N . Top , basic scheme of CerS2 promoter , indicating putative GATA - 1 binding site ( blue ) location of primers used for amplification in ChIP assays ( black arrows ) . Bottom , relative anti - GATA - 1 ChIP in OT - II WT CCR5 - / - lymphoblasts ( n = 5 ) .

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N. Top, basic scheme of the CerS2 promoter, indicating the putative GATA-1 binding site (blue) and location of the primers used for amplification in ChIP assays (black arrows). Bottom, relative anti-GATA-1 ChIP levels in OT-II WT and CCR5-/- lymphoblasts (n = 5).


A . Scheme of strategy used to form TCR proteoliposomes , of LUV generated at indicated lipid molar . Polydispersity index values are shown as black squares for each condition ( n = 3 ) . SBP , streptavidin - binding - peptide - tagged TCR .

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A. Scheme of the strategy used to form TCR proteoliposomes, and size of LUV generated at the indicated lipid molar ratio. Polydispersity index values are shown as black squares for each condition (n = 3). SBP, streptavidin-binding-peptide-tagged TCR.


B . Representative immunoblots comparing TCR nanocluster sizes via BN - PAGE anti - CD3ζ immunoblotting in TCR proteoliposomes lysed in presence of Brij - 96 or digitonin . marker is ferritin ( f1 , 440 f2 , 880 kDa forms ) .

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B. Representative immunoblots comparing TCR nanocluster sizes via BN-PAGE and anti-CD3ζ immunoblotting in TCR proteoliposomes lysed in the presence of Brij-96 or digitonin. The marker protein is ferritin (f1, 440 and f2, 880 kDa forms).


C . of nanocluster monomeric TCR in each lysis condition was quantified by densitometry from immunoblots as in B ( n ≥ 4 ) .

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C. The ratio of the nanocluster and monomeric TCR in each lysis condition was quantified by densitometry from immunoblots as in B (n ≥ 4).


D . Cer in OT ‑ II 10 - day lymphoblasts , untreated or treated with SMase ( n = 4 ) .

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D. Cer levels in OT‑II 10-day lymphoblasts, untreated or treated with SMase (n = 4).


E . Representative small field showing gold particle distribution , ( mean ± ) of gold particles in clusters of indicated in surface replicas from untreated ( gray bars ; n = 5 , 8126 particles ) SMase - treated ( 1h ) OT - II lymphoblasts ( black cyan ; n = 6 , 8457 particles ) after CD3ε labeling , as determined by EM . inset shows distribution between clusters of one , two , three , four or more than four particles .

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E. Representative small field images showing gold particle distribution, and quantification (mean ± SEM) of gold particles in clusters of the indicated size in cell surface replicas from untreated (gray bars; n = 5 cells, 8126 particles) and SMase-treated (1h) OT-II lymphoblasts (black cyan; n = 6 cells, 8457 particles) after CD3ε labeling, as determined by EM. The inset shows distribution between clusters of one, two, three, four or more than four particles.


F . GFP expression in shCtrl - ( black ) shCerS2 ( orange ) - transduced 2B4 after puromycin selection , as determined by FACS .

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F. GFP expression in shCtrl- (black) and shCerS2 (orange)-transduced 2B4 cells after puromycin selection, as determined by FACS.


G . Relative CerS2 mRNA in TAK - 779 - treated 2B4 as in F . Values were normalized to those obtained in untransduced TAK - 779 - treated 2B4 ( n = 3 ) .

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G. Relative CerS2 mRNA levels in TAK-779-treated 2B4 cells as in F. Values were normalized to those obtained in untransduced TAK-779-treated 2B4 cells (n = 3).


H . Representative immunoblot with anti - CerS2 antibody to determine CerS2 in shCtrl ShCerS2 - transduced 2B4 as in G . Filters were rehybridized with β - actin as loading control .

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H. Representative immunoblot with anti-CerS2 antibody to determine CerS2 protein levels in shCtrl and ShCerS2-transduced 2B4 cells as in G. Filters were rehybridized with β-actin as loading control.


I . TCR nanoclustering of shCtrl - shCerS2 transduced 2B4 in presence of TAK - 779 as determined by EM . Representative small field ( mean ± ) of gold particles in clusters of indicated sizes in surface replicas shCtrl ( gray black bars ; n = 6 , 12337 particles ) shCerS2 2B4 lymphoblasts ( orange ; n = 7 , 13456 particles ) . Inset , distribution between clusters of indicated statistical .

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I. TCR nanoclustering of shCtrl- and shCerS2 transduced 2B4 cells in the presence of TAK-779 as determined by EM. Representative small field images and quantification (mean ± SEM) of gold particles in clusters of indicated sizes in cell surface replicas shCtrl (gray black bars; n = 6 cells, 12337 particles) and shCerS2 2B4 lymphoblasts (orange; n = 7 cells, 13456 particles). Inset, distribution between clusters of indicated size and statistical analysis.


J . of CD69 + shCtrl ( black ) shCerS2 ( orange ) 2B4 restimulated with plate - bound anti - CD3ε antibody in presence of TAK - 779 ( n = 3 ) .

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J. Percentage of CD69+ shCtrl (black) and shCerS2 (orange) 2B4 cells restimulated with plate-bound anti-CD3ε antibody in the presence of TAK-779 (n = 3).


A . of TCR nanoclustering in lymphoblasts from healthy WT ccr5Δ32 homozygous donors by EM . Top , representative small field image showing gold particle distribution in surface replicas of anti - CD3ε - labeled ; bottom , ( mean ± ) of gold particles in clusters of indicated in WT ( gray bars ; n = 5 , 17689 particles ) Δ32 / Δ32 ( red ; n = 4 , 16938 particles ) . Insets show distribution between clusters of one , two , three , four or more than four particles , statistical .

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A. Analysis of TCR nanoclustering in lymphoblasts from healthy WT and ccr5Δ32 homozygous donors by EM. Top, representative small field image showing gold particle distribution in cell surface replicas of anti-CD3ε-labeled cells; bottom, quantification (mean ± SEM) of gold particles in clusters of the indicated size in the WT (gray bars; n = 5 cells, 17689 particles) and Δ32/Δ32 cells (light red; n = 4 cells, 16938 particles). Insets show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.


B . Normalized SM , Cer dhCer in lymphoblasts obtained as in A . A representative experiment is shown ( n = 3 donors / genotype ; n = 2 independent experiments )

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B. Normalized SM, Cer and dhCer levels in lymphoblasts obtained as in A. A representative experiment is shown (n = 3 donors/genotype; n = 2 independent experiments)


C . Relative CerS2 mRNA in day 8 WT ccr5Δ32 lymphoblasts . Each data point is average of a technical triplicate from 3 donors in two independent experiments ( n = 6 )

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C. Relative CerS2 mRNA levels in day 8 WT and ccr5Δ32 lymphoblasts. Each data point is the average of a technical triplicate from 3 donors in two independent experiments (n = 6)


OTULIN chromatograms showing homozygous single base substitution ( c . 841G > A , p . Gly281Arg , arrowhead ) . Data are representative to two independent experiments .

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OTULIN DNA sequence chromatograms showing the homozygous single base substitution (c.841G>A, p.Gly281Arg, arrowhead). Data are representative to two independent experiments.


Affinity measurements by polarisation ( FP ) with recombinant catalytically inactive OTULINC129A or OTULINC129A / G281R FlAsH - labelled M1 - linked diUb . Data represent mean ± SD of one experiment performed in technical triplicate . Data are representative of three independent experiments . a . u . , arbitrary units . K D , dissociation constant .

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Affinity measurements by fluorescence polarisation (FP) with recombinant catalytically inactive OTULINC129A or OTULINC129A/G281R and FlAsH-labelled M1-linked diUb. Data represent mean ± SD of one experiment performed in technical triplicate. Data are representative of three independent experiments. a.u., arbitrary units. KD, dissociation constant.


M1 - linked tetraUb hydrolysis by recombinant OTULINWT OTULING281R using indicated OTULIN concentrations visualised on silver - stained SDS - PAGE gels . Data are representative of three independent experiments .

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M1-linked tetraUb hydrolysis by recombinant OTULINWT and OTULING281R using the indicated OTULIN concentrations and visualised on silver-stained SDS-PAGE gels. Data are representative of three independent experiments.


First derivative of tryptophan upon thermal unfolding of recombinant OTULINcatWT OTULINcatG281R ( 1 . 0 mg / mL ) measured by nano differential scanning fluorimetry ( nanoDSF ) . Apparent melting temperatures ( Tm ) are indicated ( dashed lines ) . Data are representative to two independent experiments .

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First derivative of tryptophan fluorescence upon thermal unfolding of recombinant OTULINcatWT and OTULINcatG281R (1.0 mg/mL) measured by nano differential scanning fluorimetry (nanoDSF). Apparent melting temperatures (Tm) are indicated (dashed lines). Data are representative to two independent experiments.


Immunoblot of whole lysates from untreated healthy control fibroblasts . Data are representative of three independent experiments .

Immunoblot analysis of whole cell lysates from untreated primary healthy control and patient fibroblasts. Data are representative of three independent experiments.


Immunoblot of whole lysates from healthy control fibroblasts either left untreated or treated with proteasome inhibitor MG132 ( 10 μM ) for 24 h . Data are representative of two independent experiments .

Immunoblot analysis of whole cell lysates from primary healthy control and patient fibroblasts either left untreated or treated with the proteasome inhibitor MG132 (10 μM) for 24 h. Data are representative of two independent experiments.


Immunoblot of whole lysates from untreated healthy control or fibroblasts . Data are representative of three independent experiments .

Immunoblot analysis of whole cell lysates from untreated primary healthy control or patient fibroblasts. Data are representative of three independent experiments.


Immunoblot ( left ) densitometry ( right ) of IκBα in healthy control fibroblasts treated with cycloheximide ( CHX ) ( 50 μg / mL ) as indicated . Data are representative of three independent experiments .

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Immunoblot (left) and densitometry (right) analysis of IκBα levels in primary healthy control and patient fibroblasts treated with cycloheximide (CHX) (50 μg/mL) as indicated. Data are representative of three independent experiments.


Immunoblot of IκBα phosphorylation degradation as well as p65 / RelA phosphorylation in healthy control or OTULING281R fibroblasts fibroblasts in response to stimulation with TNF ( 10 ng / mL ) . Data are representative of three independent experiments . Immunoblot of phosphorylation of MAP kinases p38 JNK in healthy control or OTULING281R fibroblasts in response to stimulation with TNF ( 10 ng / mL ) . Data are representative of three independent experiments .

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

Immunoblot analysis of IκBα phosphorylation and degradation as well as p65/RelA phosphorylation in healthy control or OTULING281R fibroblasts primary fibroblasts in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments. Immunoblot analysis of phosphorylation of the MAP kinases p38 and JNK in healthy control or OTULING281R primary fibroblasts in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments.


Densitometry of p65 / RelA phosphorylation as presented in ( A ) from three independent experiments .

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Densitometry analysis of p65/RelA phosphorylation as presented in (A) from three independent experiments.


ELISA of IL - 8 secretion in response to TNF stimulation ( 10 ng / mL ) in healthy control or OTULING281R fibroblasts . Bars represent mean ± ( n = 4 ) .

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ELISA analysis of IL-8 secretion in response to TNF stimulation (10 ng/mL) in healthy control or OTULING281R primary fibroblasts. Bars represent mean ± SEM (n=4).


Immunoblot of native TNF - RSC purified by immunoprecipitation from healthy control or OTULING281R fibroblasts . Data are representative of three independent experiments .

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Immunoblot analysis of the native TNF-RSC purified by immunoprecipitation from healthy control or OTULING281R primary fibroblasts. Data are representative of three independent experiments.


Immunoblot of whole lysate from untreated human THP - 1 monocytes with stable expression of a non - targeting control shRNA or shRNA targeting OTULIN . Data are representative of two independent experiments .

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Immunoblot analysis of whole cell lysate from untreated human THP-1 monocytes with stable expression of a non-targeting control shRNA or and shRNA targeting OTULIN. Data are representative of two independent experiments.


Immunoblot ( left ) densitometry ( right ) of IκBα in shControl shOTULIN THP - 1 treated with cycloheximide ( CHX ) ( 50 μg / mL ) as indicated . Data are representative of three independent experiments .

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Immunoblot (left) and densitometry (right) analysis of IκBα levels in shControl and shOTULIN THP-1 cells treated with cycloheximide (CHX) (50 μg/mL) as indicated. Data are representative of three independent experiments.


ELISA of spontaneous TNF secretion over 96 h in shControl shOTULIN THP - 1 . Data represents mean ± ( n = 4 ) were analysed using two - way ANOVA of statistical significance with Sidak ' s correction for multiple comparisons .

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ELISA analysis of spontaneous TNF secretion over 96 h in shControl and shOTULIN THP-1 cells. Data represents mean ± SEM (n=4) and were analysed using the two-way ANOVA test of statistical significance with Sidak's correction for multiple comparisons.


Immunoblot of IκBα phosphorylation degradation as well as p65 / RelA phosphorylation in shControl shOTULIN THP - 1 in response to stimulation with TNF ( 10 ng / mL ) . Data are representative of three independent experiments .

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Immunoblot analysis of IκBα phosphorylation and degradation as well as p65/RelA phosphorylation in shControl and shOTULIN THP-1 cells in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments.


Densitometry of p65 / RelA phosphorylation as presented in ( D ) from three independent experiments .

Densitometry analysis of p65/RelA phosphorylation as presented in (D) from three independent experiments.


Viability of healthy control or OTULING281R fibroblasts after 24 h 6 h , respectively , of treatment with TNF ( 100 ng / mL ) , CHX ( 50 μg / mL ) , as indicated was analysed using MTT reduction assays .

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Viability of healthy control or OTULING281R primary fibroblasts after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL), as indicated was analysed using MTT reduction assays.


Viability of healthy control or OTULING281R fibroblasts after 24 h 6 h , respectively , of treatment with TNF ( 100 ng / mL ) , CHX ( 50 μg / mL ) , Q - VD - OPh ( 10 μM ) , Nec - 1 ( 10 μM ) as indicated was analysed using MTT reduction assays .

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Viability of healthy control or OTULING281R primary fibroblasts after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL), Q-VD-OPh (10 μM), and Nec-1 (10 μM) as indicated was analysed using MTT reduction assays.


Viability of shControl shOTULIN THP - 1 ( C - D ) after 24 h 6 h , respectively , of treatment with TNF ( 100 ng / mL ) , CHX ( 50 μg / mL ) ,

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Viability of shControl and shOTULIN THP-1 cells (C-D) after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL),


shControl shOTULIN THP - 1 ( C - D ) after 24 h 6 h , respectively , of treatment with TNF ( 100 ng / mL ) , CHX ( 50 μg / mL ) , Q - VD - OPh ( 10 μM ) , Nec - 1 ( 10 μM ) as indicated was analysed using MTT reduction assays .

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

shControl and shOTULIN THP-1 cells (C-D) after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL), Q-VD-OPh (10 μM), and Nec-1 (10 μM) as indicated was analysed using MTT reduction assays.


Increased apoptotic in skin of ORAS III . 2 . Serial sections of normal skin ( top panels ) a skin biopsy from III . 2 taken at an inflammatory flare ( bottom panels ) were immunostained for caspase - 3 ( centre panels ) or analysed by terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) ( right panels ) . Arrowheads indicate caspase - 3 - positive mesenchymal . None of these markers were present in healthy control skin . H & E , haematoxylin eosin . Boxes in H & E panels indicate areas magnified in caspase - 3 TUNEL panels . Scale bars , 100 μm . Data are representative of stained sections from three healthy controls one biopsy from ORAS III . 2 .

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Increased apoptotic cell death in the skin of ORAS patient III.2. Serial sections of normal skin (top panels) and a skin biopsy from patient III.2 taken at an inflammatory flare (bottom panels) were immunostained for cleaved caspase-3 (centre panels) or analysed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (right panels). Arrowheads indicate cleaved caspase-3-positive mesenchymal cells. None
of these markers were present in the healthy control skin. H&E, haematoxylin and eosin. Boxes in H&E panels indicate the areas magnified in the cleaved caspase-3 and TUNEL panels. Scale bars, 100 μm.
Data are representative of stained sections from three healthy controls and one biopsy from ORAS patient III.2.


of blood chimerism in III . 2 after HSCT . Short terminal repeat ( STR ) was used to determine of donor in peripheral blood . Arrow indicates point of relapse .

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Analysis of the blood cell chimerism in patient III.2 after HSCT. Short terminal repeat (STR) analysis was used to determine the percentage of donor cells in peripheral blood. Arrow indicates point of relapse.


Photograph of III . 2 at ~ 10 years at an episode of inflammation caused by delayed etanercept administration . Arrows indicate erythematous subcutaneous nodules ( panniculitis ) .

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Photograph of patient III.2 at age ~10 years at an episode of inflammation caused by delayed etanercept administration. Arrows indicate the erythematous subcutaneous nodules (panniculitis).


a ) Photomicrographs represent 2D optical sections from a Z stack acquisition of whole mounted skin displaying IFE harbouring multiple clones of different colours at 3 , 5 , 12 24 ( bar represents 50 μm ) . In each image a clone is circled in , illustrating progression of clone at different times . b ) Histogram charts showing relative frequency of clones according to their between 3 24 in mice induced at P21 P42 . frequency of larger clones increases over time . Chi square p < 0 . 0001 for P21 group p = 0 . 0056 for P42 group . number of clones evaluated at each time point is indicated above each histogram series . c ) d ) Correction for contiguous clones harbouring a same colour .

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a) Photomicrographs represent 2D optical sections from a Z stack acquisition of whole mounted skin displaying IFE harbouring multiple clones of different colours at 3, 5, 12 and 24 weeks (bar represents 50 μm). In each image a clone is circled in white, illustrating the progression of clone size at different times. b) Histogram charts showing the relative frequency of clones according to their size between 3 and 24 weeks in mice induced at P21 and P42. The frequency of larger clones increases over time. Chi square test p<0.0001 for P21 group and p=0.0056 for P42 group. The number of clones evaluated at each time point is indicated above each histogram series. c) and d) Correction for contiguous clones harbouring a same colour.


d ) Plots show probability for an keratinocyte to belong to a clone of a given . Experimental data are represented in green bars . Red curve represents 5 - colour simulation blue curve real simulation . Although frequency of large clones is overestimated in experimental data due to limited 5 - color palette , it is apparent that real clone progresses over time .

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d) Plots show the probability for an individual keratinocyte to belong to a clone of a given size. Experimental data are represented in green bars. Red curve represents the 5-colour simulation and the blue curve the real size simulation. Although the frequency of large clones is overestimated in experimental data due to the limited 5-color palette, it is apparent that real clone size progresses over time.


e ) Histogram charts showing distribution of clones at D1 - 3 following tamoxifen induction according to total number of number of basal .

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e) Histogram charts showing the distribution of clones at D1-3 following tamoxifen induction according to total number of cells and number of basal cells.


f ) Histogram charts showing relative frequency of clones according to their between 3 5 in groups injected at P21 P42 with a single low dose of Tamoxifen to perform a low density . frequency of larger clones increases over time . Chi square p = 0 . 0013 for P21 group p = 0 . 0296 for P42 group . data are represented as mean ± s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histogram charts showing the relative frequency of clones according to their size between 3 and 5 weeks in groups injected at P21 and P42 with a single low dose of Tamoxifen to perform a low density staining. The frequency of larger clones increases over time. Chi square test p=0.0013 for P21 group and p=0.0296 for P42 group. All data are represented as mean ± s.e.m.


a ) Clones were categorized as attached or not attached to hair follicles . Average clone of attached ( circles ) non - attached ( squares ) clones were plotted over time . T - , * * * p < 0 . 0001 , * * p < 0 . 001 . ( Number of clones : 1 week n = 271 , 3 n = 1847 , 5 n = 517 , 12 n = 706 , 24 n = 258 ) . Data are represented as mean ± s . d .

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a) Clones were categorized as attached or not attached to hair follicles. Average clone size of attached (circles) and non-attached (squares) clones were plotted over time. T-test, *** p<0.0001, ** p<0.001. (Number of clones: 1 week n = 271, 3 weeks n = 1847, 5 weeks n = 517, 12 weeks n = 706, 24 weeks n = 258). Data are represented as mean ± s.d.


b ) Photomicrographs represent 2D optical sections from a Z stack acquisition of a whole mounted back skin harbouring multiple clones of different colours at 12 . yellow blue clone in IFE immediately adjacent to a HF are considered as attached to HF . orange pink clones are not attached to HF . Limits of clones ( lines ) ( dots ) within each clone were identified on this optical section . dashed circles represent projection of hair follicle openings ( scale bar = 50 µm ) .

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b) Photomicrographs represent 2D optical sections from a Z stack acquisition of a whole mounted back skin harbouring multiple clones of different colours at 12 weeks. The yellow and blue clone in the IFE immediately adjacent to a HF are considered as attached to the HF. The orange and pink clones are not attached to the HF. Limits of clones (lines) and cells (dots) within each clone were identified on this optical section. White dashed circles represent the projection of hair follicle openings (scale bar= 50 µm).


c ) Bar charts showing relative frequency of clone between 3 24 after recombination . In P42 group , clones attached to HF were more likely to be bigger than those not attached at 3 , 5 , 12 24 . ǂǂǂǂ Chi square p < 0 . 0001 . number of clones analyzed at each time - point is indicated on graph . Of note , whether attached or not clones continued to grow over time : * * * * Chi square p < 0 . 0001 . data are represented as mean ± s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bar charts showing the relative frequency of clone size between 3 and 24 weeks after recombination. In the P42 group, clones attached to HF were more likely to be bigger than those not attached at 3, 5, 12 and 24 weeks. ǂǂǂǂ Chi square test p<0.0001.The number of clones analyzed at each time-point is indicated on the graph. Of note, whether attached or not clones continued to grow over time: ****Chi square test p<0.0001. All data are represented as mean ± s.e.m.


d ) Plot showing probability of a clone to be attached to a hair follicle as a function of its ( dashed : predicted curve on theoretical probabilities according to spacing of hair follicles at different time - points ; squares : experimental data ) . Between different time points between hair follicles varies slightly ; therefore each time point ( 3 , 5 , 12 24 ) has a distinct theoretical curve . observed frequency of clone attachment to hair follicles is at every time point above random prediction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Plot showing the probability of a clone to be attached to a hair follicle as a function of its size (dashed line: predicted curve based on theoretical probabilities according to spacing of hair follicles at different time-points; squares: experimental data). Between different time points the distance between hair follicles varies slightly; therefore each time point (3, 5, 12 and 24 weeks) has a distinct theoretical curve. The observed frequency of clone attachment to hair follicles is at every time point above the random prediction.


a ) Anagen telogen skin identified by skin pigmentation at time of collection were obtained from each Skinbowmouse at different time points . Histogram charts represent relative frequency of clone from clones attached ( top row ) or not ( bottom row ) to HF in anagen telogen phase at time of tissue collection . Clones connected to hair follicles were more likely to be larger in anagen compared to telogen ( Chi square , * * * p = 0 . 0009 at 3 , * * * p < 0 . 0001 at 5 , * p = 0 . 0193 at 12 , * * * p = 0 . 0001 at 24 ) . However no difference between two phases was observed in clones that were not attached to HFs . Clone numbers are indicated on graph . data are represented as mean ± s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Anagen and telogen skin identified by skin pigmentation at the time of collection were obtained from each Skinbowmouse at different time points. Histogram charts represent the relative frequency of clone size from clones attached (top row) or not (bottom row) to HF in anagen and telogen phase at the time of tissue collection. Clones connected to hair follicles were more likely to be larger in anagen compared to telogen (Chi square test, *** p=0.0009 at 3 weeks, *** p<0.0001 at 5 weeks, * p=0.0193 at 12 weeks, *** p=0.0001 at 24 weeks). However no difference between the two phases was observed in clones that were not attached to HFs. Clone numbers are indicated on the graph. All data are represented as mean ± s.e.m.


b ) Telogen skin was obtained from Skinbowmice induced at P42 at different time points from 1 to 24 post - induction . Each skin sample was evaluated for average number of club hair found in hair follicles reflecting its history of past hair cycling . Average clone of attached ( circles ) non - attached ( squares ) clones were plotted over number of club hair . T - , * * * * p < 0 . 0001 .

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

b) Telogen skin was obtained from Skinbowmice induced at P42 at different time points from 1 to 24 weeks post-induction. Each skin sample was evaluated for the average number of club hair found in hair follicles reflecting its history of past hair cycling. Average clone size of attached (circles) and non-attached (squares) clones were plotted over the number of club hair. T-test, **** p<0.0001.


c ) Skinbowmice were induced at a time were back skin contained both areas of anagen telogen samples collected for evaluation at 7 days . Histogram charts represent relative frequency of clone from clones attached ( top row ) or not ( bottom row ) to HF in anagen telogen phase at induction . Even at a rate of one division per 24h , 7 cycles of divisions results in 128 . Therefore findings reported are not incompatible with our claims as suggested . Observation of spontaneous recombinations if they exist are therefore a very rare event that we have never been able to observe . Such rare event is unlikely to influence our on sizes of hundreds of clones . Chi square , * * * p = 0 . 001 ; * * 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

c) Skinbowmice were induced at a time were the back skin contained both areas of anagen and telogen and samples collected for evaluation at 7 days. Histogram charts represent the relative frequency of clone size from clones attached (top row) or not (bottom row) to HF in anagen and telogen phase at induction. Even at a rate of one cell division per 24h, 7 cycles of divisions results in 128 cells. Therefore the findings reported are not incompatible with our claims as suggested. Observation of spontaneous recombinations if they exist are therefore a very rare event that we have never been able to observe. Such rare event is unlikely to influence our analysis based on sizes of hundreds of clones. Chi square test, *** p=0.001; ** p=0.01.


( a - b ) Ki67 immunohistochemistry of back skin sections at different phases of hair follicle cycle determined using a bioluminescent reporter mice of canonical Wnt signaling pathway ( a ) corresponding scatter plots . ( Scale bar 100µm ) ( b ) . focused on basal layer of IFE . Each dot represents one image . Proliferation in IFE is significantly increased during anagen phase except in mid - anagen . Unpaired t , * * * * p < 0 . 0001 , * * p = 0 . 0020 . EA n = 32 , EMA n = 13 , MA n = 15 , C n = 21 , T n = 19 from 6 mice . Black arrows : positive Ki67 .

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

(a-b) Ki67 immunohistochemistry of back skin sections at different phases of the hair follicle cycle determined using a bioluminescent reporter mice of the canonical Wnt signaling pathway (a) and corresponding scatter plots quantification. (Scale bar 100µm) (b). Analysis focused on the basal layer of the IFE. Each dot represents one image. Proliferation in the IFE is significantly increased during the anagen phase except in mid-anagen. Unpaired t test, ****p<0.0001, **p=0.0020. EA n = 32, EMA n = 13, MA n = 15, C n = 21, T n = 19 from 6 mice. Black arrows: positive Ki67 cells.


( c ) Photomicrographs represent 2D optical sections from Z stack acquisition of whole mounted back skin stained in anagen telogen with anti - Ki67 antibody or upon BrdU incorporation . Ki67 + are labelled with Alexa 647 ( red ) . density of Ki67 + increased in anagen phase ( iii iv ) compared to telogen ( i ii ) . arrows : ki67 + . ( Scale bar = 20µm ) . Similarly , short term BrdU ( 2h ) resulted in significant of anagen epidermal areas ( vi , red stain ) reinforcing hair follicle openings as compared to telogen areas .

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(c) Photomicrographs represent 2D optical sections from Z stack acquisition of whole mounted back skin stained in anagen and telogen with anti-Ki67 antibody or upon BrdU incorporation. Ki67+ cells are labelled with Alexa 647 (red). The density of Ki67+ cells increased in the anagen phase (iii and iv) compared to telogen (i and ii). White arrows: ki67+ cells. (Scale bar = 20µm). Similarly, short term BrdU pulse (2h) resulted in significant staining of anagen epidermal areas (vi, red nuclear stain) reinforcing hair follicle openings as compared to telogen areas.


a - b ) Skinbow mice were induced for 5 consecutive days pulsed with BrdU for 3 consecutive days with 2 injections per day . Skin was collected either immediately after last BrdU injection at 72hours or after periods of 1 to 5 of chase . a ) Photomicrographs show optical sections from a confocal image stack acquired on a whole mounted skin preparation immunostained with anti - BrdU antibody ( red ) . dots represent hair follicles at different chase time points .

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a-b) Skinbow mice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day. Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase. a) Photomicrographs show optical sections from a confocal image stack acquired on a whole mounted skin preparation immunostained with anti-BrdU antibody (red). White dots represent hair follicles at different chase time points.


a - b ) Skinbowmice were induced for 5 consecutive days pulsed with BrdU for 3 consecutive days with 2 injections per day . Skin was collected either immediately after last BrdU injection at 72hours or after periods of 1 to 5 of chase . b ) At 5 of chase , BrdU ( green ) in skinbowskin revealed single distant from hair follicles . dashed circles : hair follicles . : small clones . arrows : BrdU + . ( scale bar = 20 µm ) .

a-b) Skinbowmice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day. Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase. b) At 5 weeks of chase, BrdU staining (green) in skinbowskin revealed single cells distant from hair follicles. White dashed circles: hair follicles. White line: small clones. White arrows: BrdU+ cells. (scale bar = 20 µm).


c - d ) In Skinbowmice induced at P42 - 46 , TPA was applied for one day , 3 post - induction were sacrificed at 48hours following a short of BrdU ( 2h ) . c ) Photomicrographs show optical sections from a confocal image stack acquired on a 0 . 5 mm back skin section immunostained with anti - ki67 antibody . Ki67 + are labelled with Alexa 647 ( red ) . Back skin from treated untreated areas was collected 48hrs after TPA topical application .

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

c-d) In Skinbowmice induced at P42-46, TPA was applied for one day, 3 weeks post-induction and animals were sacrificed at 48hours following a short pulse of BrdU (2h). c) Photomicrographs show optical sections from a confocal image stack acquired on a 0.5 mm back skin section immunostained with anti-ki67 antibody. Ki67+ cells are labelled with Alexa 647 (red). Back skin from treated and untreated areas was collected 48hrs after TPA topical application.


c - d ) In Skinbowmice induced at P42 - 46 , TPA was applied for one day , 3 post - induction were sacrificed at 48hours following a short of BrdU ( 2h ) . d ) Multiple optical section from a Z stack acquisition of whole mount preparation of skin treated with TPA stained with anti - BrdU ( green ) . Small clones not attached to HF ( 2 examples figured by dashed lines ) can be tracked from corneocyte layer down to basement where BrdU demonstrates their proliferation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In Skinbowmice induced at P42-46, TPA was applied for one day, 3 weeks post-induction and animals were sacrificed at 48hours following a short pulse of BrdU (2h). d) Multiple optical section from a Z stack acquisition of whole mount preparation of skin treated with TPA and stained with anti-BrdU (green). Small clones not attached to HF (2 examples figured by dashed lines) can be tracked from the corneocyte layer down to the basement membrane where BrdU staining demonstrates their proliferation.


e ) ( i to iv ) Representative sections from Z stacks , show small clones ( lines ) identified in back skin treated ( i ii ) or not with TPA ( iii iv ) . ( scale bar = 100 µm ) . ( v to viii ) Maximal projection from stack acquired on whole mounted skin . A contribution of infundibulum bulge to IFE is observed in back skin treated with TPA ( v to vi ) compared to skin treated with vehicle ( vii viii ) . ( scale bars = 100 µm ) . f ) Reduction in small clone frequency in areas treated with TPA . Histogram chart showing clone distribution relative frequency of small clones not attached to HF in areas treated with TPA not treated with TPA in a same mouse . Mann Whitney , * * p = 0 . 0079 . Data pooled from 3 mice represented as median with interquartile range . number of clones analyzed is indicated on graph . * p = 0 . 0272 . Data are represented as mean ± s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (i to iv) Representative individual sections from Z stacks, show small clones (lines) identified in back skin treated (i and ii) or not with TPA (iii and iv). (scale bar = 100 µm). (v to viii) Maximal intensity projection from stack acquired on whole mounted skin. A contribution of the infundibulum and the bulge to the IFE is observed in back skin treated with TPA (v to vi) compared to skin treated with vehicle (vii and viii). (scale bars = 100 µm). f) Reduction in small clone frequency in the areas treated with TPA. Histogram chart showing the clone size distribution and relative frequency of small clones not attached to HF in areas treated with TPA and not treated with TPA in a same mouse. Mann Whitney test, ** p=0.0079. Data pooled from 3 mice represented as median with interquartile range. The number of clones analyzed is indicated on the graph. * p=0.0272. Data are represented as mean ± s.e.m.


a ) Bar charts showing relative surface of clones attached not attached between 3 24 after induction at P21 or P42 . Data are represented as mean ± s . e . m .

O O O O O O O O O O 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) Bar charts showing the relative surface of clones attached and not attached between 3 and 24 weeks after induction at P21 or P42. Data are represented as mean ± s.e.m.


A Transmigration of mouse neutrophils towards chemokine CXCL - 1 through TNF - α - stimulated VE - cadherin WT ( WT ) or VE - cadherin Y731F ( Y731F ) endothelial , each transfected with control or SHP2 - specific siRNA . transmigration rate is presented relative to that of WT transfected with control siRNA , set as 100 % . On right , Western blot of lysates from used in transmigration assays for expression of SHP2 α - tubulin .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_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 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

A Transmigration of mouse neutrophils towards the chemokine CXCL-1 through TNF-α-stimulated VE-cadherin WT (WT) or VE-cadherin Y731F (Y731F) primary endothelial cells, each transfected with control or SHP2-specific siRNA. The transmigration rate is presented relative to that of WT cells transfected with control siRNA, set as 100%. On the right, Western blot analysis of lysates from cells used in transmigration assays for the expression of SHP2 and α-tubulin.


B Transmigration of human neutrophils towards chemokine IL - 8 through TNFα - stimulated human endothelial ( HUVEC ) , transfected with control or SHP2 - specific siRNA were either not transduced ( - ) or transduced ( + ) with adenoviruses expressing human SHP2 ( Adv - SHP2 ) . transmigration rate is presented relative to that of transfected with control siRNA , set as 100 % . On right , Western blot of lysates from used in transmigration assays for expression of SHP2 α - tubulin .

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B Transmigration of human neutrophils towards the chemokine IL-8 through TNFα-stimulated human endothelial cells (HUVEC), transfected with control or SHP2-specific siRNA and were either not transduced (-) or transduced (+) with adenoviruses expressing human SHP2 (Adv-SHP2). The transmigration rate is presented relative to that of cells transfected with control siRNA, set as 100%. On the right, Western blot analysis of lysates from cells used in transmigration assays for the expression of SHP2 and α-tubulin.


C Lysates of WT or Y731F endothelial were submitted to pulldowns with GST , GST ‐ SHP2 ‐ WT , or GST ‐ SHP2 ‐ DA . Pulldowns total lysates were analyzed by Western blotting for VE - cadherin GST . Graph below represents of two independent experiments , presented as fold change of bound VE - cadherin .

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C Lysates of WT or Y731F primary endothelial cells were submitted to pulldowns with GST, GST‐SHP2‐WT, or GST‐SHP2‐DA. Pulldowns and total cell lysates were analyzed by Western blotting for VE-cadherin and GST. Graph below represents quantification of two independent experiments, presented as fold change of bound VE-cadherin.


TNFα - activated bEnd . 5 were incubated with mouse T for 0 - 30 min . SHP2 ( above ) or VE - cadherin ( below ) were immunoprecipitated ( IP ) from lysates precipitates as well as total lysates were analyzed by immunoblotting for indicated antigens . Molecular markers are indicated in kDa . Graph below represents of five independent experiments , presented as fold change of bound VE - cadherin .

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TNFα-activated bEnd.5 cells were incubated with mouse T cells for 0-30 min. SHP2 (above) or VE-cadherin (below) were immunoprecipitated (IP) from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblotting for indicated antigens. Molecular weight markers are indicated in kDa. Graph below represents quantification of five independent experiments, presented as fold change of bound VE-cadherin.


endothelial were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations immunoblots of precipitates endothelial lysates ( Iso = isotype control ) . ( A ) bEnd . 5 were incubated with antigen - stimulated mouse T for 0 - 15 min .

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

endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (A) bEnd.5 cells were incubated with antigen-stimulated mouse T cells for 0-15 min.


endothelial were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations immunoblots of precipitates endothelial lysates ( Iso = isotype control ) . ( B ) luEnd devoid of PECAM - 1 ( KO ) or re - transfected with PECAM - 1 ( RT ) were incubated with T for 30 min .

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endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (B) luEnd cells devoid of PECAM-1 (KO) or re-transfected with PECAM-1 (RT) were incubated with T cells for 30 min.


endothelial were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations immunoblots of precipitates endothelial lysates ( Iso = isotype control ) . ( C ) HUVEC transfected with control or PECAM - 1 - specific siRNA were incubated with differentiated HL60 for 20 min .

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endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (C) HUVEC transfected with control or PECAM-1-specific siRNA were incubated with differentiated HL60 cells for 20 min.


endothelial were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations immunoblots of precipitates endothelial lysates ( Iso = isotype control ) . ( D ) bEnd . 5 were pre - treated with anti - PECAM - 1 ( Mec13 . 3 ) or anti - endomucin antibodies for 30 min prior to incubation with T for 30 min .

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

endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (D) bEnd.5 cells were pre-treated with anti-PECAM-1 (Mec13.3) or anti-endomucin antibodies for 30 min prior to incubation with T cells for 30 min.


endothelial were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations above ) immunoblots of precipitates endothelial lysates ( Iso = isotype control ) . ( E ) HUVEC , siRNA depleted for endogenous PECAM - 1 , were transduced with wild - type PECAM - 1 ( PECAM - 1 WT ) or Y663 , 686F PECAM - 1 double mutant ( PECAM - 1 YF ) incubated with HL60 - derived neutrophils for 20 min . On right , immunoblot of lysates from siRNA treated before PECAM - 1 re - expression .

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

endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations above) and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (E) HUVEC, siRNA depleted for endogenous PECAM-1, were transduced with wild-type PECAM-1 (PECAM-1 WT) or Y663,686F PECAM-1 double mutant (PECAM-1 YF) and incubated with HL60-derived neutrophils for 20 min. On the right, immunoblot analysis of lysates from siRNA treated cells before PECAM-1 re-expression.


F Transmigration of human neutrophils towards IL - 8 through TNFα - stimulated HUVEC expressing PECAM - 1 WT or PECAM - 1 - Y663 , 686F . transmigration rate is presented relative to that of PECAM - 1 WT expressing , set as 100 % .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

F Transmigration of human neutrophils towards IL-8 through TNFα-stimulated HUVEC expressing PECAM-1 WT or PECAM-1-Y663,686F. The transmigration rate is presented relative to that of PECAM-1 WT expressing cells, set as 100%.


Endocytosis of VE - cadherin in 17 h TNF - α treated HUVEC was induced by adding HL60 - derived neutrophils for 20 - 30 min at 37 ° C monitored by internalization of preincubated mAb BV6 , which was visualized in fixed with a secondary antibody ( ) . Total VE - cadherin was stained with another mAb ( red ) , nuclei stained with Hoechst ( ) . ( A ) HUVEC had been transfected prior to experiment with control or PECAM - 1 - specific siRNA . lysates were analyzed for expression of PECAM - 1 tubulin ( below ) . Relative VE - cadherin internalization efficiency is given on right .

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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

Endocytosis of VE-cadherin in 17 h TNF-α treated HUVEC was induced by adding HL60-derived neutrophils for 20-30 min at 37°C and monitored by the internalization of preincubated mAb BV6, which was visualized in fixed cells with a secondary fluorescent antibody (white). Total VE-cadherin was stained with another mAb (red), nuclei stained with Hoechst (white). (A) HUVEC had been transfected prior to the experiment with control or PECAM-1-specific siRNA. Cell lysates were analyzed for expression levels of PECAM-1 and tubulin (below). Relative VE-cadherin internalization efficiency is given on the right.


Endocytosis of VE - cadherin in 17 h TNF - α treated HUVEC was induced by adding HL60 - derived neutrophils for 20 - 30 min at 37 ° C monitored by internalization of preincubated mAb BV6 , which was visualized in fixed with a secondary antibody ( ) . Total VE - cadherin was stained with another mAb ( red ) , nuclei stained with Hoechst ( ) . ( B ) PECAM - 1 siRNA treated HUVEC were transduced with WT PECAM - 1 - EGFP or PECAM - 1 - Y663 , 686F - EGFP . Expression of transduced PECAM - 1 forms of EGFP VE - cadherin are shown in immunoblots ( on right ) . Relative VE - cadherin internalization efficiency is given on right .

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

Endocytosis of VE-cadherin in 17 h TNF-α treated HUVEC was induced by adding HL60-derived neutrophils for 20-30 min at 37°C and monitored by the internalization of preincubated mAb BV6, which was visualized in fixed cells with a secondary fluorescent antibody (white). Total VE-cadherin was stained with another mAb (red), nuclei stained with Hoechst (white). (B) PECAM-1 siRNA treated HUVEC were transduced with WT PECAM-1-EGFP or PECAM-1-Y663,686F-EGFP. Expression levels of transduced PECAM-1 forms and of EGFP and VE-cadherin are shown in immunoblots (on the right). Relative VE-cadherin internalization efficiency is given on the right.


A Transmigration of mouse neutrophils towards CXCL - 1 through TNF - α - stimulated bEnd . 5 transfected with control or SHP2 - specific siRNA pre - treated with anti - endomucin ( 7C7 . 1 ) or anti - PECAM - 1 ( Mec13 . 3 ) antibodies for 30 min at 37 ° C before addition of neutrophils . transmigration rate is presented relative to that of control , set as 100 % .

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A Transmigration of mouse neutrophils towards CXCL-1 through TNF-α-stimulated bEnd.5 cells transfected with control or SHP2-specific siRNA and pre-treated with anti-endomucin (7C7.1) or anti-PECAM-1 (Mec13.3) antibodies for 30 min at 37°C before addition of neutrophils. The transmigration rate is presented relative to that of control cells, set as 100%.


B Transmigration of mouse neutrophils towards CXCL - 1 through TNF - α - stimulated WT or Y731F mouse endothelial pre - treated with isotype - control or anti - PECAM - 1 ( 2H8 ) antibodies for 30 min at 37 ° C prior to addition of neutrophils . transmigration rate is presented relative to that of control , set as 100 % .

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B Transmigration of mouse neutrophils towards CXCL-1 through TNF-α-stimulated WT or Y731F primary mouse endothelial cells pre-treated with isotype-control or anti-PECAM-1 (2H8) antibodies for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to that of control cells, set as 100%.


Extravasated leukocytes ( C ) in cremaster tissue from WT or Y731F VE - cadherin mice stimulated intrascrotally with IL - 1β treated i . v . with isotype - control or anti - PECAM - 1 ( 2H8 ) antibodies for 4 h before intravital microscopy .

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Extravasated leukocytes (C) in cremaster tissue from WT or Y731F VE-cadherin mice stimulated intrascrotally with IL-1β and treated i.v. with isotype-control or anti-PECAM-1 (2H8) antibodies for 4 h before intravital microscopy.


adherent leukocytes ( D ) in cremaster tissue from WT or Y731F VE - cadherin mice stimulated intrascrotally with IL - 1β treated i . v . with isotype - control or anti - PECAM - 1 ( 2H8 ) antibodies for 4 h before intravital microscopy .

adherent leukocytes (D) in cremaster tissue from WT or Y731F VE-cadherin mice stimulated intrascrotally with IL-1β and treated i.v. with isotype-control or anti-PECAM-1 (2H8) antibodies for 4 h before intravital microscopy.


( D ) rolling flux fraction of leukocytes ( E ) in cremaster tissue from WT or Y731F VE - cadherin mice stimulated intrascrotally with IL - 1β treated i . v . with isotype - control or anti - PECAM - 1 ( 2H8 ) antibodies for 4 h before intravital microscopy .

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(D) and rolling flux fraction of leukocytes (E) in cremaster tissue from WT or Y731F VE-cadherin mice stimulated intrascrotally with IL-1β and treated i.v. with isotype-control or anti-PECAM-1 (2H8) antibodies for 4 h before intravital microscopy.


A HUVEC were transduced with increasing titers of adenovirus expressing human SHP2 . VE - cadherin was immunoprecipitated from lysates precipitates as well as total lysates were analyzed by immunoblots for indicated antigens .

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A HUVEC were transduced with increasing titers of adenovirus expressing human SHP2. VE-cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens.


B bEnd . 3 were lysed VE - cadherin or immature VE - cadherin was immunoprecipitated respectively with C5 or VD47 antibodies . Precipitates were blotted against pY731 - VE - cadherin , β - catenin , plakoglobin , p120 , α - catenin VE - cadherin .

B bEnd.3 cells were lysed and VE-cadherin or immature VE-cadherin was immunoprecipitated respectively with C5 or VD47 antibodies. Precipitates were blotted against pY731-VE-cadherin, β -catenin, plakoglobin, p120, α -catenin and VE-cadherin.


C VE - cadherin - catenin was immunoprecipitated from HUVEC lysates was either left untreated ( no SDS ) or treated with 0 . 2 % SDS for 30 min at room , followed by removal of SDS addition of 2 μg recombinant human SHP2 for 30 min at 30 ° C . Precipitates were analyzed in immunoblots for pY731 - VE - cadherin , plakoglobin , β - catenin VE - cadherin . D of pY731 blot signals of three independent experiments ( from C ) , with signal for untreated VE - cadherin set as 1 . Molecular markers are indicated in kDa .

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C VE-cadherin-catenin complex was immunoprecipitated from HUVEC lysates and was either left untreated (no SDS) or treated with 0.2% SDS for 30 min at room temperature, followed by removal of SDS and addition of 2 μg recombinant human SHP2 for 30 min at 30°C. Precipitates were analyzed in immunoblots for pY731-VE-cadherin, plakoglobin, β-catenin and VE-cadherin. D Quantification of pY731 blot signals of three independent experiments (from C), with the signal intensity for untreated VE-cadherin set as 1. Molecular weight markers are indicated in kDa.


A Transmigration of mouse neutrophils towards chemokine CXCL - 1 through TNFα - stimulated WT or Y731F endothelial , either pre - treated with vehicle control ( DMSO ) or Ca2 + chelator ( MAPTAM ) for 30 min at 37 ° C prior to addition of neutrophils . transmigration rate is presented relative to DMSO - treated endothelial expressing WT VE - cadherin .

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A Transmigration of mouse neutrophils towards the chemokine CXCL-1 through TNFα-stimulated WT or Y731F primary endothelial cells, either pre-treated with vehicle control (DMSO) or Ca2+ chelator (MAPTAM) for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to DMSO-treated endothelial cells expressing WT VE-cadherin.


B TNFα - activated bEnd . 5 were treated with DMSO or MAPTAM , prior to incubation with antigen - stimulated mouse T for 30 min . VE - cadherin was immunoprecipitated from lysates precipitates as well as total lysates were analyzed by immunoblots for indicated antigens . of pY731 blot signals of five independent experiments are shown below , with signal for control sample ( no T , vehicle - control ) set as 100 % .

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B TNFα-activated bEnd.5 cells were treated with DMSO or MAPTAM, prior to incubation with antigen-stimulated mouse T cells for 30 min. VE-cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens. Quantification of pY731 blot signals of five independent experiments are shown below, with the signal intensity for the control sample (no T cells, vehicle-control) set as 100%.


A Transmigration of mouse neutrophils towards chemokine CXCL - 1 through TNFα - stimulated WT or Y731F endothelial , either pre - treated with vehicle control ( DMSO ) or Ca2 + chelator for 30 min at 37 ° C prior to addition of neutrophils . transmigration rate is presented relative to DMSO - treated endothelial expressing WT VE - cadherin . C Transmigration assays treatment with Blebbistatin .

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A Transmigration of mouse neutrophils towards the chemokine CXCL-1 through TNFα-stimulated WT or Y731F primary endothelial cells, either pre-treated with vehicle control (DMSO) or Ca2+ chelator for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to DMSO-treated endothelial cells expressing WT VE-cadherin. C Transmigration assays treatment with Blebbistatin.


B TNFα - activated bEnd . 5 were treated with DMSO prior to incubation with antigen - stimulated mouse T for 30 min . VE - cadherin was immunoprecipitated from lysates precipitates as well as total lysates were analyzed by immunoblots for indicated antigens . of pY731 blot signals of five independent experiments are shown below , with signal for control sample ( no T , vehicle - control ) set as 100 % . treatment with Blebbistatin .

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B TNFα-activated bEnd.5 cells were treated with DMSO prior to incubation with antigen-stimulated mouse T cells for 30 min. VE-cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens. Quantification of pY731 blot signals of five independent experiments are shown below, with the signal intensity for the control sample (no T cells, vehicle-control) set as 100%. treatment with Blebbistatin.


E HUVEC transduced with lacZ or with SHP2 were either untreated ( - ) or treated ( + ) with thrombin for 30 min followed by immunoblotting either VE - cadherin immunoprecipitates or lysates for indicated antigens on right .

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E HUVEC transduced with lacZ or with SHP2 were either untreated (-) or treated (+) with thrombin for 30 min followed by immunoblotting either VE-cadherin immunoprecipitates or cell lysates for the indicated antigens on the right.


HUVEC were transfected with control or VE - cadherin - specific siRNA transduced with VE - cad FL or VE - cad FL - TS . lysates were immunoblotted for VE - cadherin actin .

HUVEC were transfected with control or VE-cadherin-specific siRNA and transduced with VE-cad FL or VE-cad FL-TS. Cell lysates were immunoblotted for VE-cadherin and actin.


C Immunofluorescence for VE - cadherin of HUVEC transfected with control or VE ‑ cadherin specific siRNA ( VE - cad kd ) transduced with VE - cad FL or VE - cad FL - TS . D HUVEC were either transfected with control or VE - cadherin targeting siRNA transduced with VE ‑ cad FL or VE - cad FL ‑ TS . tension sensor was immunoprecipitated from lysates using an anti - GFP antibody . Precipitates as well as total lysates were analyzed by immunoblotting for indicated antigens . Molecular markers are indicated in kDa . E , of FRET efficiency ( ) in junctions of HUVEC expressing VE - cad FL or VE - cad FL - TS , after thrombin stimulation ( 1 U / ml ) or under control conditions .

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

C Immunofluorescence staining for VE-cadherin of HUVEC transfected with control or VE‑cadherin specific siRNA (VE-cad kd) and transduced with VE-cad FL or VE-cad FL-TS. D HUVEC were either transfected with control or VE-cadherin targeting siRNA and transduced with VE‑cad FL or VE-cad FL‑TS. The tension sensor was immunoprecipitated from cell lysates using an anti-GFP antibody. Precipitates as well as total cell lysates were analyzed by immunoblotting for indicated antigens. Molecular weight markers are indicated in kDa. E, Quantification of FRET efficiency (percentage) in junctions of HUVEC expressing VE-cad FL or VE-cad FL-TS, after thrombin stimulation (1 U/ml) or under control conditions.


A - C of FRET efficiency ( ) in junctions of HUVEC expressing VE - cad FL ( A , B ) or VE - cad FL - TS ( C ) . HUVEC were stimulated 4h with TNFα prior to adding IL - 8 for 4 min , followed by flow with buffer alone ( A ) or together with PMNs ( B , C ) . connected data points represent FRET efficiency of same junctions compared before after addition of flow / PMNs .

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A-C Quantification of FRET efficiency (percentage) in junctions of HUVEC expressing VE-cad FL (A, B) or VE-cad FL-TS (C). HUVEC were stimulated 4h with TNFα prior to adding IL-8 for 4 min, followed by flow with buffer alone (A) or together with PMNs (B, C). The connected data points represent FRET efficiency of same junctions compared before and after addition of flow/PMNs.


D of FRET efficiency ( ) in junctions of HUVEC expressing VE - cad FL or VE - cad FL - TS . were either exposed to flow PMNs or to flow alone , fixed using 4 % PFA washed with PBS . FLIM measurements were performed at sites of transmigration ( PMNs ) or at junctions without PMNs ( Flow ) .

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D Quantification of FRET efficiency (percentage) in junctions of HUVEC expressing VE-cad FL or VE-cad FL-TS. Cells were either exposed to flow and PMNs or to flow alone, fixed using 4 % PFA and washed with PBS. FLIM measurements were performed at sites of transmigration (PMNs) or at junctions without PMNs (Flow).


E Representative of HUVEC expressing VE - cad FL at a site of PMN transmigration ( left ) or without PMN ( right ) . Maximum projection of a Z - stack of YPet ( VE - cadherin in green ) CellTracker DeepRed ( PMN in red ) is shown for upper . part of PMN above HUVEC monolayer is encircled in yellow part underneath in blue . amplitude averaged lifetime of YPet per pixel ( FLIM measurement ) of same is shown in bottom .

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E Representative images of HUVEC expressing VE-cad FL at a site of PMN transmigration (left micrographs) or without PMN (right micrographs). Maximum intensity projection of a Z-stack of YPet fluorescence (VE-cadherin in green) and CellTracker DeepRed (PMN in red) is shown for the upper micrographs. The part of the PMN above the HUVEC monolayer is encircled in yellow and the part underneath in blue. The amplitude averaged lifetime of YPet per pixel (FLIM measurement) of the same cells is shown in the bottom micrographs.


F HUVEC were grown on collagen - coated polyacrylamide gels of varying physiologic stiffness of 0 . 2 kPa 20 kPa , stimulated with TNFα for 17 h prior to adding HL60 - derived neutrophils for 20 min . VE - cadherin was immunoprecipitated precipitates as well as total lysates were analyzed by Western blotting for indicated antigens . Relative quantifications of pY731 blot signals adjusted to amount of precipitated VE - cadherin are shown on

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F HUVEC were grown on collagen-coated polyacrylamide gels of varying physiologic stiffness of 0.2 kPa and 20 kPa, and stimulated with TNFα for 17 h prior to adding HL60-derived neutrophils for 20 min. VE-cadherin was immunoprecipitated and precipitates as well as total cell lysates were analyzed by Western blotting for indicated antigens. Relative quantifications of pY731 blot signals adjusted to the amount of precipitated VE-cadherin are shown on the right


B ) Western blot on fractions obtained during SN preparation revealed enrichment of both pre - postsynaptic markers , but not glia marker ( GFAP ) in SN fraction . markers were depleted , while markers were barely detected in SN ( n = 4 biological replicates , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ; one sample t - ; error bars indicate ) .

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B) Western blot on fractions obtained during SN preparation revealed the enrichment of both pre- and postsynaptic markers, but not glia marker (GFAP) in the SN fraction. Cytosolic markers were depleted, while nuclear markers were barely detected in the SN (n=4 biological replicates, *** p<0.001, ** p<0.01, * p<0.05; one sample t-test; error bars indicate SEM).


C ) Synaptoneurosomes were NMDAR - stimulated incubated with radioactive 35S - methionine / cysteine mix for 15 - 120 min . After labeling , were separated on SDS - PAGE newly synthesized in SN labeled with [ 35S ] were visualized by autoradiography ( left panel ) . To determine contribution of mitochondrial translation labeling was preceded by treatment with chloramphenicol ( CAP , 50 μg / ml ) or with puromycin ( PURO , 3mM ) respectively ( right panel ) . After 1 hour after stimulation , a significant increase in 35S - labelled was observed ( n = 6 biological replicates , * * * p < 0 . 001 ; repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons ) CAP treatment did not affect overall of de novo synthetized ( n = 6 biological replicates , ns ; p = 0 . 220 ) . In contrast , PURO treatment significantly inhibited de novo synthesis in synaptoneurosomes ( n = 6 biological replicates , * * p = 0 . 0091 , repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons ) . Error bars indicate .

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C) Synaptoneurosomes were NMDAR-stimulated and incubated with radioactive 35S-methionine/cysteine mix for 15-120 min. After labeling, proteins were separated on SDS-PAGE and newly synthesized proteins in SN labeled with [35S] were visualized by autoradiography (left panel). To determine the contribution of mitochondrial and cytosolic translation the labeling was preceded by treatment with chloramphenicol (CAP, 50 μg/ml) or with puromycin (PURO, 3mM) respectively (right panel). After 1 hour after the stimulation, a significant increase in 35S-labelled proteins was observed (n=6 biological replicates, *** p<0.001; repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test) and the CAP treatment did not affect the overall levels of de novo synthetized proteins (n=6 biological replicates, ns; p=0.220). In contrast, PURO treatment significantly inhibited de novo protein synthesis in synaptoneurosomes (n=6 biological replicates, ** p=0.0091, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Error bars indicate SEM.


( D - F ) Results of mass spectrometry ( label free - LFQ ) identifying with increased abundance in NMDAR - stimulated ( 20 min ) synaptoneurosomes . ( D ) Volcano plot ( Whitney - Mann , n = 4 per group ) showing abundance of identified in stimulated synaptoneurosomes as compared to unstimulated . Mitochondrial are depicted in orange . vertical defines p - value statistical significance cut - off . ( E ) Biological functions of identified annotated using DAVID . Third top functional category identified as mitochondrial ones . ( F ) Pie - chart showing share of mitochondrial among identified upregulated .

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(D-F) Results of mass spectrometry (label free quantification - LFQ) analysis identifying proteins with increased abundance in NMDAR-stimulated (20 min) synaptoneurosomes. (D) Volcano plot (Whitney-Mann test, n=4 per group) showing abundance of identified proteins in stimulated synaptoneurosomes as compared to unstimulated. Mitochondrial proteins are depicted in orange. The vertical line defines the p-value statistical significance cut-off. (E) Biological functions of identified proteins annotated using DAVID. Third top functional category identified as mitochondrial ones. (F) Pie-chart showing percentage share of mitochondrial proteins among all identified upregulated proteins.


( A - D ) Results of high - resolution quantitative mass spectrometry identifying with increased abundance in stimulated synaptoneurosomes . Volcano plots ( Whitney - Mann , Benjamini Hochberg correction , n = 5 per group ) showing abundance of identified in NMDAR - stimulated ( 20 min ) synaptoneurosomes as compared to unstimulated using iTRAQ8 - plex ( A ) TMT10 - plex ( B ) labelling methods . Mitochondrial are depicted in orange . vertical defines p - value statistical significance cut - off . For nine mitochondrial their mRNA abundance on polyribosomes was quantified , data is presented on Figure EV2 . ( C ) Biological functions of identified as significantly upregulated after NMDAR stimulation ( assembled from three proteomic analyzes : LFQ , iTRAQ8 ,

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(A-D) Results of high-resolution quantitative mass spectrometry analysis identifying proteins with increased abundance in stimulated synaptoneurosomes. Volcano plots (Whitney-Mann test, Benjamini Hochberg correction, n=5 per group) showing abundance of identified proteins in NMDAR-stimulated (20 min) synaptoneurosomes as compared to unstimulated using iTRAQ8-plex (A) and TMT10-plex (B) labelling methods. Mitochondrial proteins are depicted in orange. The vertical line defines the p-value statistical significance cut-off. For nine mitochondrial proteins their mRNA abundance on polyribosomes was quantified, the data is presented on Figure EV2. (C) Biological functions of proteins identified as significantly upregulated after NMDAR stimulation (assembled from all three proteomic analyzes: LFQ, iTRAQ8, TMT10


( E ) absorbance profiles from polyribosomal fractionation of unstimulated NMDAR - stimulated ( 20 min ) synaptoneurosomes . Upon NMDAR stimulation , a fraction of mRNAs is shifted towards heavy - polyribosomal fraction , which represents actively translating ribosomes . fraction of polyribosomes used for - seq experiment is depicted .

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(E) RNA absorbance profiles from polyribosomal fractionation of unstimulated and NMDAR-stimulated (20 min) synaptoneurosomes. Upon NMDAR stimulation, a fraction of mRNAs is shifted towards heavy-polyribosomal fraction, which represents actively translating ribosomes. The fraction of polyribosomes used for RNA-seq experiment is depicted.


( F ) Comparison of proteomic data with - seq experiments on mRNAs associated with polyribosomes from synaptoneurosomal samples . Ribosome association of synaptoneurosomal mRNAs determined by normalizing - seq values of polyribosome associated mRNA with total mRNA ( y axis ) . Green dots represent mRNAs encoding upregulated upon stimulation ( identified in MS experiments ) while orange dots represent mRNAs encoding mitochondrial . Density curves for transcripts encoding mitochondrial ( orange ) upregulated ( green ) detected by mass spectrometry are present on right side of plot . Please note that both classes represent high abundant mRNAs with good ribosome association values . Transcripts with statistically significant increased or decreased abundance on polyribosomes in response to stimulation are depicted in red .

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(F) Comparison of proteomic data with RNA-seq experiments on mRNAs associated with polyribosomes from synaptoneurosomal samples. Ribosome association of synaptoneurosomal mRNAs determined by normalizing RNA-seq values of polyribosome associated mRNA with total mRNA (y axis). Green dots represent mRNAs encoding proteins upregulated upon stimulation (identified in MS experiments) while orange dots represent mRNAs encoding mitochondrial proteins. Density curves for transcripts encoding mitochondrial proteins (orange) and upregulated proteins (green) detected by the mass spectrometry are present on the right side of the plot. Please note that both classes represent high abundant mRNAs with good ribosome association values. Transcripts with statistically significant increased or decreased abundance on the polyribosomes in response to the stimulation are depicted in red.


( A ) Respiration activity of permeabilized synaptoneurosomes ( upper panel ) as compared to permeabilized HEK293T ( lower panel ) . Oxygen ( blue ) oxygen consumption ( red ) measured using O2k oxygraph are shown .

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(A) Respiration activity of permeabilized synaptoneurosomes (upper panel) as compared to permeabilized HEK293T (lower panel). Oxygen concentration (blue line) and oxygen consumption (red line) measured using O2k oxygraph are shown.


( B ) Blue native gel electrophoresis ( BN - PAGE ) of mitochondrial respiratory chain complexes from synaptoneurosomes as compared to HEK293T mitochondria .

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(B) Blue native gel electrophoresis (BN-PAGE) analysis of mitochondrial respiratory chain complexes from synaptoneurosomes as compared to HEK293T mitochondria.


( C ) Detection of respiratory chain complexes supercomplexes in SN HEK293T mitochondria using BN - PAGE followed by Western blot using antibodies specific for : I ( Ndufb8 ) , II ( Sdha ) , III ( Uqcrc2 ) , IV ( Cox6a ) V ( Atp5a ) . Note similar composition of OXPHOS supercomplexes complexes in synaptoneurosomes as in mitochondria .

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(C) Detection of respiratory chain complexes and supercomplexes in SN and HEK293T mitochondria using BN-PAGE followed by Western blot using antibodies specific for: complex I (Ndufb8), complex II (Sdha), complex III (Uqcrc2), complex IV (Cox6a) and complex V (Atp5a). Note similar composition of OXPHOS supercomplexes and complexes in synaptoneurosomes as in cellular mitochondria.


( D ) Selected bands ( 1 - 8 ) visible in blue - native gel were excised analyzed using mass spectrometry ( LC - MS ) . Proteomic confirms presence of subunits of different respiratory chain complexes in indicated bands .

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(D) Selected bands (1-8) visible in blue-native gel were excised and analyzed using mass spectrometry (LC-MS). Proteomic analysis confirms the presence of protein subunits of different respiratory chain complexes in indicated bands.


( A ) Synaptoneurosomes were NMDAR - stimulated incubated with radioactive 35S - methionine / cysteine mix for 1h . Samples were separated using blue - native electrophoresis ( BN - PAGE ) . complexes were visualized by Coomassie ( left ) autoradiography ( right ) . Autoradiography of BN - PAGE gel shows de novo synthetized mitochondrial that are incorporated into respiratory chain complexes .

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(A) Synaptoneurosomes were NMDAR-stimulated and incubated with radioactive 35S-methionine/cysteine mix for 1h. Samples were separated using blue-native electrophoresis (BN-PAGE). Protein complexes were visualized by Coomassie staining (left) and autoradiography (right). Autoradiography of the BN-PAGE gel shows de novo synthetized mitochondrial proteins that are incorporated into respiratory chain complexes.


To block import incorporation of newly synthesized into respiratory complexes mitochondrial electrochemical potential was abolished by treatment with carbonyl cyanide m - chlorophenyl hydrazone ( 10 μM CCCP ) ( B ) complexes were separated on BN - PAGE visualized by Coomassie autoradiography . 1 hour after stimulation , significant increase in 35S - labelled was observed ( B , n = 4 biological replicates , * , * * p = 0 . 0023 ; repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons ) . Blocking import into mitochondria significantly inhibited incorporation of 35S - methionine into respiratory chain complexes when samples were incubated in presence of CCCP ( B ; n = 4 biological replicates , * p = 0 . 02 , repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons )

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To block protein import and incorporation of newly synthesized proteins into respiratory complexes the mitochondrial electrochemical potential was abolished by treatment with carbonyl cyanide m-chlorophenyl hydrazone (10 μM CCCP) (B) Protein complexes were separated on BN-PAGE and visualized by Coomassie staining and autoradiography. 1 hour after the stimulation, significant increase in 35S-labelled proteins was observed (B, n=4 biological replicates, * , ** p=0.0023; repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S-methionine into respiratory chain complexes when samples were incubated in the presence of CCCP (B; n=4 biological replicates, * p=0.02, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test)


To block import incorporation of newly synthesized into respiratory complexes mitochondrial electrochemical potential was abolished by treatment with VOA mixture ( containing 1 μM valinomycin , 20 μM oligomycin , 8 μM antimycin ) ( C ) . Additionaly , in order to block synthesis , SN were pretreated with puromycin ( PURO , 3mM ) ( C ) . complexes were separated on BN - PAGE visualized by Coomassie autoradiography . 1 hour after stimulation , significant increase in 35S - labelled was observed C , n = 5 biological replicates , * * p = 0 . 0023 ; repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons ) . Blocking import into mitochondria significantly inhibited incorporation of 35S - methionine into respiratory chain complexes when samples were incubated in presence of VOA ( C ; n = 5 biological replicates , * * p = 0 . 0044 , repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons ) . Also , significant inhibition of 35S - methionine / cysteine incorporation into mitochondrial complexes in synaptoneurosomes was observed when samples were stimulated in presence of puromycin ( C , n = 5 biological replicates , * p = 0 . 0236 , repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons ) . Error bars indicate .

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To block protein import and incorporation of newly synthesized proteins into respiratory complexes the mitochondrial electrochemical potential was abolished by treatment with VOA mixture (containing 1 μM valinomycin, 20 μM oligomycin, 8 μM antimycin) (C). Additionaly, in order to block protein synthesis, SN were pretreated with puromycin (PURO, 3mM) (C). Protein complexes were separated on BN-PAGE and visualized by Coomassie staining and autoradiography. 1 hour after the stimulation, significant increase in 35S-labelled proteins was observed C, n=5 biological replicates, ** p=0.0023; repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S-methionine into respiratory chain complexes when samples were incubated in the presence of VOA (C; n=5 biological replicates, ** p=0.0044, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Also, significant inhibition of 35S-methionine/cysteine incorporation into mitochondrial protein complexes in synaptoneurosomes was observed when samples were stimulated in the presence of puromycin (C, n=5 biological replicates, * p=0.0236, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Error bars indicate SEM.


( B ) Equal amounts of synaptoneurosomal samples isolated from mouse brains ( Fmr1 KO wild - type ) were permeabilized by saponin assayed on MitoPlates for 2 hours . Results are presented as average rate / min / µg of , + / - ( n = 3 per genotype ; two - way ANOVA , post - hoc Sidak ' s multiple comparisons ; * * * p < 0 . 0001 ) .

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(B) Equal amounts of synaptoneurosomal samples isolated from mouse brains (Fmr1 KO and wild-type littermates) were permeabilized by saponin and assayed on MitoPlates for 2 hours. Results are presented as the average rate/min/µg of protein, +/- SEM (n=3 per genotype; two-way ANOVA, post-hoc Sidak's multiple comparisons test; *** p< 0.0001).


( C ) Mitochondrial O2 consumption rates in SN were measured by high - resolution respirometry in presence of following respiratory substrates : malate , pyruvate , glutamate for Complexes I - IV , succinate for Complexes II - IV , glycerol 3 - phosphate for mGpDH - IV ascorbate TMPD for IV , normalized to content . Fmr1 KO mitochondria show elevated of respiration in presence of succinate as well as ascorbate TMPD ( * p = 0 . 0483 * p = 0 . 0360 respectively , n = 9 per genotype , paired two - t - ) . Data are presented as a box - - whiskers graph ( box extends from 25th to 75th percentiles , central horizontal is plotted at median , whiskers show 5th - 95th percentile ) .

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(C) Mitochondrial O2 consumption rates in SN were measured by high-resolution respirometry in the presence of following respiratory substrates: malate, pyruvate, and glutamate for Complexes I-IV, succinate for Complexes II-IV, glycerol 3-phosphate for mGpDH-IV and ascorbate and TMPD for complex IV, and normalized to protein content. Fmr1 KO mitochondria show elevated levels of respiration in the presence of succinate as well as ascorbate and TMPD (*p=0.0483 and *p=0.0360 respectively, n=9 per genotype, paired two-tailed t-test). Data are presented as a box-and-whiskers graph (the box extends from 25th to 75th percentiles, central horizontal line is plotted at the median, whiskers show 5th - 95th percentile).


( D ) Ultrastructure of Fmr1 KO brain sections revealed important differences in mitochondria when compared to wild - types . Mitochondria are indicated by arrows ; presynaptic part in blue , postsynaptic part in green . 3 mice per genotype were analyzed . Scale bars , 0 . 5 µm .

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(D) Ultrastructure of Fmr1 KO brain sections revealed important differences in mitochondria morphology when compared to wild-types. Mitochondria are indicated by arrows; presynaptic part in blue, postsynaptic part in green. 3 mice per genotype were analyzed. Scale bars, 0.5 µm.


( A ) bar graph represents top 10 biological pathways upregulated in gene ontology ( GO ) Reactome pathway using a set of genes induced ( 1 . 5 fold , p < 0 . 05 ( Wald ) , 3 biological replicates ) in - seq in IRGM shRNA knockdown HT29 compared to control shRNA . Heatmaps were generated for sentinel interferon - regulated genes ( three biological replicates ) using ' ComplexHeatmap ' library using ' R ' Bioconductor package where gene expression matrix was transformed into z - score . heat map was generated from common genes present in three GO terms indicated by three black lines . numbers on bars indicate p - value of that particular GO term .

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(A) The bar graph represents top 10 biological pathways upregulated in gene ontology (GO) based Reactome pathway analysis using a set of genes induced (1.5 fold, p<0.05 (Wald Test), 3 biological replicates) in RNA-seq analysis in IRGM shRNA knockdown HT29 cells compared to control shRNA cells. Heatmaps were generated for sentinel interferon-regulated genes (three biological replicates) using 'ComplexHeatmap' library using 'R' Bioconductor package where the gene expression matrix was transformed into z-score. The heat map was generated from the common genes present in the three GO terms indicated by the three black lines. The numbers on the bars indicate the p-value of that particular GO term.


( D ) qRT - PCR validation of - seq data in control IRGM KD HT29 .

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(D) The qRT-PCR validation of RNA- seq data in control and IRGM KD HT29 cells.


( E ) bar graph represents top pathways upregulated in GO - Reactome pathway using set of genes induced ( 1 . 5 fold , p < 0 . 05 ( Wald ) , 3 mice each group ) in brain of Irgm1 - / - mice compared to Irgm1 + / + wild type mice . Heatmaps were generated for sentinel interferon - regulated genes ( three biological replicates ) using ' ComplexHeatmap ' library using ' R ' Bioconductor package where gene expression matrix was transformed into z - score . heat map was generated from common genes present in three GO terms indicated by three black lines . numbers on bars indicate p - value of that particular GO term .

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(E) The bar graph represents the top pathways upregulated in GO-based Reactome pathway analysis using set of genes induced (1.5 fold, p<0.05 (Wald Test), 3 mice each group) in the brain of Irgm1-/- mice compared to Irgm1+/+ wild type mice. Heatmaps were generated for sentinel interferon-regulated genes (three biological replicates) using 'ComplexHeatmap' library using 'R' Bioconductor package where the gene expression matrix was transformed into z-score. The heat map was generated from the common genes present in the three GO terms indicated by the three black lines. The numbers on the bars indicate the p-value of that particular GO term.