( 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 .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) and nuclei ( DAPI , blue ) , scale bar : 10 μm . White boxes in the upper image are showed at higher magnification below , scale bar : 3 µm .

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

(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 ) 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 .

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

(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 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 .

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

(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 ) 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 .

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

(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 ) 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 .

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

(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 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 .

O O O O O O B-MEASURED_VAR 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O 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 B-MEASURED_VAR 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 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 O O O O O O O O O O O O O O O O O O O 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 ) 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 .

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

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

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 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 .

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

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 + / + 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 .

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

(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 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 .

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

(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 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 .

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

(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 ) 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 .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 + / + 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 .

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

(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 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 .

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

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

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

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


( 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 .

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

(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 ) 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 .

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

(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 ) 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 .

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

(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 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 .

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

(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 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 .

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

(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 , 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 .

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 B-MEASURED_VAR 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

(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 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 .

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

(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 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 .

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

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 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

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 . .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 ) .

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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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 ) .

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

(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 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 ) .

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

(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 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 .

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

(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 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 .

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

(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 body weight ( day 6 ) of the indicated groups .

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


( E ) Representative H & amp ; 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 .

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

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

(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 ) 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 .

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

(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 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 .

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

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

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

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


( 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 ) .

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

(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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 : 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 .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: 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 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 .

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

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 , 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 .

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

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 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 .

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

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 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 ) .

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 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 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

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 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 .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR 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 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 .

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

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 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 .

O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-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 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 the lifespan of adult worms in the metabolite longevity screen . All metabolites were given at a concentration 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 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 the α - KG effect on longevity .

O 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 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 .

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

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 the lifespan of ogdh - 1 RNAi worms , mveh = 21 . 2 ( n = 98 ) , mα - KG = 21 . 1 ( n = 100 ) , P = 0 . 65 ( log - rank test ) .

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 O O O O O O O O O O O O O O O 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 protein . Red arrowhead , protected band .

O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O 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 B-MEASURED_VAR 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 levels in octyl alpha - KG - treated normal humanfibroblasts ( * * P = 0 . 0016 , * * * * P 0 . 0001 ; by t - test , two - tailed , two - sample unequal variance ) ( d )

O O O O O B-MEASURED_VAR 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

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 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 .

O O O O O B-MEASURED_VAR 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

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 cells ( * * * P = 0 . 0004 , * * * * P 0 . 0001 ; by t - test , two - tailed , 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 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 - test , two - tailed , 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 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 . 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 .

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

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 O O O O 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 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 ) .

O 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 O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O 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 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 levels are increased in starved worms . * * P 0 . 01 ( t - test , two - tailed , 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 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 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 .

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

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 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 .

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

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 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 .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 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 .

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

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 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 .

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

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 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 .

O O O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR 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 ‐ 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 .

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

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 cells . The efficiency of the THEMIS KD was assessed using immunoblotting .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR 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 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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

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


A Lentiviral knock ‐ down of THEMIS expression in primary humanCD4 + T cells . Following transduction , cells were expanded on CD3 / CD28 beads in the presence of exogenous IL ‐ 2 and analysed for THEMIS KD by immunoblotting on day 4 post ‐ transduction . Data shown are representative of three independent experiments .

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

A Lentiviral knock‐down of THEMIS expression in primary humanCD4+T cells. Following transduction, cells were expanded on CD3/CD28 beads in the presence of exogenous IL‐2 and analysed for THEMIS KD by immunoblotting on day 4 post‐transduction. Data shown are representative of three independent experiments.


B pERK response in THEMIS KD humanCD4 + T cells . Transduced CD4 + cells were removed from beads , rested overnight and stimulated with CD3 mAb for the indicated time points . ERK phosphorylation was assessed by immunoblotting . Data shown are representative of three independent experiments .

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

B pERK response in THEMIS KD humanCD4+T cells. Transduced CD4+ cells were removed from beads, rested overnight and stimulated with CD3 mAb for the indicated time points. ERK phosphorylation was assessed by immunoblotting. Data shown are representative of three independent experiments.


C Expression of CD25 and CD69 surface markers of THEMIS KD humanCD4 + T cells was assessed on day 4 post ‐ transduction by flow cytometry . Data shown are representative of three independent experiments .

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

C Expression of CD25 and CD69 surface markers of THEMIS KD humanCD4+T cells was assessed on day 4 post‐transduction by flow cytometry. Data shown are representative of three independent experiments.


D CD69 up ‐ regulation on re ‐ stimulated THEMIS KD CD4 + T cells . CD3 / CD28 beads were removed on day 1 post ‐ transduction , and cells were cultured in IL ‐ 2 and IL ‐ 7 . After removal of cell debris at day 4 , cells were restimulated with CD3 / CD28 beads at the indicated bead to cell ratio . CD69 surface expression was assessed by flow cytometry 24 h later . Data shown are representative of two independent experiments . n = 3 , means ± SD are shown ; two ‐ tailed unpaired Student ' s t ‐ test , * P 0 . 05 , * * * P 0 . 001 .

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

D CD69 up‐regulation on re‐stimulated THEMIS KD CD4+T cells. CD3/CD28 beads were removed on day 1 post‐transduction, and cells were cultured in IL‐2 and IL‐7. After removal of cell debris at day 4, cells were restimulated with CD3/CD28 beads at the indicated bead to cell ratio. CD69 surface expression was assessed by flow cytometry 24 h later. Data shown are representative of two independent experiments. n = 3, means ± SD are shown; two‐tailed unpaired Student's t‐test, *P 0.05, ***P 0.001.