( B ) UCSC genome browser view of normalized TT - seq coverage on the plus and minus strand at the 2 . 8 Mbp gene desert surrounding MYC ( hg38 ; chr8 : 127 , 042 , 406 - 129 , 779 , 109 ; ( Kent et al . , 2002 ) ) in fourteen cancer cell lines . eRNAs represent the union from all cell lines . Genome - wide association studies ( GWAS ) risk loci contain GWAS single nucleotide polymorphisms ( SNPs ) for prostate cancer ( PrCa ) , acute lymphocytic leukemia ( ALL ) , breast cancer ( BrCa ) , colorectal cancer ( CRC ) , and glioblastoma ( GBM ) including SNPs in linkage disequilibrium ( r2 ≥ 0 . 8 ) . TT - seq coverage is displayed between 8 - 250 to allow for better visualization of the eRNA signal surrounding MYC . Dashed rectangle is shown at full scale on the right . Note : TT - seq reads were mapped against the hg38 reference genome and we cannot rule out the existence of genomic rearrangements ( e . g . translocations ) affecting the shown region in the used cancer cell line clones .

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

(B) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the 2.8 Mbp gene desert surrounding MYC (hg38; chr8:127,042,406-129,779,109; (Kent et al., 2002)) in fourteen cancer cell lines. eRNAs represent the union from all cell lines. Genome-wide association studies (GWAS) risk loci contain GWAS single nucleotide polymorphisms (SNPs) for prostate cancer (PrCa), acute lymphocytic leukemia (ALL), breast cancer (BrCa), colorectal cancer (CRC), and glioblastoma (GBM) including SNPs in linkage disequilibrium (r2 ≥ 0.8). TT-seq coverage is displayed between 8-250 to allow for better visualization of the eRNA signal surrounding MYC. Dashed rectangle is shown at full scale on the right. Note: TT-seq reads were mapped against the hg38 reference genome and we cannot rule out the existence of genomic rearrangements (e.g. translocations) affecting the shown region in the used cancer cell line clones.


( A ) Med1 - defined enhancers ranked by Med1 ChIP - seq signal ( Yan et al . , 2013 ) in LoVo colorectal cancer cells . Super - enhancers ( black rectangles ) were determined as enhancer regions lying above the inflection point of the

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(A) Med1-defined enhancers ranked by Med1 ChIP-seq signal (Yan et al., 2013) in LoVo colorectal cancer cells. Super-enhancers (black rectangles) were determined as enhancer regions lying above the inflection point of the curve


( B ) Increased eRNA synthesis from super - enhancers . Boxplots comparing eRNA synthesis ( TT - seq signal ) at Med1 - defined typical enhancers ( n = 2 , 111 ) and super - enhancers ( n = 130 ) in LoVo cells ( analysis was restricted to intergenic enhancers Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Notches represent 95 % confidence intervals for the median values . Outliers not shown . P - value by two - sided Mann - Whitney U - test .

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(B) Increased eRNA synthesis from super-enhancers. Boxplots comparing eRNA synthesis (TT-seq signal) at Med1-defined typical enhancers (n=2,111) and super-enhancers (n=130) in LoVo cells (analysis was restricted to intergenic enhancers Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Notches represent 95% confidence intervals for the median values. Outliers not shown. P-value by two-sided Mann-Whitney U-test.


( C ) Super - enhancers are associated with the production of long eRNAs . Boxplots showing length of TT - seq - defined eRNAs originating from Med1 - defined super - enhancers ( n = 552 ) or other enhancer regions ( n = 6 , 934 ) . Median , hinges , whiskers and notches are shown as in ( B ) . P - value by two - sided Mann - Whitney U - test .

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(C) Super-enhancers are associated with the production of long eRNAs. Boxplots showing length of TT-seq-defined eRNAs originating from Med1-defined super-enhancers (n=552) or other enhancer regions (n=6,934). Median, hinges, whiskers and notches are shown as in (B). P-value by two-sided Mann-Whitney U-test.


( D ) Barplot showing the number of TT - seq enhancer regions per Med1 - defined super - enhancer region in LoVo cells . Super - enhancers spanning a protein - coding TSS ( n = 32 ) are excluded from this comparison as regions TSS ± 1 kbp were excluded from TT - seq enhancer calling .

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(D) Barplot showing the number of TT-seq enhancer regions per Med1-defined super-enhancer region in LoVo cells. Super-enhancers spanning a protein-coding TSS (n=32) are excluded from this comparison as regions TSS ± 1 kbp were excluded from TT-seq enhancer calling.


( E ) UCSC genome browser view of normalized TT - seq coverage on the plus and minus strand at the PHLDA1 locus ( hg38 ; chr12 : 75 , 612 , 350 - 76 , 042 , 552 ; in the colorectal cancer cell line HCT 116 . TT - seq coverage is cut at 250 ( purple lines ) to allow for better visualization . eRNAs are shown between the plus and minus strand . Aggregated transcription factor ChIP - seq peak regions ( TF ChIP - seq ) comprise ChIP - seq binding profiles of 19 TFs in HCT 116 cells from CISTROME The height of the signal indicates the number of different TFs binding at a particular region . Binding regions for selected TFs ( TCF7l2 , YY1 , SP1 ) are shown below together with p300 and RNA polymerase II ( Pol II ) . TT - seq enhancer regions are shown at the bottom together with a H3K27ac - defined super - enhancer annotation , which was downloaded from SEdb 2019 ) . At date of publication no Med1 ChIP - seq data was available for HCT 116 .

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

(E) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the PHLDA1 locus (hg38; chr12:75,612,350-76,042,552; in the colorectal cancer cell line HCT 116. TT-seq coverage is cut at 250 (purple lines) to allow for better visualization. eRNAs are shown between the plus and minus strand. Aggregated transcription factor ChIP-seq peak regions (TF ChIP-seq) comprise ChIP-seq binding profiles of 19 TFs in HCT 116 cells from CISTROME The height of the signal indicates the number of different TFs binding at a particular region. Binding regions for selected TFs (TCF7l2, YY1, SP1) are shown below together with p300 and RNA polymerase II (Pol II). TT-seq enhancer regions are shown at the bottom together with a H3K27ac-defined super-enhancer annotation, which was downloaded from SEdb 2019). At date of publication no Med1 ChIP-seq data was available for HCT 116.


( A ) Unsupervised clustering of all pairwise Spearman correlations of normalized TT - seq signal for mRNAs ( left ) and eRNAs ( right ) for the union of transcripts over all fourteen cancer cell lines Grey and colored bars on top indicate gender and cancer type , respectively .

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(A) Unsupervised clustering of all pairwise Spearman correlations of normalized TT-seq signal for mRNAs (left) and eRNAs (right) for the union of transcripts over all fourteen cancer cell lines Grey and colored bars on top indicate gender and cancer type, respectively.


( B ) Heatmaps showing mRNAs ( left ) and eRNAs ( right ) as being synthesized ( red , normalized TT - seq signal FPK ≥ 15 ) or not ( blue , normalized TT - seq signal FPK < 15 ) for all fourteen cancer cell lines ( columns ) . Heatmaps show only transcripts ( rows ) with normalized FPK ≥ 30 in at least one of the cell lines ( mRNAs , n = 14 , 040 ; eRNAs n = 48 , 105 ) . Heatmaps are ordered by increasing cell type specificity , as indicated by the grey bars to the right showing the number of cell lines a transcript is observed in .

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(B) Heatmaps showing mRNAs (left) and eRNAs (right) as being synthesized (red, normalized TT-seq signal FPK ≥ 15) or not (blue, normalized TT-seq signal FPK < 15) for all fourteen cancer cell lines (columns). Heatmaps show only transcripts (rows) with normalized FPK ≥ 30 in at least one of the cell lines (mRNAs, n=14,040; eRNAs n=48,105). Heatmaps are ordered by increasing cell type specificity, as indicated by the grey bars to the right showing the number of cell lines a transcript is observed in.


( D ) Boxplots showing normalized TT - seq signal for nearest active promoters in the cell line in which enhancers are exclusively transcribed ( from left to right : n = 890 ; 500 ; 494 ; 214 ; 238 ; 239 ; 378 ; 62 ; 153 ; 879 ; 586 ; 173 ; 601 ; 232 ) compared to the same promoters in all other cell lines in which the respective enhancers are not transcribed . All pairwise comparisons are significantly different ( P - values < 1e - 9 , two - sided Mann - Whitney U - test ) . Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Notches represent 95 % confidence intervals for the median values . Outliers not shown .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplots showing normalized TT-seq signal for nearest active promoters in the cell line in which enhancers are exclusively transcribed (from left to right: n=890; 500; 494; 214; 238; 239; 378; 62; 153; 879; 586; 173; 601; 232) compared to the same promoters in all other cell lines in which the respective enhancers are not transcribed. All pairwise comparisons are significantly different (P-values < 1e-9, two-sided Mann-Whitney U-test). Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Notches represent 95% confidence intervals for the median values. Outliers not shown.


UCSC genome browser view of normalized TT - seq coverage on the plus and minus strand at the BMP4 locus ( hg38 ; chr14 : 53 , 935 , 697 - 54 , 264 , 430 ; in LoVo colorectal cancer cells . TT - seq coverage is cut at 500 ( purple lines ) to allow for better visualization . eRNAs and enhancers are shown above and below the minus strand , respectively . Aggregated transcription factor ChIP - seq peak regions ( TF ChIP - seq ) include ChIP - seq binding profiles of 326 TFs in LoVo cells The height of the signal indicates the number of different TFs binding at a particular region . Significant promoter capture Hi - C ( CHi - C ) interactions are shown in

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

UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the BMP4 locus (hg38; chr14:53,935,697-54,264,430; in LoVo colorectal cancer cells. TT-seq coverage is cut at 500 (purple lines) to allow for better visualization. eRNAs and enhancers are shown above and below the minus strand, respectively. Aggregated transcription factor ChIP-seq peak regions (TF ChIP-seq) include ChIP-seq binding profiles of 326 TFs in LoVo cells The height of the signal indicates the number of different TFs binding at a particular region. Significant promoter capture Hi-C (CHi-C) interactions are shown in purple


B . Western blots of MDI protein in wild type ( wt ) and mdi1 flies , showing that mdi1 is a protein - null mutation . Tubulin was used as loading control .

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B. Western blots of MDI protein in wild type (wt) and mdi1 flies, showing that mdi1 is a protein-null mutation. Tubulin was used as loading control.


C . Ovarioles expressing MDI - GFP stained with a mitochondrial marker , ATP synthase ( ATP - S ) . Overlapping signals ( merge ) indicate that MDI localizes to mitochondria . Note that MDI is highly expressed in germ cells at late germarium stages ( left panels ) and in egg chambers ( right panels ) . Bars , 10 m .

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C. Ovarioles expressing MDI-GFP stained with a mitochondrial marker, ATP synthase (ATP-S). Overlapping signals (merge) indicate that MDI localizes to mitochondria. Note that MDI is highly expressed in germ cells at late germarium stages (left panels) and in egg chambers (right panels). Bars, 10 m.


A - C . mtDNA replication in wt ( A ) and mdi1 ovarioles ( B , C ) as illustrated by EdU incorporation . Arrows point to mitochondrial DNA , arrowheads to nuclei . In wt ovaries ( A ) , mtDNA replication starts at germarium stage 2B and lasts into egg - chamber stage 2 ( S2 ) . In most mdi1 ovarioles ( B ) , mtDNA replication is undetectable at these stages , whereas in some ( C ) it appears delayed until germarium stage 3 . Bars , 10 m .

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A-C. mtDNA replication in wt (A) and mdi1 ovarioles (B, C) as illustrated by EdU incorporation. Arrows point to mitochondrial DNA, arrowheads to nuclei. In wt ovaries (A), mtDNA replication starts at germarium stage 2B and lasts into egg-chamber stage 2 (S2). In most mdi1 ovarioles (B), mtDNA replication is undetectable at these stages, whereas in some (C) it appears delayed until germarium stage 3. Bars, 10 m.


D . Hatching rates and mtDNA content of eggs laid by females of different mutant genotypes relative to eggs laid by wt controls . Each data point represents the mean of 3 independent replicates . Expression of mdi or hAKAP1 in mdi1 significantly restored the mtDNA level and hatching rate . N = 3 x > 100 eggs / genotype for hatching rate . The relative mtDNA level was determined as the average of 3 biological repeats . P values of comparing mdi1 ; nos > mdi to mdi1 : hatching rate , P = 1 . 1995E - 05 ; mtDNA , P = 0 . 0078 . P values of comparing mdi1 ; nos > hAKAP1 to mdi1 : hatching rate , P = 1 . 6998E - 05 ; mtDNA level , P = 0 . 0009 .

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

D. Hatching rates and mtDNA content of eggs laid by females of different mutant genotypes relative to eggs laid by wt controls. Each data point represents the mean of 3 independent replicates. Expression of mdi or hAKAP1 in mdi1 significantly restored the mtDNA level and hatching rate. N= 3 x >100 eggs / genotype for hatching rate. The relative mtDNA level was determined as the average of 3 biological repeats. P values of comparing mdi1; nos> mdi to mdi1: hatching rate, P= 1.1995E-05; mtDNA, P= 0.0078. P values of comparing mdi1; nos> hAKAP1 to mdi1: hatching rate, P= 1.6998E-05; mtDNA level, P= 0.0009.


B . Representative images of S2 cells expressing GFP fusions ( green ) of the MDI proteins diagramed in ( A ) . Staining with MitoTracker Red ( red ) to label mitochondria shows an overlapping signal ( yellow ) , indicating proper localization to mitochondria for all fusion proteins except MDIΔMTS .

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B. Representative images of S2 cells expressing GFP fusions (green) of the MDI proteins diagramed in (A). Staining with MitoTracker Red (red) to label mitochondria shows an overlapping signal (yellow), indicating proper localization to mitochondria for all fusion proteins except MDIΔMTS.


C . Submitochondrial localization of MDI . Mitochondria from cultured cells expressing an MDI - myc fusion protein were kept intact or subjected to swelling to disrupt their outer membranes , and digested or not with Protease K ( PK ) . Protein extracts were subjected to western blot analysis . Tom20 , cytochrome C ( cyt . C ) and SOD2 were used as markers of the outer membrane , inter - membrane space and matrix , respectively . MDI behaved like an outer - membrane protein .

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

C. Submitochondrial localization of MDI. Mitochondria from cultured cells expressing an MDI-myc fusion protein were kept intact or subjected to swelling to disrupt their outer membranes, and digested or not with Protease K (PK). Protein extracts were subjected to western blot analysis. Tom20, cytochrome C (cyt.C) and SOD2 were used as markers of the outer membrane, inter-membrane space and matrix, respectively. MDI behaved like an outer-membrane protein.


D . Western blots of MDI protein in wt and mdi knockout ( mdi - ko ) cells confirming the mdi - ko cells completely lacked MDI protein . Actin was used as loading control .

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D. Western blots of MDI protein in wt and mdi knockout (mdi-ko) cells confirming the mdi-ko cells completely lacked MDI protein. Actin was used as loading control.


E . Representative images of wt or mdi - ko cells expressing a Larp - GFP fusion protein and stained with MitoTracker Red to label mitochondria . The majority of Larp localized to mitochondria in wt cells ( overlapping red and green signal ) , but diffused into the cytoplasm of mdi - ko cells .

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

E. Representative images of wt or mdi-ko cells expressing a Larp-GFP fusion protein and stained with MitoTracker Red to label mitochondria. The majority of Larp localized to mitochondria in wt cells (overlapping red and green signal), but diffused into the cytoplasm of mdi-ko cells.


F . Representative images of mdi - ko cells co - expressing Larp - GFP with MDI or the deletion mutants diagramed in ( A ) fused to the mCherry . Of the 4 MDI deletions that localized to mitochondria , 2 ( MDIΔR and MDIΔTudor ) did not allow Larp - GFP to localize to mitochondria . The localization of Larp in mdi1 flies expressing MDI or MDI deletion mutants , and the hatching rate and mtDNA level in their eggs were determined . The result was summarized in ( A ) .

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F. Representative images of mdi-ko cells co-expressing Larp-GFP with MDI or the deletion mutants diagramed in (A) fused to the mCherry. Of the 4 MDI deletions that localized to mitochondria, 2 (MDIΔR and MDIΔTudor) did not allow Larp-GFP to localize to mitochondria. The localization of Larp in mdi1 flies expressing MDI or MDI deletion mutants, and the hatching rate and mtDNA level in their eggs were determined. The result was summarized in (A).


A . Co - immunoprecipitation of Larp with MDI - myc in transfected S2 cells . Tubulin was used as loading control .

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A. Co-immunoprecipitation of Larp with MDI-myc in transfected S2 cells. Tubulin was used as loading control.


B . mtDNA replication , as illustrated by EdU incorporation , in wt and larp mutant ( larp ) ovarioles . mtDNA replication is dramatically reduced in the larp ovariole . Arrows : mtDNA ; arrowheads : nuclei .

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B. mtDNA replication, as illustrated by EdU incorporation, in wt and larp mutant (larp) ovarioles. mtDNA replication is dramatically reduced in the larp ovariole. Arrows: mtDNA; arrowheads: nuclei.


C . wt and mdi1 egg chambers stained for Larp ( green ) and ATP - S ( red ) to reveal mitochondria . Larp closely associates with mitochondria in wt egg chambers . Mitochondria in mdi1 flies are clumped together , and completely lack Larp staining . Bars in ( B ) ( C ) : 10 m .

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

C. wt and mdi1 egg chambers stained for Larp (green) and ATP-S (red) to reveal mitochondria. Larp closely associates with mitochondria in wt egg chambers. Mitochondria in mdi1 flies are clumped together, and completely lack Larp staining. Bars in (B) (C): 10 m.


A . Nascent protein synthesis revealed by HpG incorporation ( green ) in wt and mdi1 egg chambers . Arrowheads point to the HpG signal on the ER in the perinuclear region and cell periphery . Arrows point to HpG signal associated with mitochondria . Mitochondria are marked by a Tom20 - mCherry ( red ) created by inserting mCherry at the endogenous Tom20 locus .

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

A. Nascent protein synthesis revealed by HpG incorporation (green) in wt and mdi1 egg chambers. Arrowheads point to the HpG signal on the ER in the perinuclear region and cell periphery. Arrows point to HpG signal associated with mitochondria. Mitochondria are marked by a Tom20-mCherry (red) created by inserting mCherry at the endogenous Tom20 locus.


B . ER location is unaltered in mdi1 egg chambers . An ER marker ( ER - GFP ) was expressed in wt and mdi1 egg chambers that were co - stained with ATP - S to mark mitochondria . ER localizes to the perinuclear region , cytoplasm and cell periphery in both wt and mdi1 egg chambers .

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

B. ER location is unaltered in mdi1 egg chambers. An ER marker (ER-GFP) was expressed in wt and mdi1 egg chambers that were co-stained with ATP-S to mark mitochondria. ER localizes to the perinuclear region, cytoplasm and cell periphery in both wt and mdi1 egg chambers.


Detection of nascent protein synthesis in the mitochondrial fraction of the ovary . A . Nascent protein synthesis was monitored by AHA incorporation , and detected by anti - biotin antibody . Tom20 served as a loading control . Note that the synthesis of nuclear - encoded mitochondrial proteins was decreased in the mdi1 background and was restored by overexpressing Tom20 - Larp ( mdi1 / TL ) .

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

Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. A. Nascent protein synthesis was monitored by AHA incorporation, and detected by anti-biotin antibody. Tom20 served as a loading control. Note that the synthesis of nuclear-encoded mitochondrial proteins was decreased in the mdi1 background and was restored by overexpressing Tom20-Larp (mdi1/TL).


Detection of nascent protein synthesis in the mitochondrial fraction of the ovary . B . Western blots of mitochondrial proteins in ovarioles of wt , mdi1 and mdi1 flies expressing Tom20 - Larp ( mdi1 / TL ) fusion protein . Note that levels of Tamas , TFAM , mRpL19 and COX4 were reduced in mdi1 flies , but restored in mdi1 / TL flies .

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

Detection of nascent protein synthesis in the mitochondrial fraction of the ovary.B. Western blots of mitochondrial proteins in ovarioles of wt, mdi1 and mdi1 flies expressing Tom20-Larp (mdi1/TL) fusion protein. Note that levels of Tamas, TFAM, mRpL19 and COX4 were reduced in mdi1 flies, but restored in mdi1/TL flies.


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 .

O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O B-TISSUE O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD B-TISSUE O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-SMALL_MOLECULE 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 protein synthesis in the mitochondrial fraction of the ovary . D . Representative profile of 254 nm absorbance of wt , mdi1 and mdi1 / TL ovary extracts .

O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O B-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 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 .

O O O O O O O B-SUBCELLULAR O O O B-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-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 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 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 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 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 .

O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD B-TISSUE O O O B-GENEPROD O O B-ORGANISM O O O O 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 protein 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 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 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 .

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

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 O O 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 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 .

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

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 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 .

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

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 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 .

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

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

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 . 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 .

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 O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O 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 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 ) .

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

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 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 O O O O O O B-EXP_ASSAY O O O B-GENEPROD O 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 O B-GENEPROD O 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 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 ) .

O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE O 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 O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O 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 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 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 ) .

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 O B-CELL_LINE O 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 O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O 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 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 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 .

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

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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 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 .

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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 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 .

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(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 , ● ) and apg16Δ cells ( YNM124 , ▴ ) were cultured in nitrogen starvation medium , and their viability was determined by phloxine B staining .

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(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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 cells ( YNM115 ) harboring both pHA ‐ APG5 and pMyc ‐ APG16 CEN plasmids . The lysates were immunoprecipitated and 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 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 .

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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 cells ( YNM124 ) transformed with pMyc ‐ APG16 ( 2μ ) and / or pHA ‐ APG16 ( 2μ ) were immunoprecipitated with anti ‐ Myc antibody and 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 ) 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 .

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

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 ) 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 .

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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 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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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 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 .

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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 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

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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 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

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

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 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

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

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 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

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

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 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

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

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 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 .

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

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

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 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

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

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

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 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

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

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 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 ,

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

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 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

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

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 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 .

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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 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

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

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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 . 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 .

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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 images of LacI - MAD1 and Cyclin B1 in U2OS cells 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 cells transfected with LacI - MAD1 - FL ( full length : aa1 - 718 ) or various LacI - MAD1 truncations ( amino 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 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 .

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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.