promoter was administered intravenously (5x1010 genomic particles/mouse, age 8 weeks). Long-term transgene expression was analyzed by in vivo bioluminescence imaging at 16 time points during a 665-day period (n = 2 animals). Original images of analyzed animals (above) and quantification of luminescence in the brain as the region of interest = ROI (bottom).


Luciferase activity vector copy numbers were determined in tissue lysates 14 days after vector administration ( 5x1010 genomic particles / mouse , 8 - 12 ) . A Vector - mediated luminescence . Transgene expression was measured in AAV - BR1 harboring luciferase gene under control of CAG promoter control vectors ( AAV - PPS wild type rAAV2 ) in brain off - target control organs ( left panel ) . Comparison of luminescence in brain mediated by AAV - BR1 control vectors ( right panel ) , * * * * p < 0 . 0001 ( BR1 : brain vs . ) , * * * p = 0 . 0002 ( PPS : heart vs . brain / muscle ) , * * * p = 0 . 0001 ( PPS : heart vs . lung / liver / spleen / kidney ) , * p = 0 . 0101 ( WT : heart vs . lung ) , * p = 0 . 0120 ( WT : heart vs . spleen ) , * p = 0 . 0148 ( WT : heart vs . kidney ) , * p = 0 . 0113 ( WT : heart vs . muscle ) , * * p = 0 . 0011 ( Brain : BR1 vs . PPS ) , * * * p = 0 . 0009 ( Brain : BR1 vs . WT ) .

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Luciferase activity and vector copy numbers were determined in tissue lysates 14 days after vector administration (5x1010 genomic particles/mouse, age 8-12 weeks).A Vector-mediated luminescence. Transgene expression was measured in AAV-BR1 harboring the luciferase gene under control of the CAG promoter and control vectors (AAV-PPS and wild type rAAV2) in brain and off-target control organs (left panel). Comparison of luminescence in the brain mediated by AAV-BR1 and control vectors (right panel), ****p < 0.0001 (BR1: brain vs. all), ***p = 0.0002 (PPS: heart vs. brain/ muscle), ***p = 0.0001 (PPS: heart vs. lung/ liver/ spleen/ kidney), *p = 0.0101 (WT: heart vs. lung), *p = 0.0120 (WT: heart vs. spleen), *p = 0.0148 (WT: heart vs. kidney), *p = 0.0113 (WT: heart vs. muscle), **p = 0.0011 (Brain: BR1 vs. PPS), ***p = 0.0009 (Brain: BR1 vs. WT).


Luciferase activity vector copy numbers were determined in tissue lysates 14 days after vector administration ( 5x1010 genomic particles / mouse , 8 - 12 ) . B Biodistribution of AAV - BR1 control vectors ( AAV - PPS wild type rAAV2 ) in brain off - target control organs , excluding spleen ( left panel ) . Genome copy numbers of AAV - BR1 control vectors ( AAV - PPS wild type rAAV2 ) in brain ( right panel ) , * * * * p < 0 . 0001 ( BR1 : brain vs . liver / kidney / muscle ) , * * * p = 0 . 0006 ( BR1 : brain vs . heart ) , * * * p = 0 . 0003 ( BR1 : brain vs . lung ) , * * p = 0 . 0025 ( PPS : kidney vs . muscle ) , * p = 0 . 0378 ( PPS : kidney vs . lung ) , * p = 0 . 0114 ( PPS : kidney vs . liver ) , * * * * * p < 0 . 0001 ( WT : liver vs . ) , * * p = 0 . 0028 ( Brain : BR1 vs . PPS / WT ) .

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Luciferase activity and vector copy numbers were determined in tissue lysates 14 days after vector administration (5x1010 genomic particles/mouse, age 8-12 weeks).B Biodistribution of AAV-BR1 and control vectors (AAV-PPS and wild type rAAV2) in brain and off-target control organs, excluding spleen (left panel). Genome copy numbers of AAV-BR1 and control vectors (AAV-PPS and wild type rAAV2) in the brain (right panel), ****p < 0.0001 (BR1: brain vs. liver/ kidney/ muscle), ***p = 0.0006 (BR1: brain vs. heart), ***p = 0.0003 (BR1: brain vs. lung), **p = 0.0025 (PPS: kidney vs. muscle), *p = 0.0378 (PPS: kidney vs. lung), *p = 0.0114 (PPS: kidney vs. liver), *****p < 0.0001 (WT: liver vs. all), **p = 0.0028 (Brain: BR1 vs. PPS/ WT).


C57BL / 6 mice ( 8 ) were injected with AAV - BR1 harboring an eGFP reporter gene under control of CAG promoter . show representative examples of n = 6 mice . A Representative from cerebellum , olfactory bulb , striatum , cerebral cortex , spinal cord , 14 days after vector injection . BR1 - eGFP - transduced ( green ) were positive for endothelial marker CD31 ( red ) . Scale bars represent 250 µm .

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C57BL/6 mice (age 8 weeks) were injected with AAV-BR1 harboring an eGFP reporter gene under control of the CAG promoter. Images show representative examples of n = 6 mice.A Representative images from cerebellum, olfactory bulb, striatum, cerebral cortex, and the spinal cord, 14 days after vector injection. BR1-eGFP-transduced cells (green) were positive for the endothelial marker CD31 (red). Scale bars represent 250 µm.


C57BL / 6mice ( 8 ) were injected with AAV - BR1 harboring an eGFP reporter gene under control of CAG promoter . show representative examples of n = 6 mice . B Higher magnification confocal microscopy . endothelial marker CD31 ( red ) colocalizes with vector - mediated eGFP expression ( green ) . Scale bars represent 50 µm ( upper panel ) or 10 µm ( lower panel ) .

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C57BL/6mice (age 8 weeks) were injected with AAV-BR1 harboring an eGFP reporter gene under control of the CAG promoter. Images show representative examples of n = 6 mice.B Higher magnification confocal microscopy images. The endothelial marker CD31 (red) colocalizes with vector-mediated eGFP expression (green). Scale bars represent 50 µm (upper panel) or 10 µm (lower panel).