( A ) Protein sequence alignment of the C - terminal region of D . melanogaster , human , and yeast Glycogen synthases . Identical residues are blue ; all other residues are red . Conserved in all three species , R593 , W609 , and S651 are underlined .

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(A) Protein sequence alignment of the C-terminal region of D. melanogaster, human, and yeast Glycogen synthases. Identical residues are blue; all other residues are red. Conserved in all three species, R593, W609, and S651 are underlined.


( B ) Western blot / Co - immunoprecipitation ( co - IP ) showing that Flag - Atg8 binds to a Venus - GlyS or Venus - GlyS ( S651A ) protein complex in response to starvation . Flag - Atg8 is unable to co - IP with either Venus - GlyS ( R593A ) or Venus - GlyS ( W609A ) . Venus - GlyS and Venus - GlyS mutants were co - IP ' d from muscle lysate from Dmef2 - Gal4 / UAS - Flag - Atg8 or UAS - Venus - GlyS ( WT or mutant ) / + ; Dmef2 - Gal4 / UAS - Flag - Atg8third instar larvae . These were fed on high - nutrientfood for 18 h , and then transferred to fresh high - nutrientfood or low - nutrientfood for 6 h .

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(B) Western blot/Co-immunoprecipitation (co-IP) showing that Flag-Atg8 binds to a Venus-GlyS or Venus-GlyS (S651A) protein complex in response to starvation. Flag-Atg8 is unable to co-IP with either Venus-GlyS (R593A) or Venus-GlyS (W609A). Venus-GlyS and Venus-GlyS mutants were co-IP'd from muscle lysate from Dmef2-Gal4/UAS-Flag-Atg8 or UAS-Venus-GlyS(WT or mutant)/+;Dmef2-Gal4/UAS-Flag-Atg8third instar larvae. These were fed on high-nutrientfood for 18 h, and then transferred to fresh high-nutrientfood or low-nutrientfood for 6 h.


( C ) Venus - GlyS was localized predominantly to the Flag - labeled autophagosomes , with weak staining in the rest of the cytoplasm . ( D ) Venus - GlyS ( R593A ) was found throughout the cytoplasm and did not colocalize with autophagosomes . ( E ) Venus - GlyS ( S651A ) was localized to the autophagosomes . ( F ) Venus - GlyS ( W609A ) did not colocalize with the Flag - labeled autophagosomes .

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(C) Venus-GlyS was localized predominantly to the Flag-labeled autophagosomes, with weak staining in the rest of the cytoplasm. (D) Venus-GlyS (R593A) was found throughout the cytoplasm and did not colocalize with autophagosomes. (E) Venus-GlyS (S651A) was localized to the autophagosomes. (F) Venus-GlyS (W609A) did not colocalize with the Flag-labeled autophagosomes.


( B ) Lignin accumulation patterns at endodermal surface or median positions with or without the 100 nM CIF2 ( Casparian strip integrity factor 2 ) ligand treatment . Lignin and cellulosic ( unmodified ) cell walls are stained with Basic Fuchsin and Calcofluor White , shown in magenta and white respectively . Schematics are indicating the position of optical sections in a 3D illustration . For each condition , at least 10 roots were tested and showed similar results in two independent experiments . White arrows indicate sites of excess lignification on the cortex facing ( outer ) side . Scale Bar = 5 µm . Data information : " inner " designates the stele - facing endodermal surface , " outer " , the cortex - facing surface .

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 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 O O O O O O O O O O O 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) Lignin accumulation patterns at endodermal surface or median positions with or without the 100 nM CIF2 (Casparian strip integrity factor 2) ligand treatment. Lignin and cellulosic (unmodified) cell walls are stained with Basic Fuchsin and Calcofluor White, shown in magenta and white respectively. Schematics are indicating the position of optical sections in a 3D illustration. For each condition, at least 10 roots were tested and showed similar results in two independent experiments. White arrows indicate sites of excess lignification on the cortex facing (outer) side. Scale Bar = 5 µm. Data information: "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.


( C , D ) Localization of SGN1 - Citrine and lignin deposition patterns in pCASP1 : : SGN1 - Citrine lines in wild - type ( Col ) and different mutant backgrounds ( sgn1 , sgn3 , cif1 cif2 ) ( C ) . myrpalm - SGN1 - Citrine localization and lignin deposition patterns in pCASP1 : : myrpalm - SGN1 - Citrine lines ( D ) . Lignin ( Basic Fuchsin ) and cell walls ( Calcoflour White ) are shown in magenta and white respectively . For this experiment , two or three independent lines were tested . From each transgenic line , 2 positions from 12 roots were observed and representative pictures are shown in the figure . Schematics are indicating the position of optical sections in a 3D illustration . White arrows in ( D ) highlight excess lignification on the pericycle - facing ( inner ) side . Scale bars = 5 µm . Data information : " inner " designates the stele - facing endodermal surface , " outer " , the cortex - facing surface .

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(C,D) Localization of SGN1-Citrine and lignin deposition patterns in pCASP1::SGN1-Citrine lines in wild-type (Col) and different mutant backgrounds (sgn1, sgn3, cif1 cif2) (C). myrpalm-SGN1-Citrine localization and lignin deposition patterns in pCASP1::myrpalm-SGN1-Citrine lines (D). Lignin (Basic Fuchsin) and cell walls (Calcoflour White) are shown in magenta and white respectively. For this experiment, two or three independent lines were tested. From each transgenic line, 2 positions from 12 roots were observed and representative pictures are shown in the figure. Schematics are indicating the position of optical sections in a 3D illustration. White arrows in (D) highlight excess lignification on the pericycle-facing (inner) side. Scale bars = 5 µm. Data information: "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.


( A ) Quantification of defects in CSD formation as number of holes per 100 µm in the CASP1 - GFP domain at around 10 cells after onset of CASP1 - GFP expression in 5 - day - old seedlings . In the box plot , boxes indicate ranges from first to third quartiles , bold central lines display median . Upper and lower whiskers extend to maximum or minimum values no further than 1 . 5 times IQR ( interquartile range , the distance between the first and third quartiles ) . One - way ANOVA was performed followed by Tukey ' s test . Different letters show significant statistical differences . ( p < 0 . 05 , One - way ANOVA and Tukey ' s test , 12 roots in total were observed for each condition in two independent assays ) .

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(A) Quantification of defects in CSD formation as number of holes per 100 µm in the CASP1-GFP domain at around 10 cells after onset of CASP1-GFP expression in 5-day-old seedlings. In the box plot, boxes indicate ranges from first to third quartiles, bold central lines display median. Upper and lower whiskers extend to maximum or minimum values no further than 1.5 times IQR (interquartile range, the distance between the first and third quartiles). One-way ANOVA was performed followed by Tukey's test. Different letters show significant statistical differences. (p<0.05, One-way ANOVA and Tukey's test, 12 roots in total were observed for each condition in two independent assays).


( B ) Propidium iodide ( PI ) penetration assay in the presence or the absence of CIF2 . CS barrier function was scored as exclusion of PI signal from the inner side of endodermal cells . In the box plot , boxes indicate ranges from first to third quartiles , bold central lines display median . Upper and lower whiskers extend to the maximum or minimum values no further than 1 . 5 times IQR . Different letters show significant statistical differences . ( p < 0 . 05 , One - way ANOVA and Tukey ' s test . During two independent experiments , 10 roots in total were tested for each condition ) .

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(B) Propidium iodide (PI) penetration assay in the presence or the absence of CIF2. CS barrier function was scored as exclusion of PI signal from the inner side of endodermal cells. In the box plot, boxes indicate ranges from first to third quartiles, bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters show significant statistical differences. (p<0.05, One-way ANOVA and Tukey's test. During two independent experiments, 10 roots in total were tested for each condition).


( C ) [ γ - 32P ] ATP radioactive in vitro kinase assay of SGN3 kinase domain against SGN1 . Autoradiograph is shown on top . Coomassie stained - gel below illustrates presence and equal loading of recombinant proteins . Note that a kinase - dead SGN1 variant was used to avoid auto - phosphorylation activity of SGN1 . Also note that trigger factor represents a very big tag protein , accounting for the high migration of TF - SGN1 . Representative result of three independent experiments is shown .

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(C) [γ-32P]ATP radioactive in vitro kinase assay of SGN3 kinase domain against SGN1. Autoradiograph is shown on top. Coomassie stained-gel below illustrates presence and equal loading of recombinant proteins. Note that a kinase-dead SGN1 variant was used to avoid auto-phosphorylation activity of SGN1. Also note that trigger factor represents a very big tag protein, accounting for the high migration of TF-SGN1. Representative result of three independent experiments is shown.


( A ) Localization of Citrine - RBOHD ( left ) or Citrine - RBOHF ( right ) in endodermal ( en ) cells . Both proteins were expressed under the control of pCASP1 , an endodermis - specific promoter . Representative pictures are shown , 2 positions from 10 roots for each transgenic line were inspected . Scale bar = 5 µm .

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(A) Localization of Citrine-RBOHD (left) or Citrine-RBOHF (right) in endodermal (en) cells. Both proteins were expressed under the control of pCASP1, an endodermis-specific promoter. Representative pictures are shown, 2 positions from 10 roots for each transgenic line were inspected. Scale bar = 5 µm.


( B ) Lignin accumulation in WT and rbohD and rbohF single mutants and a double mutant with or without 2 - hour 100 nM CIF2 peptide treatment . Arrowheads indicate excess lignification . Pictures are shown as overviews ( maximum projection ) or median sections . Lignin and cell walls are shown with magenta ( stained with Basic Fuchsin ) and gray ( stained with Calcofluor White ) respectively . Representative pictures are shown , 12 roots ( overview ) and 2 positions in 12 roots ( median section ) were inspected . Scale bars = 20 µm ( lignin overviews ) , 5 µm ( median sections ) . " inner " designates the stele - facing endodermal surface , " outer " , the cortex - facing surface .

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(B) Lignin accumulation in WT and rbohD and rbohF single mutants and a double mutant with or without 2-hour 100 nM CIF2 peptide treatment. Arrowheads indicate excess lignification. Pictures are shown as overviews (maximum projection) or median sections. Lignin and cell walls are shown with magenta (stained with Basic Fuchsin) and gray (stained with Calcofluor White) respectively. Representative pictures are shown, 12 roots (overview) and 2 positions in 12 roots (median section) were inspected. Scale bars = 20 µm (lignin overviews), 5 µm (median sections). "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.


( A ) Co - visualization of pCASP1 : : SGN1 - mCherry and pCASP1 : : SGN3 - GFP . Note that their respective localization at the PM has only restricted overlap at the cortical side of the CS domain ( Arrow ) . Representative picture is shown , 3 - 6 roots in 4 different transgenic lines were inspected . Scale bar = 10 µm . " inner " designates the stele - facing endodermal surface , " outer " , the cortex - facing surface .

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(A) Co-visualization of pCASP1::SGN1-mCherry and pCASP1::SGN3-GFP. Note that their respective localization at the PM has only restricted overlap at the cortical side of the CS domain (Arrow). Representative picture is shown, 3-6 roots in 4 different transgenic lines were inspected. Scale bar = 10 µm. "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.


( B , C ) Overview of endodermal cells after with or without 1 µM CIF2 treatment for 24 hours . Red arrowheads are indicating ROS production sites and boxes in dotted lines are corresponding to the regions in ( sky blue and creme boxes in C and black boxes in D ) . Similar patterns were obtained in 39 or 41 cells from 5 roots with or without the peptide treatment . Scale bars = 500 nm .

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(B,C) Overview of endodermal cells after with or without 1 µM CIF2 treatment for 24 hours. Red arrowheads are indicating ROS production sites and boxes in dotted lines are corresponding to the regions in (sky blue and creme boxes in C and black boxes in D). Similar patterns were obtained in 39 or 41 cells from 5 roots with or without the peptide treatment. Scale bars = 500 nm.


( D ) In situ H2O2 detection at Casparian strips in WT , sgn3 , sgn1 , rbohF , rbohD and rbohDF with or without 24 - hour treatment of 1 µM CIF2 . Brackets and arrowheads indicate Casparian strips ( seen as uniformly whitish cell wall areas ) and H2O2 production sites ( black area ) respectively . Scale bar = 1000 nm .

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(D) In situ H2O2 detection at Casparian strips in WT, sgn3, sgn1, rbohF, rbohD and rbohDF with or without 24-hour treatment of 1 µM CIF2. Brackets and arrowheads indicate Casparian strips (seen as uniformly whitish cell wall areas) and H2O2 production sites (black area) respectively. Scale bar = 1000 nm.


( E ) Quantification of ROS production as a number of dark pixels area ( n = 23 - 44 sites from 5 roots of each condition in one experiment . ) . In the box plot , boxes indicate ranges from first to third quartiles , bold central lines display median . Upper and lower whiskers extend to the maximum or minimum values no further than 1 . 5 times IQR . Different letters mean significant statistical differences . ( p < 0 . 01 , One - way ANOVA and Tukey ' s test . )

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(E) Quantification of ROS production as a number of dark pixels area (n = 23 - 44 sites from 5 roots of each condition in one experiment.). In the box plot, boxes indicate ranges from first to third quartiles, bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters mean significant statistical differences. (p<0.01, One-way ANOVA and Tukey's test.)


( A ) [ γ - 32P ] ATP radioactive in vitro kinase assay of TF - SGN1 against GST - N - terminal cytoplasmic domains of RBOHD or F . Autoradiograph is shown on top . Coomassie stained - gel below illustrates presence and equal loading of recombinant proteins . Experiments were done independently three times with similar results .

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(A) [γ-32P]ATP radioactive in vitro kinase assay of TF-SGN1 against GST-N-terminal cytoplasmic domains of RBOHD or F. Autoradiograph is shown on top. Coomassie stained-gel below illustrates presence and equal loading of recombinant proteins. Experiments were done independently three times with similar results.


( B ) HEK293Tcell based NOX activation assay . Cells were transfected with the indicated plasmid combinations . The phosphatase inhibitor Calyculin A was added directly before the start of the measurements . Each data point represents the mean of six wells analyzed in parallel , bars indicate S . D . . Experiments were repeated three

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(B) HEK293Tcell based NOX activation assay. Cells were transfected with the indicated plasmid combinations. The phosphatase inhibitor Calyculin A was added directly before the start of the measurements. Each data point represents the mean of six wells analyzed in parallel, bars indicate S.D.. Experiments were repeated three times


( A ) CASP1 - GFP and lignin deposition in WT , sgn3 , rbohD , rbohF and rbohDF . CASP1 - GFP and lignin ( fuchsin ) are presented in green and magenta respectively . Pictures were obtained from more than 10 roots from each background with similar results . Scale bar = 10 µm .

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(A) CASP1-GFP and lignin deposition in WT, sgn3, rbohD, rbohF and rbohDF. CASP1-GFP and lignin (fuchsin) are presented in green and magenta respectively. Pictures were obtained from more than 10 roots from each background with similar results. Scale bar = 10 µm.


( B ) Time lapse imaging of single - or cotreatment of 10 nM CIF2 with 25 µM cycloheximide ( CHX ) on CASP1 - GFP in cif1 cif2 . Seedlings were pretreated with or without CHX for 30 min and transferred onto each medium . Scale bar = 10 µm ( See also movie EV1 ) . ( C ) Quantification of ( B ) . Relative numbers of holes in CASP1 - GFP domain after single - or co - treatment with CIF2 or CHX from the pictures in ( B ) . Bars are S . D . * indicates statistical significance from all other conditions ( p < 0 . 01 ) after one - way ANOVA and Tukey test . 6 roots in total for each condition were observed during two independent tests .

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(B) Time lapse imaging of single- or cotreatment of 10 nM CIF2 with 25 µM cycloheximide (CHX) on CASP1-GFP in cif1 cif2. Seedlings were pretreated with or without CHX for 30 min and transferred onto each medium. Scale bar = 10 µm (See also movie EV1). (C) Quantification of (B). Relative numbers of holes in CASP1-GFP domain after single- or co-treatment with CIF2 or CHX from the pictures in (B). Bars are S.D. * indicates statistical significance from all other conditions (p<0.01) after one-way ANOVA and Tukey test. 6 roots in total for each condition were observed during two independent tests.


( D ) Fold - change of 930 genes ( p < 0 . 05 and log2 ( Fold change ) ≥ 1 or ≤ - 1 at least one time point in one genotype ) after CIF2 treatment at indicated time points in WT , cif1 , 2 and sgn3 . Degree of the fold changes are shown in color code as indicated .

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(D) Fold-change of 930 genes (p<0.05 and log2(Fold change) ≥1 or ≤ -1 at least one time point in one genotype) after CIF2 treatment at indicated time points in WT, cif1,2 and sgn3. Degree of the fold changes are shown in color code as indicated.


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


( a ) Lipid phosphatase activity of Paladin , wild - type 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 .

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(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 .

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(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 .

(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 .

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(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 .

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(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 .

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


( c ) Internalized pool of VEGFR2 after VEGF - A treatment ( 50 ng / ml ) of non - transfected HDMEC ( " NT " ) or HDMEC transfected with PALD1 siRNA ( # 1 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 .

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(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 .

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(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 .

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(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O B-CONTROLLED_VAR 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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


( B - C ) Represented 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O 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 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

(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 .

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(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 O O O O O O O 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. (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 O O O O O O O O O O O O O O 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 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

(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 .

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(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 ) .

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


E , F : EM 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O 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: 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 O O O O O O O O O O O O O O O O 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: 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.