( E ) Relative expression of PER15 to CLATHRIN control in each genotype with or without 2 - hour CIF2 treatment . Bars are S . D . ( n = 3 ) . Different characters indicate statistically significant differences ( p < 0 . 01 , ANOVA Tukey ) . ( F ) Fold changes of PER15 in each genotype with or without 2 - hour CIF2 treatment . Bars are S . D . ( n = 3 ) . Different characters indicate statistical significance differences ( p < 0 . 01 , ANOVA Tukey ) .

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(E) Relative expression levels of PER15 to CLATHRIN control in each genotype with or without 2-hour CIF2 treatment. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test). (F)Fold changes of PER15 in each genotype with or without 2-hour CIF2 treatment. Bars are S.D. (n=3). Different characters indicate statistical significance differences (p < 0.01, ANOVA and Tukey test).


( G ) Relative expression of PER49 to CLATHRIN control in each genotype with or without 2 - hour CIF2 treatment . Bars are S . D . ( n = 3 ) . Different characters indicate statistically significant differences ( p < 0 . 01 , ANOVA Tukey ) . ( H ) Fold changes of PER49 in each genotype with or without 2 - hour CIF2 treatment . Bars are S . D . ( n = 3 ) . Different characters indicate statistically significant differences ( p < 0 . 01 , ANOVA Tukey ) .

(G) Relative expression levels of PER49 to CLATHRIN control in each genotype with or without 2-hour CIF2 treatment. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test). (H) Fold changes of PER49 in each genotype with or without 2-hour CIF2 treatment. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test).


( I ) Relative fold changes of PER15 in pCASP1 : : SGN1 - Citrine pCASP1 : : myrpalm - SGN1 - Citrine compared to expression level in WT . Bars are S . D . ( n = 3 ) . Different characters indicate statistically significant differences ( p < 0 . 01 , ANOVA Tukey ) . ( J ) Relative fold changes of PER49 in pCASP1 : : SGN1 - Citrine pCASP1 : : myrpalm - SGN1 - Citrine compared to expression level in WT . Bars are S . D . ( n = 3 ) . Different characters indicate statistically significant differences ( p < 0 . 01 , ANOVA Tukey ) .

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(I)Relative fold changes of PER15 in pCASP1::SGN1-Citrine and pCASP1::myrpalm-SGN1-Citrine compared to the expression level in WT. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test). (J)Relative fold changes of PER49 in pCASP1::SGN1-Citrine and pCASP1::myrpalm-SGN1-Citrine compared to the expression level in WT. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test).


A Immunostaining of HeLa probed for FITC ‐ labeled streptavidin merge streptavidin ( red ) ( blue ) . surface was labeled using amine biotinylation . Scale bar , 10 μm .

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A Immunostaining of HeLa cells probed for FITC‐labeled streptavidin and DNA and the merge streptavidin (red) and DNA (blue). Cell surface was labeled using amine biotinylation. Scale bar, 10 μm.


B Western blot of surface preparation . surface ‐ biotinylated were lysed using a detergent containing lysis buffer , incubated with then eluted from streptavidin beads ( i . e . , sur . fraction ) . surface fraction whole ‐ lysate were blotted against a surface , EGFR , a , actin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot analysis of cell surface preparation. The surface‐biotinylated cells were lysed using a detergent containing lysis buffer, incubated with and then eluted from streptavidin beads (i.e., cell sur. fraction). Cell surface fraction and the whole‐cell lysate were blotted against a cell surface protein, EGFR, and a cytoplasmic protein, actin.


B Western blot confirmation of mitosis ‐ selective surface localization of PCDH7 . Western blotting of surface preparation from mitosis interphase . whole ‐ lysate elution from streptavidin beads were blotted against TFRC , EGFR , actin , phospho ‐ Histone H3 . TFRC was enriched in interphase surface preparation in comparison with mitosis preparation , while PCDH7 was enriched in mitotic surface preparation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot confirmation of the mitosis‐selective cell surface localization of PCDH7. Western blotting analysis of cell surface protein preparation from mitosis and interphase cells. The whole‐cell lysate and elution from streptavidin beads were blotted against TFRC, EGFR, actin, and phospho‐Histone H3. TFRC protein was enriched in interphase cell surface preparation in comparison with mitosis preparation, while PCDH7 protein was enriched in mitotic cell surface preparation.


C Western blot confirmation of mitosis ‐ selective surface localization of PCDH1 . Whole ‐ lysate samples were subjected to SDS - PAGE blotted with anti ‐ PCDH1 , anti ‐ β ‐ actin , anti ‐ phospho ‐ Histone H3 antibodies , as indicated on left . surface fraction samples were blotted with anti ‐ PCDH1 anti ‐ EGFR antibodies .

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C Western blot confirmation of the mitosis‐selective cell surface localization of PCDH1. Whole‐cell lysate samples were subjected to SDS-PAGE and blotted with the anti‐PCDH1, anti‐β‐actin, and anti‐phospho‐Histone H3 antibodies, as indicated on the left. Cell surface fraction samples were blotted with the anti‐PCDH1 and anti‐ EGFR antibodies.


A Tubulin PCDH7immunostaining of unsynchronized HeLa : tubulin , green ; PCDH7 , red ; , blue . PCDH7 localizes to mitotic surface ( arrows ) . PCDH7 is barely detectable in surrounding interphase . Scale bar , 5 μm . B of PCDH7 signal on surface of immunostained in mitosis ( n = 6 , red ) interphase ( n = 7 , blue ) . Bars show mean ± .

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A Tubulin and PCDH7immunostaining of unsynchronized HeLa cells: tubulin, green; PCDH7, red; DNA, blue. PCDH7 localizes to the mitotic cell surface (arrows). PCDH7 staining is barely detectable in the surrounding interphase cells. Scale bar, 5 μm.B Quantification of PCDH7 signal on the cell surface of immunostained cells in mitosis (n = 6, red) and interphase (n = 7, blue). Bars show mean ± SEM.


C PCDH7 ‐ EGFP ‐ transfected unsynchronized U2OS , stably expressing mCherry ‐ tubulin , at mitosis ( top ) interphase ( bottom ) . PCDH7 , green ; tubulin , red ; , blue . Scale bar , 10 μm .

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C PCDH7‐EGFP‐transfected unsynchronized U2OS cells, stably expressing mCherry‐tubulin, at mitosis (top) and interphase (bottom). PCDH7, green; tubulin, red; DNA, blue. Scale bar, 10 μm.


D Immunostaining of HeLa Kyoto stably expressing PCDH7 ‐ LAP ‐ tagged BAC transgenes ( green ) probed for tubulin ( red ) ( blue ) . Top panel shows mitotic those were treated with 5 μM STC for 10 h , bottom panel shows interphase . Scale bar , 10 μm .

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D Immunostaining of HeLa Kyoto cells stably expressing PCDH7‐LAP‐tagged BAC transgenes (green) probed for tubulin (red) and DNA (blue). Top panel shows mitotic cell those were treated with 5 μM STC for 10 h, and bottom panel shows interphase cells. Scale bar, 10 μm.


A Confocal microscopy representing localization of PCDH7 . U2OS expressing full ‐ ( WT ) PCDH7 at mitosis ( top ) interphase ( middle ) . deleted ( CytΔ ) PCDH7 ‐ expressing at mitosis ( middle ) interphase ( bottom ) . PCDH7 , green ; , blue . Scale bar , 20 μm . B of PCDH7 : : GFP ( WT CytΔ ) localization on surface in mitosis [ WT ( n = 11 ) , CytΔ ( n = 6 ) , red ] interphase [ WT ( n = 25 ) , CytΔ ( n = 20 ) , blue ] . Interphase cytΔ PCDH7 localizes to surface more than WT ( full ) PCDH7 ( insets ) . Bars show mean ± .

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A Confocal microscopy images representing the localization of PCDH7. U2OS cells expressing full‐length (WT) PCDH7 at mitosis (top) and interphase (middle). Cytoplasmic deleted (CytΔ) PCDH7‐expressing cells at mitosis (middle) and interphase (bottom). PCDH7, green; DNA, blue. Scale bar, 20 μm.B Quantification of PCDH7::GFP (WT and CytΔ) protein localization on the cell surface in mitosis [WT (n = 11), CytΔ (n = 6), red] and interphase [WT (n = 25), CytΔ (n = 20), blue]. Interphase cytΔ PCDH7 localizes to the cell surface more than WT (full length) PCDH7 (insets). Bars show mean ± SEM.


C In interphase co ‐ localization between PCDH7 ER marker KDEL . HeLa were transiently transfected with PCDH7 : : GFP ( green ) ( ectopic ) , fixed stained for ER marker KDEL ( red ) ( interphase , top panel ) . Interphase ( middle panel ) mitotic ( bottom panel ) HeLa were fixed stained against ( endogenous ) PCDH7 KDEL . Scale bar , 5 μm .

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C In interphase co‐localization between PCDH7 and ER marker KDEL. HeLa cells were transiently transfected with PCDH7::GFP (green) (ectopic), fixed and stained for ER marker KDEL (red) (interphase, top panel). Interphase (middle panel) and mitotic (bottom panel) HeLa cells were fixed and stained against (endogenous) PCDH7 and KDEL. Scale bar, 5 μm.


D PCDH7 PCDH1 enriched on endoplasmic reticulum fraction . Whole ‐ lysate , , endoplasmic , mitochondrial fractions were blotted against an endoplasmic reticulum , KDEL , a , actin , a mitochondrial Cox ‐ 2 PCDH7 , PCDH1 .

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D PCDH7 and PCDH1 enriched on endoplasmic reticulum fraction. Whole‐cell lysate, cytoplasmic, endoplasmic, and mitochondrial fractions were blotted against an endoplasmic reticulum protein, KDEL, a cytoplasmic protein, actin, a mitochondrial protein Cox‐2 and PCDH7, PCDH1.


A PCDH7 antibody detects a single band of ˜ 110 kDa in luciferase RNAi ( luc ) , PCDH1 RNAi HeLa lysates , but this band is not detectable in PCDH7 PCDH1 PCDH7 RNAi . α ‐ tubulin was used as a loading control ( left ) . HeLa Kyoto stably expressing PCDH7 ‐ LAP ‐ tagged BAC transgenes treated with luciferase RNAi ( control ) 150 nM ( 1 × ) 300 nM ( 2 × ) PCDH1 esiRNA blotted against anti ‐ GFP , anti ‐ PCDH1 , anti ‐ actin antibodies . PCDH1 bands were reduced in PCDH1 RNAi , whereas mouse GFP : : PCDH1 was not affected by esiRNA treatment ( right ) .

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A PCDH7 antibody detects a single band of ˜110 kDa in luciferase RNAi (luc), PCDH1 RNAi HeLa cell lysates, but this band is not detectable in PCDH7 and PCDH1 and PCDH7 RNAi cells. α‐tubulin was used as a loading control (left). HeLa Kyoto cells stably expressing PCDH7‐LAP‐tagged BAC transgenes treated with luciferase RNAi (control) and 150 nM (1×) and 300 nM (2×) PCDH1 esiRNA and blotted against anti‐GFP, anti‐PCDH1, and anti‐actin antibodies. PCDH1 bands were reduced in PCDH1 RNAi cells, whereas mouse GFP::PCDH1 was not affected by the esiRNA treatment (right).


B Rounding pressure plots of MYH9 ( positive control , n = 7 ) , luc ( negative control , n = 12 ) , PCDH7 ( n = 11 ) , PCDH1 ( n = 12 ) , PCDH1 PCDH7 ( n = 11 ) RNAi . Bars show mean ± ( left ) . Rounding pressure plots HeLa Kyoto stably expressing PCDH7 ‐ LAP ‐ tagged BAC transgenes treated with MYH9 ( positive control , n = 7 ) , luc ( negative control , n = 14 ) , PCDH1 ( n = 13 ) esiRNA . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 ; ns , P > 0 . 05 ( non ‐ significant ) .

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B Rounding pressure plots of MYH9 (positive control, n = 7), luc (negative control, n = 12), PCDH7 (n = 11), PCDH1 (n = 12), and PCDH1 and PCDH7 (n = 11) RNAi cells. Bars show mean ± SEM (left). Rounding pressure plots HeLa Kyoto cells stably expressing PCDH7‐LAP‐tagged BAC transgenes treated with MYH9 (positive control, n = 7), luc (negative control, n = 14), PCDH1 (n = 13) esiRNA. *P 0.05; **P 0.01; ***P 0.001; ns, P > 0.05 (non‐significant).


B Flow cytometry of HeLa treated with anti ‐ PCDH7 antibody , fixed stained for phospho ‐ Histone H3 . Forty ‐ two of unsorted HeLa cella are in G2 / M phase . When are sorted on their phospho ‐ Histone H3 ( middle ) expression , phospho ‐ Histone H3 + ( high expression ) are preferentially mitotic ( 87 . 3 % in G2 / M phase ) 42 % of those are PCDH7 + . Similarly , PCDH7 + ( high expression ) are preferentially mitotic ( 82 . 1 % in G2 / M phase ) 79 % of those are phospho ‐ Histone H3 + ( bottom ) .

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B Flow cytometry analysis of HeLa cells treated with anti‐PCDH7 antibody, fixed and stained for phospho‐Histone H3 and DNA. Forty‐two percent of unsorted HeLa cella are in G2/M phase. When cells are sorted based on their phospho‐Histone H3 (middle) expression, phospho‐Histone H3+ (high expression) cells are preferentially mitotic (87.3% in G2/M phase) and 42% of those are PCDH7+. Similarly, PCDH7+ (high expression) cells are preferentially mitotic (82.1% in G2/M phase) and 79% of those are phospho‐Histone H3+ (bottom).


( a ) Lipid phosphatase activity of Paladin , wild - type phosphatase - dead C / S mutant , towards PI ( 4 , 5 ) P2 PI ( 3 , 4 , 5 ) P3 substrates . Positive control ; wild - type phosphatase tensin homolog ( PTEN ) ; negative control ; lipid phosphatase - dead C124S PTEN . Mean ± , Paired t - , n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(a) Lipid phosphatase activity of Paladin, wild-type and phosphatase-dead C/S mutant, towards PI(4,5)P2 and PI(3,4,5)P3 substrates. Positive control; wild-type phosphatase and tensin homolog (PTEN); negative control; lipid phosphatase-dead C124S PTEN. Mean±SEM, Paired t-test, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( b ) Representative confocal image of HDMEC stained for Paladin ( green ) , Vascular Endothelial Cadherin ( VEC , red ) nuclei ( DAPI , blue ) , scale bar : 10 μm . boxes in upper image are showed at higher magnification below , scale bar : 3 µm .

(b) Representative confocal image of HDMEC stained for Paladin (green), Vascular Endothelial Cadherin (VEC, red) and nuclei (DAPI, blue), scale bar: 10 μm. White boxes in the upper image are showed at higher magnification below, scale bar: 3 µm.


( c ) HDMEC stained for VEGFR2 ( green ) , Paladin ( red ) nuclei ( DAPI , blue ) with or without stimulation of 50 ng / ml VEGF - A for 0 , 2 or 10 min . Scale bar : 10 µm . ( d ) of VEGFR2 - Paladin co - before after VEGF - A stimulation as shown in ( C ) . Mean ± , One - way ANOVA . n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(c) HDMEC stained for VEGFR2 (green), Paladin (red) and nuclei (DAPI, blue) and with or without stimulation of 50 ng/ml VEGF-A for 0, 2 or 10 min. Scale bar: 10 µm. (d) Quantification of VEGFR2-Paladin co-staining before and after VEGF-A stimulation as shown in (C). Mean±SEM, One-way ANOVA. n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( e ) HDMEC analysed using Proximity Ligand ( PLA ) for Paladin VEGFR2 . Green dots indicate formation , VE - cadherin ( red ) nuclei ( DAPI , blue ) . stimulated with 50 ng / ml VEGF - A for 0 , 2 or 10 min . Scale bar : 10 µm . ( f ) of ( e ) , number of PLA Paldin - VEGFR2 complexes per at 0 , 2 10 minutes after VEGF - A stimulation ( top ) . of ( e ) , average for PLA complexes to nearest VE - cadherin positive junction at 0 , 2 10 minutes after VEGF - A stimulation ( bottom ) . Mean ± , One - way ANOVA . n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(e) HDMEC analysed using Proximity Ligand Assay (PLA) for Paladin and VEGFR2. Green dots indicate complex formation, VE-cadherin (red) and nuclei (DAPI, blue). Cells stimulated with 50 ng/ml VEGF-A for 0, 2 or 10 min. Scale bar: 10 µm. (f) Quantification of (e), the number of PLA Paldin-VEGFR2 complexes per cell at 0, 2 and 10 minutes after VEGF-A stimulation (top). Quantification of (e), average distance for the PLA complexes to the nearest VE-cadherin positive junction at 0, 2 and 10 minutes after VEGF-A stimulation (bottom). Mean±SEM, One-way ANOVA. n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( g ) HDMEC stained for Paladin ( green ) EEA1 ( red ) , colocalization in yellow , nuclei ( DAPI , blue ) after VEGF - A stimulation for 0 , 2 or 10 minutes , a representative image from a single confocal plane is shown , scale bar : 10 µm . boxes in upper image are showed at higher magnification below , scale bar 3 µm . ( h ) of ( g ) , EEA1 / Paladin double positive particles per field of view at 0 , 2 10 minutes after VEGF - A stimulation . Mean ± , One - way ANOVA . n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(g) HDMEC stained for Paladin (green) and EEA1 (red), colocalization in yellow, and nuclei (DAPI, blue) after VEGF-A stimulation for 0, 2 or 10 minutes, a representative image from a single confocal plane is shown, scale bar: 10 µm. White boxes in the upper image are showed at higher magnification below, scale bar 3 µm. (h) Quantification of (g), EEA1/Paladin double positive particles per field of view at 0, 2 and 10 minutes after VEGF-A stimulation. Mean±SEM, One-way ANOVA. n= 3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( a ) surface VEGFR2 detected by surface biotinylation , using thiol - cleavable sulfo - NHS - SS - biotin , of HDMEC transfected with PALD1 siRNA ( # 1 # 2 ) or non - targeting control ( " c " ) siRNA , followed by VEGF - A stimulation ( 50 ng / ml ) for indicated time periods . Total lysates ( input ) streptavidin ( SA ) pull down , immunoblotted for VEGFR2 , Paladin , actin . ' No biotin ctrl ' , not treated with sulfo - NHS - SS - biotin . ( b ) of data in ( a ) . VEGFR2 surface ( data pooled for indicated time points ) normalized to total VEGFR2 compared between control siRNA treated HDMEC . n = 4 for each time point , biological replicates , Mean ± . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(a) Cell surface VEGFR2 levels detected by cell surface biotinylation, using thiol-cleavable sulfo-NHS-SS-biotin, of HDMEC transfected with PALD1 siRNA (#1 and #2) or non-targeting control ("c") siRNA, followed by VEGF-A stimulation (50 ng/ml) for indicated time periods. Total lysates (input) and streptavidin (SA) pull down, immunoblotted for VEGFR2, Paladin, and actin. 'No biotin ctrl', cells not treated with sulfo-NHS-SS-biotin. (b) Quantification of data in (a). VEGFR2 surface levels (data pooled for the indicated time points) normalized to total VEGFR2 levels and compared between control and siRNA treated HDMEC. n=4 for each time point, biological replicates, Mean±SEM. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( c ) Internalized pool of VEGFR2 after VEGF - A treatment ( 50 ng / ml ) of non - transfected HDMEC ( " NT " ) or HDMEC transfected with PALD1 siRNA ( # 1 # 2 ) or non - targeting control siRNA ( " c " ) . surface biotinylation was performed prior to VEGF - A stimulation at indicated time points , remaining surface biotin was stripped internalized pool of VEGFR2 was collected by SA pull down . Immunoblotting of total lysate ( input ) SA pull down fraction for VEGFR2 , Paladin actin . ( d ) of data in ( c ) . Data were normalized to total VEGFR2 in lysate after subtraction of signals in biotinylated stripped samples . Mean ± , unpaired t - for indicated time points , normalized to cntrl siRNA sample . n = 3 for each time point , biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(c) Internalized pool of VEGFR2 after VEGF-A treatment (50 ng/ml) of non-transfected HDMEC ("NT") or HDMEC transfected with PALD1 siRNA (#1 and #2) or non-targeting control siRNA ("c"). Cell surface biotinylation was performed prior to VEGF-A stimulation and at indicated time points, remaining cell surface biotin was stripped and the internalized pool of VEGFR2 was collected by SA pull down. Immunoblotting of the total lysate (input) and SA pull down fraction for VEGFR2, Paladin and actin. (d) Quantification of data in (c). Data were normalized to total VEGFR2 levels in the lysate after subtraction of signals in biotinylated and stripped samples. Mean±SEM, unpaired t-test for indicated time points, normalized to cntrl siRNA sample. n=3 for each time point, biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( e ) of EEA1 VEGFR2 vesicles following PALD1 knockdown . Representative of VEGFR2 ( green ) / EEA1 ( red ) double - positive ( yellow ) vesicles in negative control siRNA , PALD1 KD # 2 siRNA - silenced HDMEC at 0 , 2 10 min of VEGF - A stimulation ( 50 ng / ml ) . DAPI in blue , scale bar : 10 µm . Inset shows only EEA1 channel , scale bar : 10 µm . ( f ) of ( e ) , number of EEA1 positive ( top ) or VEGFR2 - EEA1 double - positive vesicles ( bottom ) per field of view . Mean ± , Two - way ANOVA , n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(e) Analysis of EEA1 and VEGFR2 vesicles following PALD1 knockdown. Representative images of VEGFR2 (green)/EEA1 (red) double-positive (yellow) vesicles in negative control siRNA, and PALD1 KD#2 siRNA-silenced HDMEC at 0, 2 and 10 min of VEGF-A stimulation (50 ng/ml). DAPI in blue, scale bar: 10 µm. Inset shows only EEA1 channel, scale bar: 10 µm. (f) Quantification of (e), number of EEA1 positive (top) or VEGFR2-EEA1 double-positive vesicles (bottom) per field of view. Mean±SEM, Two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( g ) HDMEC stained for PI ( 4 , 5 ) P2 ( cyan ) , VE - cadherin ( red ) Paladin ( yellow in inset ) following treatment using negative control or PALD1 ( KD # 2 ) siRNA . VEGF - A stimulation for 0 , 2 or 10 minutes ( 50 ng / ml ) . DAPI in blue . Scale bar : 10 µm ( h ) of ( g ) , Total PI ( 4 , 5 ) P2 signal ( top ) or intracellular PI ( 4 , 5 ) P2 not overlapping with VE - cadherin ( bottom ) . Mean ± , two - way ANOVA , n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(g) HDMEC stained for PI(4,5)P2 (cyan), VE-cadherin (red) and Paladin (yellow in inset) following treatment using negative control or PALD1 (KD#2) siRNA. VEGF-A stimulation for 0, 2 or 10 minutes (50 ng/ml). DAPI in blue. Scale bar: 10 µm (h) Quantification of (g), Total PI(4,5)P2 signal (top) or intracellular PI(4,5)P2 not overlapping with VE-cadherin (bottom). Mean±SEM, two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


Signalling downstream of VEGF - A / VEGFR2 assessed in HDMEC , untreated ( NT , non - transfected ) , transfected with non - targeting siRNA ( c / cntrl ) , or with two different PALD1 - targeting siRNAs ( KD # 1 KD # 2 ) , treated with VEGF - A for 0 - 10 0 - 20 min , respectively . Immunoblotting of lysates for phosphorylated ( p ) VEGFR2 ( pY1175 ) , total VEGFR2 Actin served as loading control ( a PALD1 knockdown was verified by blotting for Paladin . of pVEGFR2 ( normalized to total VEGFR2 ) ( b ) Mean ± , two - way ANOVA , n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 .

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Signalling downstream of VEGF-A/VEGFR2 assessed in HDMEC, untreated (NT, non-transfected), transfected with non-targeting siRNA (c/cntrl), or with two different PALD1-targeting siRNAs (KD#1 and KD#2), and treated with VEGF-A for 0-10 and 0-20 min, respectively. Immunoblotting of cell lysates for phosphorylated (p) VEGFR2 (pY1175), total VEGFR2 Actin served as loading control (a PALD1 knockdown was verified by blotting for Paladin. Quantification of pVEGFR2 (normalized to total VEGFR2) (b) Mean±SEM, two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01.


Signalling downstream of VEGF - A / VEGFR2 assessed in HDMEC , untreated ( NT , non - transfected ) , transfected with non - targeting siRNA ( c / cntrl ) , or with two different PALD1 - targeting siRNAs ( KD # 1 KD # 2 ) , treated with VEGF - A for 0 - 10 0 - 20 min , respectively . Immunoblotting of lysates for phosphorylated Erk1 / 2 ( pT202 pY204 ) , total Erk . Actin served as loading control c ) . PALD1 knockdown was verified by blotting for Paladin . of pErk1 / 2 ( normalized to total Erk1 / 2 ) ( d ) . Mean ± , two - way ANOVA , n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 .

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Signalling downstream of VEGF-A/VEGFR2 assessed in HDMEC, untreated (NT, non-transfected), transfected with non-targeting siRNA (c/cntrl), or with two different PALD1-targeting siRNAs (KD#1 and KD#2), and treated with VEGF-A for 0-10 and 0-20 min, respectively. Immunoblotting of cell lysates for phosphorylated Erk1/2 (pT202 and pY204), and total Erk. Actin served as loading control c). PALD1 knockdown was verified by blotting for Paladin. Quantification of pErk1/2 (normalized to total Erk1/2) (d). Mean±SEM, two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01.


( e - f ) Immunoblotting on total heart lysates from adult Pald1 + / + Pald1 - / - mice , tail - vein injected with VEGF - A ( 0 . 25µg / g ) or PBS for indicated time points , for Paladin , phosphorylated total of VEGFR2 , Erk1 / 2 β2 - microglobulin ( β 2 - MG , loading control ) . ( f ) of pVEGFR pY1173 normalized to total VEGFR2 ( n = 3 ) , total VEGFR2 normalized to total loading control ( n = 4 ) , pT202 / pY204 Erk1 / 2 normalized to Erk1 / 2 ( n = 5 ) . Mean ± , multiple t - with Holm - Sidak correction ( total VEGFR2 ) , two - way ANOVA ( others ) . Data information : * p < 0 . 05 , * * p < 0 . 01 .

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(e-f) Immunoblotting on total heart lysates from adult Pald1+/+ and Pald1-/- mice, tail-vein injected with VEGF-A (0.25µg/g body weight) or PBS for the indicated time points, for Paladin, phosphorylated and total levels of VEGFR2, Erk1/2 and β2-microglobulin (β 2-MG, loading control). (f) Quantification of pVEGFR pY1173 normalized to total VEGFR2 (n=3), total VEGFR2 levels normalized to total loading control (n=4), pT202/pY204 Erk1/2 normalized to Erk1/2 (n=5). Mean±SEM, multiple t-test with Holm-Sidak correction (total VEGFR2), two-way ANOVA (others). Data information: *p < 0.05, **p < 0.01.


( a , b ) Delayed vascular outgrowth hyperdense vascular front in isolectinB4 - stained P5 retina from Pald1 - / - mouse compared with Pald1 + / + ( a ) . Orange arrow indicates radial expansion of vascular plexus in Pald1 + / + retina . Scale bar : 1 mm . of radial expansion ( b ) as shown in ( a ) normalized to wild type litter mates . Mean ± , unpaired t - . n = 14 litters , 18 wild type 23 knock - out pups . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(a, b) Delayed vascular outgrowth and hyperdense vascular front in isolectinB4-stained P5 retina from Pald1-/- mouse compared with Pald1+/+ (a). Orange arrow indicates radial expansion of the vascular plexus in the Pald1+/+ retina. Scale bar: 1 mm. Quantification of radial expansion (b) as shown in (a) normalized to wild type litter mates. Mean±SEM, unpaired t-test. n=14 litters, 18 wild type and 23 knock-out pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


( c , d ) Increased filopodia number in Pald1 - / - mouse retina at vascular front , visualized by isolectinB4 ( c ) . Scale bar : 50 μm . of filopodia per 100 µm at vascular front ( d ) . Mean ± , unpaired t - . n = 3 litters , 7 wild type 5 knock - out pups . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(c, d) Increased filopodia number in Pald1-/- mouse retina at vascular front, visualized by isolectinB4 staining (c). Scale bar: 50 μm. Quantification of filopodia per 100 µm at the vascular front (d). Mean±SEM, unpaired t-test. n=3 litters, 7 wild type and 5 knock-out pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


( e , f ) P5 retina vascular front ( isolectinB4 , green ) endothelial nuclei ( Erg , red ) ( e ) . Scale bar : 100 μm . Magenta stippled square quantified in ( f ) . Vascular density was determined in capillary bed ( left ) ( 11 wild type , 12 knock - out ) in around veins ( right ) ( 10 wild type , 9 knock - out , biological replicates ) . Mean ± , unpaired t - . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(e, f) P5 retina vascular front (isolectinB4, green) and endothelial nuclei (Erg, red) (e). Scale bar: 100 μm. Magenta stippled square area quantified in (f). Vascular density was determined in the capillary bed (left) (11 wild type, 12 knock-out) and in area around veins (right) (10 wild type, 9 knock-out, biological replicates). Mean±SEM, unpaired t-test. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


( g , h ) pT202 / pY204 Erk1 / 2 immunostaining ( black ) in Pald1 + / + Pald1 - / - P5 pups . IsolectinB4 ( ) visualizes entire vasculature . Scale bar : 100 μm . of pT202 / pY204 Erk1 / 2 ( h ) as in black stippled square in ( g ) ( 400 µm from retina rim ) , normalized to isolectinB4 . Mean ± , unpaired t - . n = 5 litters , 14 wild type 10 knock - out retinas per genotype . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

(g, h) pT202/pY204 Erk1/2 immunostaining (black) in Pald1+/+ and Pald1-/- P5 pups. IsolectinB4 (white) visualizes the entire vasculature. Scale bar: 100 μm. Quantification of pT202/pY204 Erk1/2 area (h) as in the black stippled square in (g) (400 µm from the retina rim), normalized to isolectinB4 area. Mean±SEM, unpaired t-test. n=5 litters, 14 wild type and 10 knock-out retinas per genotype. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


( i ) Quantitative real - time PCR of P4 - P5 retinas from Pald1 + / + Pald1 - / - pups . Ccnd1 transcript , normalized to Gapdh . Mean ± , unpaired t - . n = 10 Pald1 + / + 14 Pald1 - / - pups . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(i) Quantitative real-time PCR analysis of P4-P5 retinas from Pald1+/+ and Pald1-/- pups. Ccnd1 transcript levels, normalized to Gapdh. Mean±SEM, unpaired t-test. n=10 Pald1+/+ and 14 Pald1-/- pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


( j ) Cyclin D1 ( black ) isolectinB4 ( ) of P5 retinas from Pald1 + / + Pald1 - / - pups . Scale bar : 100 µm .

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(j) Cyclin D1 (black) and isolectinB4 (white) staining of P5 retinas from Pald1+/+ and Pald1-/-pups. Scale bar: 100 µm.


( k ) of number of Cyclin D1 positive nuclei normalized for isolectinB4 in retinas from Pald1 + / + Pald1 - / P5 pups . Mean ± , unpaired t - . n = 7 Pald1 + / + 6 Pald1 - / - pups . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(k) Quantification of number of Cyclin D1 positive nuclei normalized for isolectinB4 area in retinas from Pald1+/+ and Pald1-/ P5 pups. Mean±SEM, unpaired t-test. n=7 Pald1+/+ and 6 Pald1-/- pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


( l ) of relative radial expansion in vehicle ( n = 2 litters , 5 Pald1 + / + 4 Pald1 - / - pups ) MEK inhibitor ( U0126 ) - treated pups ( n = 4 litters , 7 Pald1 + / + 5 Pald1 - / - pups ) . MEK inhibitor / vehicle was administered twice at 12h interval at P4 eyes collected at P5 . Each dot is one mouse . Mean ± , one - way ANOVA , n = 4 - 7 . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(l) Quantification of relative radial expansion in vehicle (n=2 litters, 5 Pald1+/+ and 4 Pald1-/- pups) and MEK inhibitor (U0126)-treated pups (n=4 litters, 7 Pald1+/+ and 5 Pald1-/- pups). MEK inhibitor/vehicle was administered twice at 12h interval at P4 and eyes collected at P5. Each dot is one mouse. Mean±SEM, one-way ANOVA, n=4-7. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


( m ) of tip number in vehicle - ( n = 3 litters , 5 Pald1 + / + 5 Pald1 - / - pups ) MEK inhibitor ( U0126 ) - treated pups ( n = 3 litters , 5 Pald1 + / + 4 Pald1 - / - pups ) . MEK inhibitor / vehicle administered twice at P5 at 2 - h intervals , eyes collected 2 h after second injection . Each dot is one mouse . Mean ± , one - way ANOVA . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(m) Quantification of the tip cell number in vehicle- (n=3 litters, 5 Pald1+/+ and 5 Pald1-/- pups) and MEK inhibitor (U0126)-treated pups (n=3 litters, 5 Pald1+/+ and 4 Pald1-/- pups). MEK inhibitor/vehicle administered twice at P5 at 2-h intervals, and eyes collected 2 h after the second injection. Each dot is one mouse. Mean±SEM, one-way ANOVA. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


( a ) Eyes from Pald1 + / LacZ mice collected at P17 from untreated or with oxygen - induced retinopathy ( OIR ) . Pald1 - promoter driven LacZ expression X - gal generated signals in capillaries , veins arteries in OIR retina at P17 compared to normoxia control with predominantly arterial LacZ expression . Scale bar : 1 mm .

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(a) Eyes from Pald1+/LacZ mice collected at P17 from untreated animals or animals with oxygen-induced retinopathy (OIR). Pald1-promoter driven LacZ expression and X-gal staining generated signals in capillaries, veins and arteries in the OIR retina at P17 compared to the normoxia control with predominantly arterial LacZ expression. Scale bar: 1 mm.


( b ) Paladin in human umbilical vein endothelial ( HUVEC ) untreated or treated for 24 h with VEGF - A ( 50 ng / ml ) , FGF2 ( 50 ng / ml ) , or SDF1α ( 30 ng / ml ) , quantified by immunoblotting for Paladin β - actin ( loading control ) . Mean ± , one way - ANOVA , n = 5 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 .

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(b) Paladin levels in primary human umbilical vein endothelial cells (HUVEC) untreated or treated for 24 h with VEGF-A (50 ng/ml), FGF2 (50 ng/ml), or SDF1α (30 ng/ml), quantified by immunoblotting for Paladin and β-actin (loading control). Mean±SEM, one way-ANOVA, n=5 biological replicates. Data information: *p < 0.05, **p < 0.01.


( c ) Eyes from adult Pald1LacZ / + mice collected at 72 h after single - bolus intravitreal injection of 1 μg VEGF - A , PBS in eye , followed by X - gal . Arrow indicates Pald1 promoter activity in veins ( outlined by dashed ) specifically after VEGF - A treatment . A , artery ; V , vein . Scale bar : 100 μm .

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(c) Eyes from adult Pald1LacZ/+ mice collected at 72 h after single-bolus intravitreal injection of 1 μg VEGF-A, and PBS in the contralateral eye, followed by X-gal staining. Arrow indicates Pald1 promoter activity in veins (outlined by dashed line) specifically after VEGF-A treatment. A, artery; V, vein. Scale bar: 100 μm.


( d - f ) Representative of isolectinB4 - stained P17 retinas from OIR - challenged Pald1 + / + Pald1 - / - mice ( detailed view in insets ) ( d ) . Scale bar : 1 mm ( inset 250 µm ) . of neovascular tuft ( e ) avascular ( f ) . Each dot represents mean of both retinas per mouse . Mean ± , unpaired t - , n = 3 litters , 4 Pald1 + / + 3 Pald1 - / - pups . Data information : * p < 0 . 05 , * * p < 0 . 01 .

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(d-f) Representative images of isolectinB4-stained P17 retinas from OIR-challenged Pald1+/+ and Pald1-/- mice (detailed view in the insets) (d). Scale bar: 1 mm (inset 250 µm). Quantification of neovascular tuft area (e) and avascular area (f). Each dot represents the mean of both retinas per mouse. Mean±SEM, unpaired t-test, n=3 litters, 4 Pald1+/+ and 3 Pald1-/- pups. Data information: *p < 0.05, **p < 0.01.


g ) Representative of retina vasculature immunostained for isolectinB4 pT202 / pY204 Erk1 / 2 ( pErk1 / 2 ) . pErk1 / 2 within vessels is also visualized using a 16 - color to display . Scale bar : 30 µm ( inset 10µm ) . ( h ) of pErk1 / 2 immunostaining as shown in ( g ) within isolectinB4 - positive vessels , as fold - change of pErk1 / 2 stained . Each dot represents mean of both retinas per mouse . Mean ± , unpaired t - , n = 3 litters , 5 Pald1 + / + 5 Pald1 - / - pups . Data information : * p < 0 . 05 , * * p < 0 . 01 .

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g) Representative images of retina vasculature immunostained for isolectinB4 and pT202/pY204 Erk1/2 (pErk1/2). pErk1/2 staining within the vessels is also visualized using a 16-color heatmap to display staining intensity. Scale bar: 30 µm (inset 10µm). (h) Quantification of pErk1/2 immunostaining as shown in (g) within isolectinB4-positive vessels, as fold-change of pErk1/2 stained area. Each dot represents the mean of both retinas per mouse. Mean±SEM, unpaired t-test, n=3 litters, 5 Pald1+/+ and 5 Pald1-/- pups. Data information: *p < 0.05, **p < 0.01.


A ) Time course of biomass . B ) Time course of glucose . Data information : means for n = 3 biologically independent samples are shown . error bars represent ± SD . . Note : although no nitrogen source was present in medium , some minor growth ( especially of control wild type strains ) remained ( A ) , which is a known phenomenon within first hours of cultivation after nitrogen

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A) Time course of biomass concentration. B) Time course of glucose concentration. Data information: The means for n = 3 biologically independent samples are shown. The error bars represent ± SD.. Note: although no nitrogen source was present in the medium, some minor growth (especially of the control and wild type strains) remained (A), which is a known phenomenon within the first hours of cultivation after nitrogen depletion


C ) Specific glucose ( Glc ) uptake rate specific productivity for ethanol ( Eth ) , acetate ( Ace ) , formate ( For ) , lactate ( Lac ) , succinate ( Suc ) . Data information : reaction rates in C were calculated from beginning of cultivation until last sampling time point where glucose was still present in medium . means data for n = 3 biologically independent samples are shown . error bars represent ± SD . .

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

C) Specific glucose (Glc) uptake rate and specific productivity for ethanol (Eth), acetate (Ace), formate (For), lactate (Lac), and succinate (Suc). Data information: The reaction rates in C were calculated from the beginning of cultivation until the last sampling time point where glucose was still present in the medium. The means and individual data for n = 3 biologically independent samples are shown. The error bars represent ± SD..


For growth regimes tested , glucose uptake rate shows a biphasic response curve to increasing ATPase . Data information : means for n = 3 biologically independent samples are shown . error bars represent ± SD .

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For all growth regimes tested, the glucose uptake rate shows a biphasic response curve to increasing ATPase levels. Data information: The means for n = 3 biologically independent samples are shown. The error bars represent ± SD.


Simulation of PFK rate with varying ADP / ( ATP + ADP ) ratios . Data information : PFK flux was simulated as single ( isolated ) reaction with following fixed metabolite concentrations relevant for kinetic rate law of PFK PEP : 0 . 27 µmol / gDW ; F6P : 0 . 91 µmol / gDW ; FBP : 9 . 74 µmol / gDW ; total ATP + ADP : 2 . 67 µmol / gDW .

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Simulation of the PFK rate with varying ADP/(ATP+ADP) ratios. Data information: The PFK flux was simulated as single (isolated) reaction with the following fixed metabolite concentrations relevant for the kinetic rate law of the PFK PEP: 0.27 µmol/gDW; F6P: 0.91 µmol/gDW; FBP: 9.74 µmol/gDW ; total concentration ATP+ADP: 2.67 µmol/gDW.


A ) Comparison of simulations of kinetic model version 1 version 2 with measured exchange fluxes . Data information : For measurements , means for n = 3 biologically independent samples are shown error bars represent ± SD .

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A) Comparison of the simulations of the kinetic model version 1 and version 2 with the measured exchange fluxes. Data information: For the measurements, the means for n = 3 biologically independent samples are shown and the error bars represent ± SD.


B ) Comparison of simulations of kinetic model version 1 version 2 with measured metabolite concentrations . Data information : For measurements , means for n = 3 biologically independent samples are shown error bars represent ± SD .

B) Comparison of the simulations of the kinetic model version 1 and version 2 with the measured metabolite concentrations. Data information: For the measurements, the means for n = 3 biologically independent samples are shown and the error bars represent ± SD.


A ) Time course of biomass . B ) Time course of glucose . C Data information : means for n = 3 biologically independent samples are shown . error bars represent ± SD .

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A) Time course of biomass concentration. B) Time course of glucose concentration. C Data information: The means for n = 3 biologically independent samples are shown. The error bars represent ± SD.


C ) Specific glucose ( Glc ) uptake rate specific productivity for ethanol ( Eth ) , acetate ( Ace ) , formate ( For ) , lactate ( Lac ) , succinate ( Suc ) . D ) Yield of Eth , Ace , For , Lac , Suc . Data information : reaction rates in C were calculated for exponential phase under assumption of quasi - steady state . means ( means data for n = 3 biologically independent samples are shown . error bars represent ± SD .

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C) Specific glucose (Glc) uptake rate and specific productivity for ethanol (Eth), acetate (Ace), formate (For), lactate (Lac), and succinate (Suc). D) Yield of Eth, Ace, For, Lac, and Suc. Data information: The reaction rates in C were calculated for the exponential phase under assumption of quasi-steady state. The means ( and the means and individual data for n = 3 biologically independent samples are shown. The error bars represent ± SD.


( A - C ) Representative of localization of TLR2 - p scrTLR2 - p peptides in BM - derived macrophages were observed using confocal microscopy . ( A ) were probed with anti TLR2 antibody following by with APC - labeled secondary antibody or ( B ) with TLR1 antibody following by with FITC - labeled secondary antibody ( left panels , red ) . For , were labelled with lipid dye , DiD ( C ) . Then rhodamine - labeled peptide was added to ( middle panels , green ) . Merged are shown in right panel . Scale bars 10µm . mean Pearson correlation coefficient observed of TLR2 - p or scrTLR2 - p peptides with TLR2 TLR1 DiD presents as mean of 2 independent experiments ± S . EM ( n = 19 - 61 ) .

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(A-C) Representative images of Cellular localization of TLR2-p and scrTLR2-p peptides in BM-derived macrophages were observed using confocal microscopy. (A) Cells were probed with anti TLR2 antibody following by staining with APC-labeled secondary antibody or (B) with TLR1 antibody following by staining with FITC-labeled secondary antibody (left panels, red). For membrane staining, cells were labelled with lipid dye, DiD (C). Then rhodamine-labeled fluorescent peptide was added to the cells (middle panels, green). Merged images are shown in the right panel. Scale bars 10µm. The mean Pearson correlation coefficient observed of TLR2-p or scrTLR2-p peptides with TLR2 TLR1 and DiD presents as mean of 2 independent experiments ± S.EM (n=19-61).


( A ) Biochemical analyses of peptide interaction with TLR . BM - derived macrophages were incubated with 1µM fluorescently labeled rhodamine ( Rho ) - TLR2 - p or its mutant Rho - ScrTLR2 - p peptide . After 1h of incubation at 370C were lysated . Soluble fraction was used for immunoprecipitation with indicated TLR antibodies . samples were resolved by SDS - PAGE presence of labeled peptide was detected with a scanner . Subsequently , gel was transferred to a subjected to Western blotting for TLR6 , 1 , 2 4 in appropriate samples . Equal loading was measured by detecting of anti - tubulin in lysate . results are presented as mean of from two independent experiments . Non - specific binding of peptides to G beads was subtracted . results are mean ± S . E . M of at least two independent experiments .

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(A) Biochemical analyses of peptide interaction with TLR. BM-derived macrophages were incubated with 1µM fluorescently labeled rhodamine (Rho)-TLR2-p or its mutant Rho-ScrTLR2-p peptide. After 1h of incubation at 370C the cells were lysated. Soluble fraction was used for immunoprecipitation with the indicated TLR antibodies. Proteins samples were resolved by SDS-PAGE and the presence of the labeled peptide was detected with a fluorescent scanner. Subsequently, the gel was transferred to a membrane and subjected to Western blotting for TLR6,1,2 and 4 in the appropriate samples. Equal loading was measured by detecting of anti-tubulin in the cell lysate. The results are presented as the mean of fluorescent intensity from two independent experiments. Non-specific binding of the peptides to G protein beads was subtracted. The results are mean± S.E.M of at least two independent experiments.


( B ) of secreted pro - inflammatory cytokines , IL - 6 TNF - α from CD115 + . were incubated with 20µM of TLR2 - p or scrTLR2 - p peptide for 0 . 5h , then washed incubated with LTA ( 5μg / ml ) , PAM3CSK ( 0 . 5μg / ml ) or LPS ( 0 . 2μg / ml ) at 370C for 5h ( for TNF - α detection ) 22h ( for IL - 6 detection ) . results are mean ± S . E . M of two independent experiments ( n = 8 ) .

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(B) Proteins analysis of secreted pro-inflammatory cytokines, IL-6 and TNF-α from primary CD115+ cells. Cells were incubated with 20µM of TLR2-p or scrTLR2-p peptide for 0.5h, then washed and incubated with LTA (5μg/ml), PAM3CSK (0.5μg/ml) or LPS (0.2μg/ml) at 370C for 5h (for TNF-α detection) and 22h (for IL-6 detection). The results are mean± S.E.M of two independent experiments (n=8).


( B - C ) Represented of interaction between TLR2 TLR6 ( A ) between F4 / 80 TLR2 ( B ) in BM - derived macrophages with indicated treatments were observed by Resonance Energy Transfer ( FRET ) using ImageStreamX . Scale bars 10μm . were incubated with 20µM of TLR2 - p or scrTLR2 - p peptide for 0 . 5h , then washed incubated with 500ng / ml LTA for another 0 . 5h at 370C . were probed with anti TLR6 - PE or anti F4 / 80 - PE conjugated antibody ( donor ) anti TLR2 antibody following by with APC - labeled secondary antibody ( acceptor ) . PE ( middle panel ) FRET ( right panel ) were measured via ImageStreanX imaging flow cytometer . FRET shown are mean ± S . E . M of two independent experiments ( n = 2579 - 15567 ) .

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(B-C) Represented images of cellular interaction between TLR2 and TLR6 (A) and between F4/80 and TLR2 (B) in BM-derived macrophages with the indicated treatments were observed by Fluorescence Resonance Energy Transfer (FRET) using ImageStreamX. Scale bars 10μm. Cells were incubated with 20µM of TLR2-p or scrTLR2-p peptide for 0.5h, then washed and incubated with 500ng/ml LTA for another 0.5h at 370C. Cells were probed with anti TLR6-PE or anti F4/80-PE conjugated antibody (donor) and anti TLR2 antibody following by staining with APC-labeled secondary antibody (acceptor). PE intensity (middle panel) and FRET intensity (right panel) were measured via ImageStreanX imaging flow cytometer. The FRET intensity shown are mean ± S.E.M of two independent experiments (n=2579-15567).


( D ) Represented image of ERK1 , 2 phosphorylation in CD115 + BM monocytes upon LTA exposure . were pre - treated for 0 . 5h with 20µM of TLR2 - p peptide , scrTLR2 - p peptide or untreated , then washed incubated with 500ng / ml LTA for indicated times . ERK1 , 2 phosphorylation total ERK1 , 2 were detected by western blotting . Equal loading was detected by measuring anti tubulin . Results are representative data of 2 independent experiments . bands was quantified for p - ERK after 15min of LTA exposure . Results are normalized to tubulin are mean of 2 experiments ± S . D . M .

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(D) Represented image of ERK1,2 phosphorylation levels in CD115+BM monocytes cells upon LTA exposure. Cells were pre-treated for 0.5h with 20µM of TLR2-p peptide, scrTLR2-p peptide or untreated, then washed and incubated with 500ng/ml LTA for the indicated times. ERK1,2 phosphorylation levels and total ERK1,2 levels were detected by western blotting. Equal loading was detected by measuring anti tubulin. Results are representative data of 2 independent experiments. The bands intensity was quantified for p-ERK levels after 15min of LTA exposure. Results are normalized to tubulin levels and are the mean of 2 experiments ± S.D.M.


( B ) Representative colonoscopy of indicated treatments ( day 7 ) . C57BL mice were treated with 2 % of dextran sulfate sodium ( DSS ) in drinking water for 7 days . An amount of 5mg / Kg of TLR2 - p scrTLR2 - p peptides were injected IP every following day from day one . ( C ) A graphical summary of endoscopic colitis grades assessed on day 7 after treated with DSS only , DSS with TLR2 - p peptide , DSS with scrTLR2 - p peptide untreated mice ( n = 19 , 21 , 22 19 respectively ) . Results represent 3 independent experiments , scale bars 100µm 1000μm .

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(B) Representative colonoscopy images of the indicated treatments (day 7). C57BL mice were treated with 2% of dextran sulfate sodium (DSS) in drinking water for 7 days. An amount of 5mg/Kg of TLR2-p and scrTLR2-p peptides were injected IP every following day from day one.(C) A graphical summary of endoscopic colitis grades assessed on day 7 after treated with DSS only, DSS with TLR2-p peptide, DSS with scrTLR2-p peptide and untreated mice (n= 19, 21, 22 and 19 respectively). Results represent 3 independent experiments, scale bars 100µm and 1000μm.


( D ) A graphical summary of changes in ( day 6 ) of indicated groups .

(D) A graphical summary of changes in body weight (day 6) of the indicated groups.


( E ) Representative H & amp ; amp ; E histological for distal colon section ( day 7 ) of 9 - 10 sections from 2 independent experiments . Black asterisk indicates on large collection of blood in lumen , arrowheads indicate on widespread mucosal collapse ulceration arrows indicates on edema in sub mucosa . ( F ) A graphical summary of histological severity score of indicated treatments .

(E) Representative H&amp;E histological images for distal colon section (day 7) of 9-10 sections from 2 independent experiments. Black asterisk indicates on large collection of blood in the lumen, arrowheads indicate on widespread mucosal collapse and ulceration and arrows indicates on edema in the sub mucosa.(F) A graphical summary of histological severity score of the indicated treatments.


( A - B ) samples of colon tissue were extracted from mice with indicated treatments . ( A ) Cytokine of IL - 6 , IL - 1β INF - λ were determined by ELISA ( n = 4 - 7 ) . Results represent two or more independent experiments .

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(A-B) Proteins and RNA samples of colon tissue were extracted from mice with the indicated treatments. (A) Cytokine levels of IL-6, IL-1β and INF-λ were determined by ELISA (n=4-7). Results represent two or more independent experiments.


( A - B ) samples of colon tissue were extracted from mice with indicated treatments . ( B ) mRNA expression of IL - 6 , IL - 12p35 IL - 23p19 were assessed by quantitative RT - PCR normalized to TBP ( n = 4 - 6 ) . Results represent two or more independent experiments .

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(A-B) Proteins and RNA samples of colon tissue were extracted from mice with the indicated treatments. (B) mRNA expression levels of IL-6, IL-12p35 and IL-23p19 were assessed by quantitative RT-PCR and normalized to TBP (n=4-6). Results represent two or more independent experiments.


( A ) Flow cytometry of colonic lamina propria CD11c - CD11b + LY6Chi MHCII - monocytes from steady - state DSS day 7 treated with TLR2 peptide or scrTLR2 peptide , showing comparable monocytes infiltrates in to colitic colon . Plots were pre gated on live CD45 + . Results are representative of three independent experiments ( n = 3 per group ) . ( B ) Graphical summary of monocytes presented as % out of CD11b + .

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(A) Flow cytometry analysis of colonic lamina propria CD11c- CD11b+LY6Chi MHCII-monocytes from steady-state and DSS day 7 treated with TLR2 peptide or scrTLR2 peptide, showing comparable monocytes infiltrates in to the colitic colon. Plots were pre gated on live CD45+ cells. Results are representative of three independent experiments (n=3 per group). (B) Graphical summary of monocytes presented as % out of CD11b + cells.


( B ) loss of DSS challenged mice on day 6 .

(B) Weight loss of DSS challenged mice on day 6.


( C ) Real - time PCR showing mRNA expression of IL - 6 , IL - 12p35 IL - 23p19 of FACS isolated monocytes collected from mice with indicated treatments . Data are obtained from two to three independent experiments ( n = 6 - 7 mice pooled per group ) .

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(C) Real-time PCR analysis showing mRNA expression levels of IL-6, IL-12p35 and IL-23p19 of FACS isolated monocytes collected from mice with the indicated treatments. Data are obtained from two to three independent experiments (n=6-7 mice pooled per group).


E , F : EM of negatively stained Dam1c alone ( E ) in presence of Bim1 ( F ) , at low salt conditions . final concentrations of Dam1c Bim1 were 0 . 3 µM 0 . 1 µM , respectively . scale bars are 100 nm , boxes mark representative complexes shown as magnified inserts .

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E, F: EM images of negatively stained Dam1c alone (E) and in the presence of Bim1 (F), at low salt conditions. The final concentrations of Dam1c and Bim1 were 0.3 µM and 0.1 µM, respectively. The scale bars are 100 nm, boxes mark representative complexes shown as magnified inserts.


B : Chromatogram SDS - PAGE of elution fractions from gel filtration . Binding of Bim1 to Dam1 ∆ SxIPc lacking SxIP motif in C - terminus of Duo1 was examined by analytical exclusion chromatography . Elution fractions were analyzed by SDS - PAGE .

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B: Chromatogram and SDS-PAGE of elution fractions from gel filtration. Binding of Bim1 to Dam1∆SxIPc lacking the SxIP motif in the C-terminus of Duo1 was examined by analytical size exclusion chromatography. Elution fractions were analyzed by SDS-PAGE.


C : Solid phase binding to for binding of different Dam1 complexes to Bim1 . Recombinant GST - Bim1185 - 344 was immobilized on glutathione sepharose beads incubated with either recombinant Dam1WTc or Dam1 ∆ SxIPc . Input pull down samples were analyzed by SDS - PAGE .

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C: Solid phase binding assay to test for binding of different Dam1 complexes to Bim1. Recombinant GST-Bim1185-344 was immobilized on glutathione sepharose beads and incubated with either recombinant Dam1WTc or Dam1∆SxIPc. Input and pull down samples were analyzed by SDS-PAGE.


D : Pull down to compare binding of Dam1WTc Dam1 ∆ SxIPc to immobilized GST - Bim1185 - 344 . Soluble lysates from yeast strains expressing respective Duo1 - 6xFlag alleles were incubated with GST - Bim1185 - 344 immobilized on beads . Duo1 - 6xFlag bound to GST - Bim1185 - 344 was analyzed by western blot . An anti - Pgk1 antibody was used to confirm equal amounts in input samples .

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D: Pull down assay to compare binding of Dam1WTc and Dam1∆SxIPc to immobilized GST-Bim1185-344. Soluble cell lysates from yeast strains expressing the respective Duo1-6xFlag alleles were incubated with GST-Bim1185-344 immobilized on beads. Duo1-6xFlag bound to GST-Bim1185-344 was analyzed by western blot. An anti-Pgk1 antibody was used to confirm equal protein amounts in input samples.


C : Representative from live microscopy of Dad1 - GFP strains with Duo1WT - 6xFlag or Duo1 ∆ SxIP - 6xFlag . Displayed are in either metaphase ( left ) or anaphase ( right ) . Scale bar : 2 μm . D : of signal intensities of Dad1 - GFP at metaphase anaphase kinetochore clusters of Duo1WT - 6xFlag Duo1 ∆ SxIP - 6xFlag strains . Bars represent mean + / - standard deviation . Each spot represents value measured for a single kinetochore cluster . Values were normalized to mean value of metaphase clusters of Duo1WT - 6xFlag . A one - way ANOVA with Sidak ' s multiple comparisons was used to calculate p values . n ≥ 76 kinetochore clusters .

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C: Representative images from fluorescence live cell microscopy of Dad1-GFP strains with Duo1WT-6xFlag or Duo1∆SxIP-6xFlag. Displayed cells are in either metaphase (left) or anaphase (right). Scale bar: 2 μm. D: Quantification of signal intensities of Dad1-GFP at metaphase and anaphase kinetochore clusters of Duo1WT-6xFlag and Duo1∆SxIP-6xFlag strains. Bars represent the mean +/- standard deviation. Each spot represents the value measured for a single kinetochore cluster. Values were normalized to the mean value of metaphase clusters of the Duo1WT-6xFlag strain. A one-way ANOVA test with Sidak's multiple comparisons test was used to calculate p values. n ≥ 76 kinetochore clusters.


F : of cycle progression of yeast strains with different Duo1 alleles . Accumulation degradation of Pds1 after release from α factor arrest was analyzed by western blot ( left ) . An antibody against Pgk1 was used to confirm equal amounts in samples . content of population at indicated time points after release from α factor arrest was analyzed by FACS ( right ) . in G1 phase mitosis are characterized by a 1C 2C content , respectively . An intermediate content is detected for in S phase .

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F: Analysis of cell cycle progression of yeast strains with different Duo1 alleles. Accumulation and degradation of Pds1 after release from α factor arrest was analyzed by western blot (left). An antibody against Pgk1 was used to confirm equal protein amounts in samples. The DNA content of the cell population at indicated time points after release from α factor arrest was analyzed by FACS (right). Cells in G1 phase and mitosis are characterized by a 1C and 2C DNA content, respectively. An intermediate DNA content is detected for cells in S phase.


B : Representative from live microscopy of Dam1WT dam1 - 19 strains with Dad1 - GFP . Displayed are in metaphase ( left ) or anaphase ( right ) . Scale bar : 2 μm . C : of Dad1 - GFP signal intensities at metaphase anaphase kinetochore clusters of Dam1WT dam1 - 19 strains . Bars represent mean + / - standard deviation . Each spot represents value measured for a single kinetochore cluster . Values were normalized to mean value of metaphase clusters of Dam1WT . p - values were obtained by a one - way ANOVA with Sidak ' s multiple comparisons . n ≥ 86 kinetochore clusters .

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B: Representative images from live cell microscopy of Dam1WT and dam1-19 strains with Dad1-GFP. Displayed cells are in metaphase (left) or anaphase (right). Scale bar: 2 μm. C: Quantification of Dad1-GFP signal intensities at metaphase and anaphase kinetochore clusters of Dam1WT and dam1-19 strains. Bars represent the mean +/- standard deviation. Each spot represents the value measured for a single kinetochore cluster. Values were normalized to the mean value of metaphase clusters of the Dam1WT strain. p-values were obtained by a one-way ANOVA test with Sidak's multiple comparisons test. n ≥ 86 kinetochore clusters.


E , F : GST - Bim1185 - 344 pull down assays . Binding of immobilized GST - Bim1185 - 344 to Duo1 - 6xFlag was analyzed in context of ( E ) selective inhibition of Mps1 ( mps1 - 737 ) or overexpression of Mps1 from a galactose - inducible promotor ( F ) . For inhibition of Mps1 , were grown at restrictive of 37 ° C . Overexpression of Mps1 was achieved by growing in medium containing galactose . GST - Bim1185 - 344 immobilized on glutathione sepharose beads were incubated with soluble lysates of respective strains grown under mentioned conditions . Binding of Duo1 was analyzed by western blot . Pgk1 indicates equal amounts in samples .

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E, F: GST-Bim1185-344 pull down assays. Binding of immobilized GST-Bim1185-344 to Duo1-6xFlag was analyzed in context of (E) selective inhibition of Mps1 (mps1-737) or overexpression of Mps1 from a galactose-inducible promotor (F). For inhibition of Mps1, cells were grown at the restrictive temperature of 37 °C. Overexpression of Mps1 was achieved by growing cells in medium containing galactose. GST-Bim1185-344 immobilized on glutathione sepharose beads were incubated with soluble cells lysates of the respective strains grown under the mentioned conditions. Binding of Duo1 was analyzed by western blot. Pgk1 indicates equal protein amounts in all samples.


G : Live microscopy of a Dad1 - GFP under conditions of Mps1 overexpression . localization of Dam1c was visualized by Dad1 - GFP in either an Mps1WT or Mps1 overexpression setup ( pGal - Mps1 ) . Ndc80 - mRuby2 was used as reference point for localization of kinetochores . show representative metaphase from both strains . in dashed box is shown as magnification in lower left corner . arrowheads point at microtubules decorated by Dad1 - GFP . Please note that brightness contrast of Dad1 - GFP image in lower row were optimized to make microtubules clearly visible . Scale bar : 2 μm .

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G: Live cell microscopy of a Dad1-GFP strain under conditions of Mps1 overexpression. The localization of Dam1c was visualized by Dad1-GFP in either an Mps1WT or Mps1 overexpression setup (pGal-Mps1). Ndc80-mRuby2 was used as reference point for the localization of kinetochores. Images show representative metaphase cells from both strains. The area in the white dashed box is shown as magnification in the lower left corner. White arrowheads point at nuclear microtubules decorated by Dad1-GFP. Please note that brightness and contrast of the Dad1-GFP image in the lower row were optimized to make the nuclear microtubules clearly visible. Scale bar: 2 μm.


A , B : 5 µM , 7 . 5 µM or 10 µM Dam1WTc alone or in combination with respective concentrations of Bim1 - Bik1 were analyzed by exclusion chromatography . SDS - PAGE of elution fractions is shown in B . High molecular species of Dam1WTc were eluted after 0 . 9 - 1 . 0 ml ( see chromatogram in A ) .

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A, B: 5 µM, 7.5 µM or 10 µM Dam1WTc alone or in combination with respective concentrations of Bim1-Bik1 complex were analyzed by size exclusion chromatography. SDS-PAGE analysis of elution fractions is shown in B. High molecular weight species of Dam1WTc were eluted after 0.9 - 1.0 ml (see chromatogram in A).


B : Pull down for of Dam1c - Bim1 binding to Ndc80c . Ndc80c immobilized on beads was incubated with either Dam1c or Bim1 alone or with pre - formed Dam1c - Bim1 with increasing amounts of Bim1 . Input elution samples were analyzed by SDS - PAGE . LC HC denote heavy chains of M2 anti - Flag antibody used for immobilization of Ndc80c on beads . names in blue , red green refer to Dam1c , Ndc80c Bim1 , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Pull down assay for analysis of Dam1c-Bim1 binding to Ndc80c. Ndc80c immobilized on beads was incubated with either Dam1c or Bim1 alone or with pre-formed Dam1c-Bim1 complex with increasing amounts of Bim1. Input and elution samples were analyzed by SDS-PAGE. LC and HC denote the light and heavy chains of the M2 anti-Flag antibody used for immobilization of Ndc80c on beads. Protein names in blue, red and green refer to Dam1c, Ndc80c and Bim1, respectively.


D : Serial dilution of yeast strains with different Duo1 Ndc80 alleles in an Ndc80 - FRB - GFP background . Ndc80WT or Ndc80 ∆ 490 - 510 integration constructs were integrated at LEU2 locus combined with either Duo1WT or Duo1 ∆ SxIP allele ( at DUO1 locus ) . were spotted on YEPD medium without with 1 µg / ml Rapamycin incubated at indicated temperatures .

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

D: Serial dilution assay of yeast strains with different Duo1 and Ndc80 alleles in an Ndc80-FRB-GFP background. Ndc80WT or Ndc80∆490-510 integration constructs were integrated at the LEU2 locus and combined with either the Duo1WT or Duo1∆SxIP allele (at DUO1 locus). Cells were spotted on YEPD medium without and with 1 µg/ml Rapamycin and incubated at the indicated temperatures.


B : Serial dilution of yeast strains with 6xFlag - tagged Duo1WT or Duo1 ∆ SxIP Ask1WT , Ask12A or Ask12D . were grown at indicated temperatures were taken two days ( 25 ° C , 30 ° C 37 ° C ) or four days ( 16 ° C ) after spotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Serial dilution assay of yeast strains with 6xFlag-tagged Duo1WT or Duo1∆SxIP and Ask1WT, Ask12A or Ask12D. Cells were grown at the indicated temperatures and images were taken two days (25 °C, 30 °C and 37 °C) or four days (16 °C) after spotting.


a , alpha - KG extends lifespan of adult worms in metabolite longevity screen . metabolites were given at a of 8 mM .

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

a, alpha-KG extends the lifespan of adult worms in the metabolite longevity screen. All metabolites were given at a concentration of 8 mM.


c , Dose - response curve of α - KG effect on longevity .

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

c, Dose-response curve of the α-KG effect on longevity.


d , e , α - KG extends lifespan of worms fed that have been ampicillin arrested , mean lifespan ( days of adulthood ) with vehicle treatment ( mveh ) = 19 . 4 ( n = 80 tested ) , mα - KG = 25 . 1 ( n = 91 ) , P 0 . 0001 ( log - rank ) ( d ) ; or γ - irradiation - killed , mveh = 19 . 0 ( n = 88 ) , mα - KG = 23 . 0 ( n = 46 ) , P 0 . 0001 ( log - rank ) ( e ) . OP50 , E . coli OP50 .

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

d, e, α-KG extends the lifespan of worms fed bacteria that have been ampicillin arrested, mean lifespan (days of adulthood) with vehicle treatment (mveh) = 19.4 (n = 80 animals tested), mα-KG = 25.1 (n = 91), P  0.0001 (log-rank test) (d); or γ-irradiation-killed, mveh = 19.0 (n = 88), mα-KG = 23.0 (n = 46), P  0.0001 (log-rank test) (e). OP50, E. coli OP50 strain.


f alpha - KG does not further extend lifespan of ogdh - 1 RNAi worms , mveh = 21 . 2 ( n = 98 ) , mα - KG = 21 . 1 ( n = 100 ) , P = 0 . 65 ( log - rank ) .

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

f alpha-KG does not further extend the lifespan of ogdh-1 RNAi worms, mveh = 21.2 (n = 98), mα-KG = 21.1 (n = 100), P = 0.65 (log-rank test).


a , DARTS identifies ATP5B as an alpha - KG - binding . Red arrowhead , protected band .

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

a, DARTS identifies ATP5B as an alpha-KG-binding protein. Red arrowhead, protected band.


b , DARTS confirms alpha - KG binding specifically to ATP5B . IB , immunoblot .

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

b, DARTS confirms alpha-KG binding specifically to ATP5B. IB, immunoblot.


c , Inhibition of ATP synthase by alpha - KG ( released from octyl alpha - KG ; Supplementary Notes ) . This inhibition was reversible ( data not shown ) .

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

c, Inhibition of ATP synthase by alpha-KG (released from octyl alpha-KG; Supplementary Notes). This inhibition was reversible (data not shown).


d , e , Reduced ATP in octyl alpha - KG - treated normal humanfibroblasts ( * * P = 0 . 0016 , * * * * P 0 . 0001 ; by t - , two - , two - sample unequal variance ) ( d )

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

d, e, Reduced ATP levels in octyl alpha-KG-treated normal humanfibroblasts (**P = 0.0016, ****P  0.0001; by t-test, two-tailed, two-sample unequal variance) (d)


d , e , Reduced ATP in octyl alpha - KG - treated worms ( day 2 , P = 0 . 969 ; day 8 , * P = 0 . 012 ; by t - , two - , two - sample unequal variance ) ( e ) . RLU , relative luminescence units .

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

d, e, Reduced ATP levels in octyl alpha-KG-treated worms (day 2, P = 0.969; day 8, *P = 0.012; by t-test, two-tailed, two-sample unequal variance) (e). RLU, relative luminescence units.


f , g , Decreased oxygen consumption rate ( OCR ) in octyl alpha - KG - treated ( * * * P = 0 . 0004 , * * * * P 0 . 0001 ; by t - , two - , two - sample unequal variance ) ( f )

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

f, g, Decreased oxygen consumption rate (OCR) in octyl alpha-KG-treated cells (***P = 0.0004, ****P  0.0001; by t-test, two-tailed, two-sample unequal variance) (f)


f , g , Decreased oxygen consumption rate ( OCR ) in octyl alpha - KG - treated worms ( P 0 . 0001 ; by t - , two - , two - sample unequal variance ) ( g ) .

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

f, g, Decreased oxygen consumption rate (OCR) in octyl alpha-KG-treated worms (P  0.0001; by t-test, two-tailed, two-sample unequal variance) (g).


h , alpha - KG , released from octyl alpha - KG ( 800 µM ) , decreases state 3 , but not state 4o or 3u ( P = 0 . 997 ) , respiration in mitochondria isolated from mouse liver . respiratory control is decreased in octyl alpha - KG - ( 3 . 1 ± 0 . 6 ) versus vehicle - treated mitochondria ( 5 . 2 ± 1 . 0 ) ( * P = 0 . 015 ; by t - , two - , two - sample unequal variance ) . Oligo , oligomycin ; FCCP , carbonyl cyanide - 4 - ( trifluoromethoxy ) phenylhydrazone ; AA , antimycin A .

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

h, alpha-KG, released from octyl alpha-KG (800 µM), decreases state 3, but not state 4o or 3u (P = 0.997), respiration in mitochondria isolated from mouse liver. The respiratory control ratio is decreased in the octyl alpha-KG- (3.1 ± 0.6) versus vehicle-treated mitochondria (5.2 ± 1.0) (*P = 0.015; by t-test, two-tailed, two-sample unequal variance). Oligo, oligomycin; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; AA, antimycin A.


i , Eadie - Hofstee plot of steady - state inhibition kinetics of ATP synthase by alpha - KG ( produced by in situ hydrolysis of octyl alpha - KG ) . [ S ] is substrate ( ADP ) , V is initial velocity of ATP synthesis in presence of 200 µM octanol ( vehicle control ) or octyl alpha - KG . alpha - KG ( produced from octyl alpha - KG ) decreases apparent Vmax ( 53 . 9 to 26 . 7 ) Km ( 25 . 9 to 15 . 4 ) , by nonlinear regression least - squares fit . c - i , Results were replicated in two independent experiments . Mean ± standard deviation ( s . d . ) is plotted in cases .

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

i, Eadie-Hofstee plot of steady-state inhibition kinetics of ATP synthase by alpha-KG (produced by in situ hydrolysis of octyl alpha-KG). [S] is the substrate (ADP) concentration, and V is the initial velocity of ATP synthesis in the presence of 200 µM octanol (vehicle control) or octyl alpha-KG. alpha-KG (produced from octyl alpha-KG) decreases the apparent Vmax (53.9 to 26.7) and Km (25.9 to 15.4), by nonlinear regression least-squares fit. c-i, Results were replicated in two independent experiments. Mean ± standard deviation (s.d.) is plotted in all cases.


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

a-g, Effect of alpha-KG on the lifespan of mutant or RNAi worms. a, atp-2 RNAi, mveh = 22.8 (n = 97), mα-KG = 22.5 (n = 94), P = 0.35; or RNAi control, mveh = 18.6 (n = 94), mα-KG = 23.4 (n = 91), P  0.0001. b, daf-2(e1370), mveh = 38.0 (n = 72), mα-KG = 47.6 (n = 69), P  0.0001. c, eat-2(ad1116), mveh = 22.8 (n = 59), mα-KG = 22.9 (n = 40), P = 0.79. d, let-363 RNAi, mveh = 25.1 (n = 96), mα-KG = 25.7 (n = 74), P = 0.95; or gfp RNAi control, mveh = 20.2 (n = 99), mα-KG = 27.7 (n = 81), P  0.0001. e, daf-16(mu86), mveh = 13.4 (n = 71), mα-KG = 17.4 (n = 72), P  0.0001; or N2, mveh = 13.2 (n = 100), mα-KG = 22.3 (n = 104), P  0.0001. f, pha-4(zu225), mveh = 14.2 (n = 94), mα-KG = 13.5 (n = 109), P = 0.55. g, hif-1(ia4), mveh = 20.5 (n = 85), mα-KG = 26.0 (n = 71), P  0.0001; or N2, mveh = 21.5 (n = 101), mα-KG = 24.6 (n = 102), P  0.0001. P values were determined by the log-rank test. Number of independent experiments: RNAi control (6), atp-2 (2), let-363 (3), N2 (5), daf-2 (2), eat-2 (2), pha-4 (2), daf-16 (2), hif-1 (5).


a , Decreased phosphorylation of mammalian TOR substrates in U87 treated with octyl alpha - KG or oligomycin . Similar results were obtained in HEK - 293 , normal human fibroblasts mouse embryonic fibroblasts ( data not shown ) . P , phospho .

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

a, Decreased phosphorylation of mammalian TOR substrates in U87 cells treated with octyl alpha-KG or oligomycin. Similar results were obtained in HEK-293 cells, normal human fibroblasts and mouse embryonic fibroblasts (data not shown). P, phospho.


b , Increased autophagy in treated with alpha - KG or RNAi for atp - 2 or let - 363 . were taken at × 100 magnification . c , GFP : : LGG - 1 quantified using ImageJ ( Methods ) . Data show results of 2 - 3 independent experiments . Bars indicate mean . * * * * P 0 . 0001 ; NS , not significant ( t - , two - , two - sample unequal variance ) .

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

b, Increased autophagy in animals treated with alpha-KG or RNAi for atp-2 or let-363. Photographs were taken at ×100 magnification. c, GFP::LGG-1 puncta quantified using ImageJ (Methods). Data show results of 2-3 independent experiments. Bars indicate the mean. ****P  0.0001; NS, not significant (t-test, two-tailed, two-sample unequal variance).


d , alpha - KG are increased in starved worms . * * P 0 . 01 ( t - , two - , two - sample unequal variance ) . Mean ± s . d . is plotted .

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

d, alpha-KG levels are increased in starved worms. **P  0.01 (t-test, two-tailed, two-sample unequal variance). Mean ± s.d. is plotted.


A SHP1immunoprecipitation ( IP ) from Jurkat 1G4 ‐ CD8 . Upper panel of immunoblots shows expression in input lysates , isolated complexes are shown at bottom . Rabbit IgGIP ( isotype ) is shown as control .

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

A SHP1immunoprecipitation (IP) from Jurkat 1G4‐CD8 cells. Upper panel of immunoblots shows expression levels in the input lysates, and isolated protein complexes are shown at the bottom. Rabbit IgGIP (isotype) is shown as control.


B Lentiviral knock ‐ down / re ‐ expression constructs for THEMIS ‐ wt ‐ Strep THEMIS ‐ dPRR1 ‐ Strep in 1G4 ‐ CD8 . Knock ‐ down / re ‐ expression constructs are on shTHEMIS ‐ 128476 . Red arrowheads indicate specific signal for endogenous THEMIS re ‐ expressed THEMIS ‐ Strep . higher molecular band observed in lanes is a non ‐ specific signal .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Lentiviral knock‐down/re‐expression constructs for THEMIS‐wt‐Strep and THEMIS‐dPRR1‐Strep in 1G4‐CD8 cells. Knock‐down/re‐expression constructs are based on shTHEMIS‐128476. Red arrowheads indicate the specific signal for endogenous THEMIS and re‐expressed THEMIS‐Strep. The higher molecular weight band observed in all lanes is a non‐specific signal.


C Streptactinpull ‐ downs ( PDs ) of THEMIS ‐ Strep from 1G4 ‐ CD8 . as described in ( B ) were stimulated with 6V tetramers , followed by Streptactin PDs of THEMIS immunoblot of isolated complexes . Separate experiments for SHP1 SHP2 are shown .

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

C Streptactinpull‐downs (PDs) of THEMIS‐Strep from 1G4‐CD8 cells. Cells as described in (B) were stimulated with 6V tetramers, followed by Streptactin PDs of THEMIS and immunoblot analysis of isolated protein complexes. Separate experiments for SHP1 and SHP2 are shown.


D SHP1IP in presence absence of SHP1 tail phosphorylation . SHP1 was immunoprecipitated from J . CaM1 . 6LCK ‐ Tet . Where indicated , LCK expression was induced with doxycycline prior to experiment or Src ‐ kinase inhibitor PP2 was used to abolish residual SHP1 ‐ pY564 . upper panel of immunoblots shows effect of LCK activity on SHP1 ‐ pY564 . red arrowhead indicates residual pY564 . Isolated complexes are shown in lower panels .

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D SHP1IP in the presence and absence of SHP1 tail phosphorylation. SHP1 was immunoprecipitated from J.CaM1.6LCK‐Tet cells. Where indicated, LCK expression was induced with doxycycline prior to the experiment or Src‐kinase inhibitor PP2 was used to abolish residual SHP1‐pY564. The upper panel of immunoblots shows the effect of LCK activity on SHP1‐pY564 levels. The red arrowhead indicates residual pY564. Isolated protein complexes are shown in the lower panels.


E In vitro phosphatase treatment of THEMIS : GRB2 : SHP1 . Bead ‐ bound THEMIS ‐ Strep complexes from 1G4 ‐ CD8 were incubated in alkaline phosphatase ( AP ) buffer in absence ( lane 1 ) or presence of AP ( lane 2 ) , or left completely untreated ( lane 3 ) , prior to washing elution .

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

E In vitro phosphatase treatment of the THEMIS:GRB2:SHP1 complex. Bead‐bound THEMIS‐Strep complexes from 1G4‐CD8 cells were incubated in alkaline phosphatase (AP) buffer in the absence (lane 1) or presence of AP (lane 2), or left completely untreated (lane 3), prior to washing and elution.


F Anti ‐ HAIP from HEK293 transfected with HA ‐ SHP1 GRB2 ‐ Myc constructs . GRB2 mutants used : W36K , N ‐ SH3 mutant ; W193K , C ‐ SH3 mutant . Upper panels show expression in input ; isolated complexes are shown at bottom . Relative amounts of GRB2 ‐ Myc are normalized to bait HA ‐ SHP1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Anti‐HAIP from HEK293 cells transfected with HA‐SHP1 and GRB2‐Myc constructs. GRB2 mutants used: W36K, N‐SH3 mutant; W193K, C‐SH3 mutant. Upper panels show expression levels in the input; isolated proteins complexes are shown at the bottom. Relative amounts of GRB2‐Myc are normalized to the bait HA‐SHP1.


G Far ‐ Western blot of GRB2 ‐ SH3 domains binding to full ‐ SHP1 . HA IPs from empty vector or HA ‐ SHP1 ‐ transfected HEK293 were subjected to far ‐ Western blotting using recombinant GST ‐ tagged N ‐ or C ‐ SH3 domains of GRB2 . GST alone served as a background control . Blots were re ‐ probed for SHP1 loading for normalization of anti ‐ GST signals . Data shown are representative of three independent experiments .

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

G Far‐Western blot of GRB2‐SH3 domains binding to full‐length SHP1. HA IPs from empty vector or HA‐SHP1‐transfected HEK293 cells were subjected to far‐Western blotting using recombinant GST‐tagged N ‐or C‐SH3 domains of GRB2. GST alone served as a background control. Blots were re‐probed for SHP1 loading for normalization of anti‐GST signals. Data shown are representative of three independent experiments.


A Lentiviral knock ‐ down ( KD ) of THEMIS expression in 1G4 ‐ CD8 . efficiency of THEMIS KD was assessed using immunoblotting .

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

A Lentiviral knock‐down (KD) of THEMIS expression in 1G4‐CD8 cells. The efficiency of the THEMIS KD was assessed using immunoblotting.


B Phospho ‐ flow cytometry of pERK responses in THEMIS KD . 1G4 ‐ CD8 control THEMIS KD were stimulated with NY ‐ ESO ‐ 1 pMHC tetramers analysed for ERK phosphorylation using flow cytometry . Data shown are representative of three independent experiments . C of pERK ‐ positive from ( B ) . Data from three independent experiments were used in . n = 3 , means ± are shown ; two ‐ unpaired Student ' s t ‐ , * P 0 . 05 , * * P 0 . 01 .

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

B Phospho‐flow cytometry analysis of pERK responses in THEMIS KD cells. 1G4‐CD8 control and THEMIS KD cells were stimulated with NY‐ESO‐1 pMHC tetramers and analysed for ERK phosphorylation using flow cytometry. Data shown are representative of three independent experiments.C Quantification of pERK‐positive cells from (B). Data from three independent experiments were used in the analysis. n = 3, means ± SEM are shown; two‐tailed unpaired Student's t‐test, *P 0.05, **P 0.01.


D Phospho ‐ flow cytometry of pERK responses against titrated concentrations of 9V tetramer . Time point was fixed at 60 s . Data shown are representative of three independent experiments . E of pERK ‐ positive from ( D ) . Data from three independent experiments were used in . n = 3 , means ± are shown .

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D Phospho‐flow cytometry analysis of pERK responses against titrated concentrations of 9V tetramer. Time point was fixed at 60 s. Data shown are representative of three independent experiments.E Quantification of pERK‐positive cells from (D). Data from three independent experiments were used in the analysis. n = 3, means ± SEM are shown.


A 1G4 ‐ CD8 control THEMIS KD were stimulated with NY ‐ ESO ‐ 1 pMHC tetramers analysed for pY142 ‐ CD3 ‐ ζ phosphorylation by flow cytometry . Data shown are representative of three independent experiments . B of pY142 ‐ CD3 ‐ ζ responses of from ( A ) . Data from three independent experiments were used in . Responses are expressed as of positive control sodium pervanadate . n = 3 , means ± are shown ; two ‐ unpaired Student ' s t ‐ , * * P 0 . 01 .

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

A 1G4‐CD8 control and THEMIS KD cells were stimulated with NY‐ESO‐1 pMHC tetramers and analysed for pY142‐CD3‐ζ phosphorylation by flow cytometry. Data shown are representative of three independent experiments.B Quantification of pY142‐CD3‐ζ responses of cells from (A). Data from three independent experiments were used in the analysis. Responses are expressed as percentage of the positive control sodium pervanadate. n = 3, means ± SEM are shown; two‐tailed unpaired Student's t‐test, **P 0.01.