( D ) Dot plot showing average V5 pixel intensities at parent centrioles in A549 of indicated genotypes , either transduced with indicated lentiviral vectors or left untransduced ( mock ) . N > 50 centrosomes were assessed for each condition in as many ; a . u . = arbitrary units . ( E ) Dot plot showing average V5 pixel intensities at parent centrioles in A549 of indicated genotypes , either transduced with indicated lentiviral vectors or left untransduced ( mock ) . N > 50 centrosomes were assessed for each condition in as many ; a . u . = arbitrary units .

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(D) Dot plot showing the average V5 pixel intensities at individual parent centrioles in A549 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. (E) Dot plot showing the average V5 pixel intensities at individual parent centrioles in A549 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units.


( F ) Representative of A549 of indicated genotypes . were either left untransduced ( mock ) or transduced with PIDD1 - V5 lentiviral vectors expressing PIDD1 non - cleavable derivative ( PIDD1S446A - S588A ) or truncations thereof . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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(F) Representative fluorescence micrographs of A549 cells of the indicated genotypes. Cells were either left untransduced (mock) or transduced with PIDD1-V5 lentiviral vectors expressing the PIDD1 non-cleavable derivative (PIDD1S446A-S588A) or truncations thereof. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( A ) A549 of indicated genotypes were either left untransduced or transduced with a lentiviral vector expressing PIDD1 - V5 in its wild type form . were treated either with DMSO or with ZM447439 for 24h subjected to immunoblotting . N = 2 independent experiments .

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(A) A549 cells of the indicated genotypes were either left untransduced or transduced with a lentiviral vector expressing PIDD1-V5 in its wild type form. Cells were treated either with DMSO or with ZM447439 for 24h and subjected to immunoblotting. N = 2 independent experiments.


( B , C ) A549 of indicated genotypes were either left untransduced ( mock ) , or transduced with lentiviral vectors expressing PIDD1 - V5 in its wild type form or carrying indicated point mutations . were treated either with DMSO or with ZM447439 for 24h subjected to immunoblotting . N = 2 independent experiments .

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(B,C) A549 cells of the indicated genotypes were either left untransduced (mock), or transduced with lentiviral vectors expressing PIDD1-V5 in its wild type form or carrying the indicated point mutations. Cells were treated either with DMSO or with ZM447439 for 24h and subjected to immunoblotting. N = 2 independent experiments.


( D ) of A549 of indicated genotypes . were either left untransduced ( mock ) or transduced with PIDD1 - V5 lentiviral vectors carrying indicated point mutations , stained with indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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(D) Fluorescence micrographs of A549 cells of the indicated genotypes. Cells were either left untransduced (mock) or transduced with PIDD1-V5 lentiviral vectors carrying the indicated point mutations, and stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( A ) Representative across indicated cycle phases from RPE1 stably expressing CETN1 - GFP . Centrosomal antigens were stained with indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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(A) Representative fluorescence micrographs across the indicated cell cycle phases from RPE1 cells stably expressing CETN1-GFP. Centrosomal antigens were stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


centrioles subjected to measurement in ( B ) are labelled with numbers . ( B ) of PIDD1 average pixel at centrioles across indicated cycle phases of RPE1 stained N > 50 centrioles were assessed for each phase , a . u . = arbitrary units .

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The centrioles subjected to fluorescence intensity measurement in (B) are labelled with numbers. (B) Quantification of the PIDD1 average pixel intensity at individual centrioles across the indicated cell cycle phases of RPE1 cells stained N > 50 centrioles were assessed for each phase, a.u. = arbitrary units.


( C ) of RPE1 stably expressing CETN1 - GFP treated either with DMSO or with DHCB for 24h . Blow - ups 33342 are magnified 2 . 5X . Scale bar : 5 μm . ( D ) Dot plot showing PIDD1 average pixel intensities at parent centrioles calculated from as in ( C ) . Mean values ( red lines ) ± s . e . m . are reported . N > 50 centrosomes were assessed for each condition , a . u . = arbitrary units .

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(C) Fluorescence micrographs of RPE1 cells stably expressing CETN1-GFP treated either with DMSO or with DHCB for 24h. Blow-ups 33342 are magnified 2.5X. Scale bar: 5 μm. (D) Dot plot showing PIDD1 average pixel intensities at individual parent centrioles calculated from images as in (C). Mean values (red lines) ± s.e.m. are reported. N > 50 centrosomes were assessed for each condition, a.u. = arbitrary units.


( E ) Movie stills of a representative RPE1 stably expressing CETN1 - GFP treated with DHCB subjected to time - lapse video microscopy in presence of SiR - . Time is expressed in minutes , relative to anaphase onset . dashed indicates plasma of of interest , arrowheads indicate centrosomes ' position .

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(E) Movie stills of a representative RPE1 cell stably expressing CETN1-GFP treated with DHCB and subjected to time-lapse video microscopy in the presence of SiR-DNA. Time is expressed in minutes, relative to anaphase onset. The dashed line indicates the plasma membrane of the cell of interest, arrowheads indicate the centrosomes' position.


( F ) Centrosomal over time in RPE1 stably expressing CETN1 - GFP treated with DHCB . Time zero corresponds to frame preceding anaphase onset . Coloured dots ( lower panel ) summarize clustering time for each , mean ± standard deviation in black . Data calculated from four - dimensional imaging as in ( E ) . N = 10 .

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(F) Centrosomal distance over time in RPE1 cells stably expressing CETN1-GFP and treated with DHCB. Time zero corresponds to the frame preceding anaphase onset. Coloured dots (lower panel) summarize the clustering time for each cell, mean ± standard deviation in black. Data calculated from four-dimensional imaging as in (E). N = 10 cells.


( A ) Dot plot showing between parent centrioles pairs in A549 following indicated treatments ( ZM = ZM447439 ; REV = reversine ) . Nocodazole concentrations are 0 . 03 μM , 0 . 1 μM , 0 . 33 μM , 1 μM , 3 . 3 μM . Median ( red ) 95 % confidence interval thereof ( black ) are shown . N > 50 were analysed . ( B ) Immunoblot of A549 subjected to indicated treatments for 24h as in ( A ) . N = 3 independent experiments . ( C ) content of A549 subjected to indicated treatments as in ( A ) either for 24h ( left panels ) or 48h ( right panels ) . N = 2 independent experiments .

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(A) Dot plot showing the distance between parent centrioles pairs in A549 cells following the indicated treatments (ZM = ZM447439; REV = reversine). Nocodazole concentrations are 0.03 μM, 0.1 μM, 0.33 μM, 1 μM, 3.3 μM. Median (red) and 95% confidence interval thereof (black) are shown. N > 50 cells were analysed. (B) Immunoblot of A549 cells subjected to the indicated treatments for 24h as in (A). N = 3 independent experiments. (C) DNA content analysis of A549 cells subjected to the indicated treatments as in (A) either for 24h (left panels) or 48h (right panels). N = 2 independent experiments.


(E) Immunoblots of RPE1 cells synchronized


( F ) Representative of RPE1 synchronized Centrosomal antigens were stained with indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2X . Scale bar : 5 μm .

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(F) Representative fluorescence micrographs of RPE1 cells synchronized Centrosomal antigens were stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2X. Scale bar: 5 μm.


( A ) A549 of indicated genotypes were treated for 24h as indicated ( CPT = camptothecin , ZM = ZM447439 ) . Samples were subjected to immunoblotting ; n = 3 independent experiments . ( B ) A549 treated as in ( A ) were subjected to microscopy centrosome abundance was assessed by visually scoring γ - tubulin - positive centrioles per . N = 3 , ≥ 50 from each independent experiment .

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(A) A549 cells of the indicated genotypes were treated for 24h as indicated (CPT = camptothecin, ZM = ZM447439). Samples were subjected to immunoblotting; n = 3 independent experiments. (B) A549 cells treated as in (A) were subjected to fluorescence microscopy and centrosome abundance was assessed by visually scoring γ-tubulin-positive centrioles per cell. N = 3, ≥ 50 cells from each independent experiment.


( C ) RT - qPCR of PIDD1 mRNA expression in A549 upon treatment with increasing doses of Nutlin 3a ( i . e . 3 . 3 μM or 10 μM ) using two independent probes . average fold of induction ± standard deviation is shown . N = 3 biological replicates with two technical replicates each . Comparisons were performed between treatments of every genotype corresponding treatment of wild type ( WT )

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(C) RT-qPCR analysis of PIDD1 mRNA expression in A549 cells upon treatment with increasing doses of Nutlin 3a (i.e. 3.3 μM or 10 μM) using two independent probes. The average fold of induction ± standard deviation is shown. N = 3 biological replicates with two technical replicates each. Comparisons were performed between treatments of every genotype and the corresponding treatment of wild type (WT) cells


with increasing doses of Nutlin 3a ( i . e . 3 . 3 μM or 10 μM ) using two independent probes . ( D ) Immunoblot of samples treated N = 3 independent experiments .

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with increasing doses of Nutlin 3a (i.e. 3.3 μM or 10 μM) using two independent probes. (D) Immunoblot analysis of samples treated N = 3 independent experiments.


( A ) Recombinant c10orf76 directly binds to PI4KB in vitro . His - pulldown assays of baculovirus / Sf9 produced 6xHis - c10orf76 ( 3 μM ) were carried out with untagged PI4KB ( 2 . 5 μM ) .

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(A) Recombinant c10orf76 directly binds to PI4KB in vitro. His-pulldown assays of baculovirus/Sf9 produced 6xHis-c10orf76 (3 μM) were carried out with untagged PI4KB (2.5 μM).


( B ) PI4KB c10orf76 form a stable . of c10orf76 - PI4KB eluted from a S200 superdex 10 / 300 GL increase gel filtration column ( GE ) at a volume consistent with a heterodimer ( 169 kDa ) , while c10orf76 alone eluted at a volume consistent with a monomer ( 79 kDa ) . Lines with MW values indicate elution of MW standards ( 158 kDa aldolase , 75 kDa conalbumin ) .

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(B) PI4KB and c10orf76 form a stable complex. The complex of c10orf76-PI4KB eluted from a S200 superdex 10/300 GL increase gel filtration column (GE) at a volume consistent with a heterodimer (169 kDa), while c10orf76 alone eluted at a volume consistent with a monomer (79 kDa). Lines with MW values indicate elution of MW standards (158 kDa aldolase, 75 kDa conalbumin).


( C ) PI4KB is potently inhibited by c10orf76 in a dose - dependent manner in vitro . Kinase assays of PI4KB ( 20 nM ) in presence of varying concentrations of c10orf76 ( 1 . 6 nM - 1 μM ) were carried out on pure PI lipid vesicles ( 0 . 5 mg / L ) in presence of 100 μM ATP . data was normalized to kinase activity of PI4KB alone . IC50 values were determined by one binding site , nonlinear regression ( curve fit ) using Graphpad . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(C) PI4KB is potently inhibited by c10orf76 in a dose-dependent manner in vitro. Kinase assays of PI4KB (20 nM) in the presence of varying concentrations of c10orf76 (1.6 nM-1 μM) were carried out on pure PI lipid vesicles (0.5 mg/L) in the presence of 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. IC50 values were determined by one binding site, nonlinear regression (curve fit) using Graphpad. Error bars represent standard deviation of independent technical replicates (n=3).


( D ) PI4KB is potently inhibited by c10orf76 on pure PI vesicles vesicles mimicking Golgi composition . Kinase assays of PI4KB c10orf76 were carried out on lipid substrate composed of pure PI vesicles ( 0 . 5 mg / mL ) with 100 μM ATP , Golgi mimic vesicles ( 0 . 5 mg / ml , 10 % PS , 20 % PI , 25 % PE , 45 % PC ) with 10 μM ATP . PI4KB was present at 20 300 nM in PI Golgi substrate assays respectively , with c10orf76 present at 500 nM in both experiments . data is normalized to kinase activity of PI4KB alone . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(D) PI4KB is potently inhibited by c10orf76 on pure PI vesicles and vesicles mimicking Golgi composition. Kinase assays of PI4KB and c10orf76 were carried out on lipid substrate composed of pure PI vesicles (0.5 mg/mL) with 100 μM ATP, and Golgi mimic vesicles (0.5 mg/ml, 10% PS, 20% PI, 25% PE, 45% PC) with 10 μM ATP. PI4KB was present at 20 and 300 nM in the PI and Golgi substrate assays respectively, with c10orf76 present at 500 nM in both experiments. The data is normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).


( E ) Changes in deuterium incorporation PI4KB in presence of c10orf76 showed a profound ordering of kinase domain N - lobe linker smaller changes in helical domain C - lobe of kinase domain . sum of difference mapped as difference in number of deuterons incorporated for PI4KB ( 400 nM ) in presence absence of c10orf76 ( 400 nM ) over time points ( 3s at 1 oC ; 3s , 30s , 300s at 23 oC ) . Each dot represents a peptide graphed on x - axis according to central residue . red boxes highlight key regions that showed significant changes ( > 7 % decrease in exchange , > 0 . 5 Da difference , unpaired two - student t - p < 0 . 05 ) . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(E) Changes in deuterium incorporation PI4KB in the presence of c10orf76 showed a profound ordering of the kinase domain N-lobe linker and smaller changes in the helical domain and C-lobe of the kinase domain. The sum of the difference mapped as the difference in number of deuterons incorporated for PI4KB (400 nM) in the presence and absence of c10orf76 (400 nM) over all time points (3s at 1 oC; 3s, 30s, and 300s at 23 oC). Each dot represents a peptide graphed on the x-axis according to the central residue. The red boxes highlight key regions that showed significant changes (>7% decrease in exchange, >0.5 Da difference, and unpaired two-tailed student t-test p<0.05). Error bars represent standard deviation of independent technical replicates (n=3).


( G ) Changes in deuterium incorporation of c10orf76 in presence of PI4KB . H / D exchange reactions displayed as sum of difference in HDX in number of deuterons for c10orf76 ( 400 nM ) in presence of PI4KB ( 400 nM ) at time points ( 3s at 1 oC ; 3s , 30s , 300s at 23 oC ) analyzed . Red boxes highlight regions that showed significant changes ( > 7 % decrease in exchange , > 0 . 5 Da difference , unpaired two - student t - p < 0 . 05 ) . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(G) Changes in the deuterium incorporation of c10orf76 in the presence of PI4KB. H/D exchange reactions displayed as the sum of the difference in HDX in the number of deuterons for c10orf76 (400 nM) in the presence of PI4KB (400 nM) at all time points (3s at 1 oC; 3s, 30s, and 300s at 23 oC) analyzed. Red boxes highlight regions that showed significant changes (>7% decrease in exchange, >0.5 Da difference, and unpaired two-tailed student t-test p<0.05). Error bars represent standard deviation of independent technical replicates (n=3).


( H ) PI4KB N - lobe linker undergoes a disorder - to - order transition upon binding c10orf76 . Selected peptides ( including , domain information , numbering ) of both PI4KB c10orf76 displayed as % deuteration incorporation over time . Error bars represent standard deviation of independent technical replicates ( n = 3 ) , are typically smaller than of point on graph .

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(H) The PI4KB N-lobe linker undergoes a disorder-to-order transition upon binding c10orf76. Selected peptides (including the sequence, domain information, and numbering) of both PI4KB and c10orf76 displayed as the % deuteration incorporation over time. Error bars represent standard deviation of independent technical replicates (n=3), and are typically smaller than the size of the point on the graph.


( B ) N - lobe kinase linker of PI4KB can be efficiently phosphorylated by PKA . Recombinant PKA at different concentrations ( 0 , 7 , 34 , 168 , or 840 ng ) was incubated with recombinant ( E . coli ) wild - type PI4KB ( 20 μg ) for 1 hour with 200 μM ATP amount of phosphorylation was followed using mass spectrometry . Relative abundance of Ser496 phosphorylated PI4KB was calculated using relative ( total ) of phosphorylated vs non - phosphorylated peptide ( 486 - 506 ) .

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(B) The N-lobe kinase linker of PI4KB can be efficiently phosphorylated by PKA. Recombinant PKA at different concentrations (0, 7, 34, 168, or 840 ng) was incubated with recombinant (E. coli) wild-type PI4KB (20 μg) for 1 hour with 200 μM ATP and the amount of phosphorylation was followed using mass spectrometry. Relative abundance of Ser496 phosphorylated PI4KB was calculated using the relative intensity (total area) of the phosphorylated vs non-phosphorylated peptide (486-506).


( C ) PI4KB phosphorylation by PKA alters affinity for c10orf76 . kinase activity of different variants of PI4KB ( 15 nM ) was measured in presence of varying amounts of c10orf76 ( 23 nM - 1 . 5μM ) with 100 % PI lipid substrate ( 0 . 5 mg / L ) 100 μM ATP . data was normalized to kinase activity of PI4KB alone Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(C) PI4KB phosphorylation by PKA alters the affinity for c10orf76. The kinase activity of different variants of PI4KB (15 nM) was measured in the presence of varying amounts of c10orf76 (23 nM-1.5μM) with 100% PI lipid substrate (0.5 mg/L) and 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone Error bars represent standard deviation of independent technical replicates (n=3).


( D ) PI4KB has same kinase activity when Ser496 is phosphorylated or mutated to alanine . Kinase of PI4KB non - phosphorylated , phos - Ser496 or S496A ( 15 nM ) on pure PI lipid vesicles ( 0 . 5 mg / L ) with 100 μM ATP . data was normalized to kinase activity of WT PI4KB . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(D) PI4KB has the same kinase activity when Ser496 is phosphorylated or mutated to alanine. Kinase assay of PI4KB non-phosphorylated, phos-Ser496 or S496A (15 nM) on pure PI lipid vesicles (0.5 mg/L) with 100 μM ATP. The data was normalized to the kinase activity of WT PI4KB. Error bars represent standard deviation of independent technical replicates (n=3).


( E ) Engineered RL494EA PI4KB mutant shows decreased binding to c10orf76 . His - pulldown assays of 6xHis - c10orf76 ( 3 μM ) with wild - type or RL494EA PI4KB ( 1 - 2 μM ) .

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(E) Engineered RL494EA PI4KB mutant shows decreased binding to c10orf76. His-pulldown assays of 6xHis-c10orf76 (3 μM) with wild-type or RL494EA PI4KB (1-2 μM).


( F ) RL494EA PI4KB activity is not inhibited by c10orf76 . Kinase assays of either wild type or mutant RL494EA PI4KB ( 40 nM ) were carried out with varying concentrations of c10orf76 ( 3 . 9 nM - 2μM ) with 100 % PI lipid vesicles ( 0 . 5 mg / L ) 100 μM ATP . data was normalized to kinase activity of PI4KB alone . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(F) RL494EA PI4KB activity is not inhibited by c10orf76. Kinase assays of either wild type or mutant RL494EA PI4KB (40 nM) were carried out with varying concentrations of c10orf76 (3.9 nM-2μM) with 100% PI lipid vesicles (0.5 mg/L) and 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).


( G ) Wild - type PI4KB RL494EA PI4KB mutant have same lipid kinase activity . Kinase assays of either wild - type mutant PI4KB ( 10 nM ) were carried out with 100 % PI lipid vesicles ( 0 . 5 mg / L ) , 100 μM ATP , PI4KB ( 300 nM ) on Golgi - mimic vesicles ( 0 . 5 mg / mL ) with 10 μM ATP . data was normalized to kinase activity of WT PI4KB . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(G) Wild-type PI4KB and RL494EA PI4KB mutant have the same lipid kinase activity. Kinase assays of either wild-type and mutant PI4KB (10 nM) were carried out with 100% PI lipid vesicles (0.5 mg/L), 100 μM ATP, and PI4KB (300 nM) on Golgi-mimic vesicles (0.5 mg/mL) with 10 μM ATP. The data was normalized to the kinase activity of WT PI4KB. Error bars represent standard deviation of independent technical replicates (n=3).


( H ) FLH409AAA - c10orf76 mutant shows decreased affinity for PI4KB . His - pulldown assays of 6xHis - c10orf76 ( 1 μM ) with wild - type PI4KB ( 1 μM ) . Samples washed a total of 4 times .

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(H) FLH409AAA-c10orf76 mutant shows decreased affinity for PI4KB. His-pulldown assays of 6xHis-c10orf76 (1 μM) with wild-type PI4KB (1 μM). Samples washed a total of 4 times.


( I ) Kinase shows FLH409AAA c10orf76 inhibition of PI4KB is greatly reduced . Kinase of PI4KB ( 40 nM ) a curve of c10orf76 ( 3 . 9 nM - 2μM ) on pure PI lipid vesicles ( 0 . 5 mg / L ) with 100 μM ATP . data was normalized to kinase activity of PI4KB alone . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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

(I) Kinase assay shows FLH409AAA c10orf76 inhibition of PI4KB is greatly reduced. Kinase assay of PI4KB (40 nM) and a concentration curve of c10orf76 (3.9 nM-2μM) on pure PI lipid vesicles (0.5 mg/L) with 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).


( A ) Transfections of HEK293 revealed that both wild - type GFP - PI4KB RL494EA GFP - PI4KB localize to Golgi .

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(A) Transfections of HEK293 cells revealed that both wild-type GFP-PI4KB and RL494EA GFP-PI4KB localize to the Golgi.


( B ) WT c10orf76 also localized to Golgi , however , PI4KB binding deficient mutant of c10orf76 ( FLH409AAA ) predominantly localized to cytosol .

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(B) WT c10orf76 also localized to the Golgi, however, the PI4KB binding deficient mutant of c10orf76 (FLH409AAA) predominantly localized to the cytosol.


( D ) Mitochondria recruitment experiment with wild - type PI4KB c10orf76 . Left : AKAP1 - FRB - CFP is localized to mitochondria before ( top ) 5 minutes after rapamycin ( 100 nM ) treatment ( bottom ) . Middle : mRFP - FKBP12 - PI4KB at high expression level is located in cytosol before rapamycin ( top ) translocates to mitochondria after rapamycin induction ( bottom ) . Right : eGFP - c10orf76 at a high expression level is located in cytosol before rapamycin ( top ) translocates to mitochondria after rapamycin induction ( bottom ) .

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(D) Mitochondria recruitment experiment with wild-type PI4KB and c10orf76. Left: AKAP1-FRB-CFP is localized to the mitochondria before (top) and 5 minutes after rapamycin (100 nM) treatment (bottom). Middle: mRFP-FKBP12-PI4KB at high expression level is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). Right: eGFP-c10orf76 at a high expression level is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom).


( F ) Mitochondria recruitment experiment with mutant PI4KB WT c10orf76 . Left : AKAP1 - FRB - CFP is localized to mitochondria before ( top ) 5 minutes after ( bottom ) rapamycin treatment . Middle : mRFP - FKBP12 - PI4KB ( RL494EA ) is located in cytosol before rapamycin ( top ) translocates to mitochondria after rapamycin induction ( bottom ) . Right : eGFP - c10orf76 is located in cytosol before ( top ) after ( bottom ) rapamycin induction .

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

(F) Mitochondria recruitment experiment with mutant PI4KB and WT c10orf76. Left: AKAP1-FRB-CFP is localized to the mitochondria before (top) and 5 minutes after (bottom) rapamycin treatment. Middle: mRFP-FKBP12-PI4KB(RL494EA) is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). Right: eGFP-c10orf76 is located in the cytosol before (top) and after (bottom) rapamycin induction.


( A HAP1 were fixed stained with antibodies examining PI4P PI4KB Data information : Nuclei were stained with DAPI ( blue ) . Scale bars represent 10 μm .

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(A HAP1 cells were fixed and stained with antibodies examining PI4P and PI4KB Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.


B ) HAP1 were fixed stained with antibodies examining Golgi markers ( B ) . ( C ) Western blot of GBF1 , PI4KB B - Actin in both wild - type c10orf76 KO HAP1 . Data information : Nuclei were stained with DAPI ( blue ) . Scale bars represent 10 μm .

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B) HAP1 cells were fixed and stained with antibodies examining Golgi morphology markers (B). (C) Western blot of GBF1, PI4KB and B-Actin levels in both wild-type and c10orf76 KO HAP1 cells. Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.


( D ) Markers of Arf1 activation - coatomer COPIα / γ βCOP act as a readout for GTP - bound Arf1 , while native coatomer was detected with CM1 antibody . Data information : Nuclei were stained with DAPI ( blue ) . Scale bars represent 10 μm .

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(D) Markers of Arf1 activation - the coatomer proteins COPIα/γ and βCOP act as a readout for GTP-bound Arf1, while the native coatomer was detected with the CM1 antibody. Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.


( A ) Viral infection assays determining viral titers by end - point titration at 0 hours 8 hours in HAP1 wild - type or c10orf76 knockout . Left : Coxsackievirus A10 infection . Middle : Poliovirus - 1 infection . Right : Coxsackievirus B3 infection . Titre values for c10orf76 knockout at 8 hours were statistically evaluated compared to c10orf76 knockout titres at 0 hours wild - type titres at 8 hours using a one - way ANOVA . * * , P < 0 . 01 ; N . S . , P > 0 . 05 .

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(A) Viral infection assays determining viral titers by end-point titration at 0 hours and 8 hours in HAP1 wild-type or c10orf76 knockout cells. Left: Coxsackievirus A10 infection. Middle: Poliovirus-1 infection. Right: Coxsackievirus B3 infection. Titre values for c10orf76 knockout cells at 8 hours were statistically evaluated compared to c10orf76 knockout titres at 0 hours and wild-type titres at 8 hours using a one-way ANOVA. **, P<0.01; N.S., P>0.05.


( B ) Viral infection assays determining titers by end point titration at 8 hours in HeLa wild - type PI4KB knockout upon transfection of wild - type PI4KB , - disrupting RL494EA PI4KB mutant or kinase dead D674A PI4KB mutant . Left : Coxsackievirus A10 infection . Middle : Poliovirus - 1 infection . Right : Coxsackievirus B3 infection . Values were statistically evaluated compared to GalT control using a one - way ANOVA . * * , P < 0 . 01 ; * , P < 0 . 05 ; N . S . , P > 0 . 05 .

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

(B) Viral infection assays determining virus titers by end point titration at 8 hours in HeLa wild-type and PI4KB knockout cells upon transfection of wild-type PI4KB, the complex-disrupting RL494EA PI4KB mutant or the kinase dead D674A PI4KB mutant. Left: Coxsackievirus A10 infection. Middle: Poliovirus-1 infection. Right: Coxsackievirus B3 infection. Values were statistically evaluated compared to the GalT control using a one-way ANOVA. **, P<0.01; *, P<0.05; N.S., P>0.05.


Overall structure of 2019 - nCoV RBD bound with ACE2 . ACE2 is colored green . 2019 - nCoV RBD is colored cyan RBM is colored red . Disulfide bonds in 2019 - nCoV RBD are shown as stick indicated by yellow arrows . N - terminal helix of ACE2 responsible for binding is labeled .

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Overall structure of 2019-nCoV RBD bound with ACE2. ACE2 is colored green. 2019-nCoV RBD core is colored cyan and RBM is colored red. Disulfide bonds in the 2019-nCoV RBD are shown as stick and indicated by yellow arrows. The N-terminal helix of ACE2 responsible for binding is labeled.


Alignment of 2019 - nCoV RBD ( in cyan RBM in red ) SARS - CoV RBD ( in orange RBM in blue ) structures .

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Alignment of the 2019-nCoV RBD (core in cyan and RBM in red) and SARS-CoV RBD (core in orange and RBM in blue) structures.


Structural alignment of 2019 - nCoV RBD / ACE2 SARS - CoV RBD / ACE2 complexes . 2019 - nCoV RBD is colored cyan red , its interacting ACE2 is colored green . SARS - CoV RBD is colored orange blue , its interacting ACE2 is colored salmon . PDB code for SARS - CoV RBD / ACE2 : 2AJF .

Structural alignment of 2019-nCoV RBD/ACE2 and SARS-CoV RBD/ACE2 complexes. 2019-nCoV RBD is colored cyan and red, its interacting ACE2 is colored green. SARS-CoV RBD is colored orange and blue, its interacting ACE2 is colored salmon. The PDB code for SARS-CoV RBD/ACE2 complex: 2AJF.


Contacting residues shown as stick at 2019 - nCoV RBD / ACE2 SARS - CoV RBD / ACE2 interfaces . Positions in both RBDs involved in ACE2 binding are indicated by red labels .

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

Contacting residues shown as stick at the 2019-nCoV RBD/ACE2 and SARS-CoV RBD/ACE2 interfaces. Positions in both RBDs involved in ACE2 binding are indicated by red labels.


( B - K ' ) Drosophila posterior midguts containing Su ( H ) - Gal4 > UAS - CD8 : GFP ( Su ( H ) > GFP ) with sucrose ( Suc , control ) ( B - C ' ) , P . e for 36 h ( D - F ' ) , Ecc15 for 36 h ( G - I ' ) DSS for 36 h ( J - K ' ) were immunostained for GFP ( green ) , PH3 ( red ) DAPI ( blue ) . ( C - C ' , E - E ' , H - H ' K - K ' ) Magnification of selected areas containing PH3 + GFP ─ from B , D , G J . EB close to PH3 + ( arrow ) in C - C ' is out of focus . ( F - F ' I - I ' ) Magnification of selected areas containing PH3 + EBs ( GFP + ) from D G . arrowheads red arrows indicate PH3 in GFP ─ GFP + , respectively . ( L M ) of PH3 GFP ─ ISCs ( L ) PH3 + GFP + EBs ( M ) with given treatments . n = 21 guts for each treatment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K') The Drosophila posterior midguts containing Su(H)-Gal4>UAS-CD8:GFP (Su(H)>GFP) with sucrose (Suc, control) (B-C'), P.e for 36 h (D-F'), Ecc15 for 36 h (G-I') and DSS for 36 h (J-K') were immunostained for GFP (green), PH3 (red) and DAPI (blue). (C-C', E-E', H-H' and K-K') Magnification of selected areas containing PH3+GFP─ cells from B, D, G and J. The EB cell close to the PH3+ cell (white arrow) in C-C' is out of focus. (F-F' and I-I') Magnification of selected areas containing PH3+ EBs (GFP+) from D and G. White arrowheads and red arrows indicate PH3 in GFP─ and GFP+ cells, respectively. (L and M) Quantification of PH3 GFP─ ISCs (L) and PH3+GFP+ EBs (M) with given treatments. n = 21 guts for each treatment.


( D - F ' ' ) When ISCs were marked by esgGal4 , su ( H ) - Gal80 > UAS - GFP , PH3 expression was found in GFP + ISCs with feeding of Suc ( control ) ( D - D ' ' ) , P . e ( E - E ' ' ) Dss ( F - F ' ' ) . However , PH3 expression is present in EBs with GFP ─ Pros ─ small nuclei upon P . e infection ( E - E ' ' , red arrows ) . arrows indicate GFP + with PH3 expression . ( G ) of PH3 + GFP ─ Pros ─ EBs with given treatments . n = 11 guts for each treatment . Data information : Three independent experiments were performed , error bars are ± . * * * , P < 0 . 001 ( Student ' s t - ) ( G ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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'') When the ISCs were marked by esgGal4, su(H)-Gal80>UAS-GFP, PH3 expression was found in GFP+ ISCs with feeding of Suc (control) (D-D''), P.e (E-E'') and Dss (F-F''). However, PH3 expression is present in EBs with GFP─Pros─ and small nuclei upon P.e infection (E-E'', red arrows). The white arrows indicate GFP+ cells with PH3 expression. (G) Quantification of PH3+GFP─Pros─ EBs with given treatments. n = 11 guts for each treatment. Data information: Three independent experiments were performed, and error bars are ± SEM. ***, P < 0.001 (Student's t-test) (G).


( D - N ' ) Drosophila midguts expressing Su ( H ) ts > GFP with control ( D - E ' ) , UAS - RasV12 for 6 days ( F - H ' ) , EGFRA887T for 4 days ( I - K ' ) UAS − λTop for 5 days ( L - N ' ) were immunostained for GFP ( green ) , PH3 ( red ) DAPI ( blue ) . ( E - E ' , G - G ' , J - J ' M - M ' ) Magnification of selected areas containing PH3 + GFP ─ from D , F , I L . ( H - H ' , K - K ' N - N ' ) Magnification of selected areas containing PH3 + GFP + from F , I L . arrowheads red arrows indicate PH3 in GFP ─ ISCs GFP + EBs , respectively . ( O , P ) of PH3 + GFP ─ ( P ) PH3 + GFP + ( O ) in midguts with indicated genotypes . n = 13 guts for each genotype . ( Q ) of PH3 + EBs ( GFP + ) with given genotypes in response to P . e infection . n = 11 guts for each genotype .

O O O 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 B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-N') The Drosophila midguts expressing Su(H)ts>GFP with control (D-E'), UAS-RasV12 for 6 days (F-H'), EGFRA887T for 4 days (I-K') and UAS−λTop for 5 days (L-N') were immunostained for GFP (green), PH3 (red) and DAPI (blue). (E-E', G-G', J-J' and M-M') Magnification of selected areas containing PH3+GFP─ cells from D, F, I and L. (H-H', K-K' and N-N') Magnification of selected areas containing PH3+GFP+ cells from F, I and L. White arrowheads and red arrows indicate PH3 in GFP─ ISCs and GFP+ EBs, respectively. (O, P) Quantification of PH3+GFP─ (P) and PH3+GFP+ (O) in midguts with indicated genotypes. n = 13 guts for each genotype. (Q) Quantification of PH3+ EBs (GFP+) with the given genotypes in response to P. e infection. n = 11 guts for each genotype.


( B - C ' ' ' ) Flies expressing UAS - Flp ; Su ( H ) ts UAS - CD8 : GFP ; ActP > Stop > LacZ were fed with Suc ( B - B ' ' ' ) or P . e ( C - C ' ' ' ) for 36h their midguts were dissected immunostained with antibodies against GFP , β - gal Dl . Blue arrowheads indicate Dl + ISCs ; arrows indicate GFP + EBs ; red arrowheads indicate EB progeny ( LacZ + GFP ─ ) . ( D ) of Dl + ISC - like from EBs ( GFP ─ LacZ + Dl + ) in control , P . e infected midguts , n = 50 . ( E ) of Dl + ISC - like from EBs ( GFP ─ LacZ + Dl + ) in control with Suc or P . e , or knockdown of EGFR with Suc or P . e infection , n = 50 .

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

(B-C''') Flies expressing UAS-Flp; Su(H)ts UAS-CD8:GFP; ActP>Stop>LacZ were fed with Suc (B-B''') or P.e (C-C''') for 36h and their midguts were dissected and immunostained with antibodies against GFP, β-gal and Dl. Blue arrowheads indicate Dl+ ISCs; white arrows indicate GFP+ EBs; and red arrowheads indicate EB progeny cells (LacZ+GFP─). (D) Quantification of Dl+ ISC-like cells from EBs (GFP─LacZ+Dl+) in the control, P. e infected midguts, n=50. (E) Quantification of Dl+ ISC-like cells from EBs (GFP─LacZ+Dl+) in the control with Suc or P. e, or knockdown of EGFR with Suc or P. e infection, n=50.


( A - C ' ' ' ) midguts from adult flies expressing UAS - Flp ; Su ( H ) ts UAS - CD8 : GFP ; ActP > Stop > LacZ with ( B - B ' ' ' ) or without ( A - A ' ' ' ) UAS - RasV12 or with UAS - RasV12 UAS - stg - RNAi ( C - C ' ' ' ) were dissected immunostained with antibodies against GFP , β - gal Dl . bigger GFP ─ nucleus with LacZ expression ( only red arrowheads ) indicate differentiating or mature ECs . Blue arrowheads indicate Dl + ISCs ; arrows indicate GFP + EBs ; red arrowheads indicate EB progeny ( LacZ + GFP ─ ) . ( D ) of Dl + ISC - like from EBs ( GFP ─ LacZ + Dl + ) in indicated genotypes , n = 50 .

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

(A-C''') The midguts from adult female flies expressing UAS-Flp; Su(H)ts UAS-CD8:GFP; ActP>Stop>LacZ with (B-B''') or without (A-A''') UAS-RasV12 or with UAS-RasV12 and UAS-stg-RNAi (C-C''') were dissected and immunostained with antibodies against GFP, β-gal and Dl. The bigger GFP─ nucleus with LacZ expression (only red arrowheads) indicate differentiating or mature ECs. Blue arrowheads indicate Dl+ ISCs; white arrows indicate GFP+ EBs; and red arrowheads indicate EB progeny cells (LacZ+GFP─). (D) Quantification of Dl+ ISC-like cells from EBs (GFP─LacZ+Dl+) in the indicated genotypes, n=50.


( C - E ' ' ) representative twin clones from midguts when EGFRA887T is overexpressed in EBs ( D - D ' ' , red green arrows ) or adult flies were fed with P . e for 36h ( E - E ' ' , red green arrows ) , but no twin clone is found in control ( C - C ' ' ) . ( F ) of different types of twin clones in control midguts , or in midguts with EGFRA887T overexpression or P . e infection . n = 10 guts for each genotype .

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

(C-E'') The representative twin clones from midguts when EGFRA887T is overexpressed in EBs (D-D'', red and green arrows) or adult flies were fed with P.e for 36h (E-E'', red and green arrows), but no twin clone is found in the control (C-C''). (F) Quantification of different types of twin clones in the control midguts, or in the midguts with EGFRA887T overexpression or P.e infection. n = 10 guts for each genotype.


( A - C ' ' ) ISCs from EBs upon RasV12 expression or P . e infection could differentiate to generate EE or pre - EE ( B - C ' ' , LacZ + Pros + GFP ─ , blue arrowheads ) , but no EE or pre - EE from EBs are found in control ( A - A ' ' , arrowheads ) . ( D ) of EE from progenies of EBs with indicated genotypes or treatments . n = 9 guts for each genotype or treatment .

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 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

(A-C'') ISCs from EBs upon RasV12 expression or P. e infection could differentiate to generate EE or pre-EE cells (B-C'', LacZ+Pros+GFP─, blue arrowheads), but no EE or pre-EE cells from EBs are found in the control (A-A'', white arrowheads). (D) Quantification of EE cells from progenies of EBs with indicated genotypes or treatments. n = 9 guts for each genotype or treatment.


Flow cytometric of γδ T in RORγtCRE - STAT3F / F ( Cre + ) littermate control mice ( Cre − ) . In graphs , each symbol represents a mouse median . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 using Mann - Whitney . Numbers of γδT17 in LN ( A ) skin ( B ) before ( steady - state ) after ( IMQ ) IMQ - induced psoriasis . Steady - state : n = 8 ; 4 experiments , IMQ : n = 11 - 12 ; 4 experiments .

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

Flow cytometric analysis of γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse and line the median. *p < 0.05, **p < 0.01, ***p < 0.001 using Mann-Whitney test. Numbers of γδT17 cells in the LN (A) and skin (B) before (steady-state) and after (IMQ) IMQ-induced psoriasis. Steady-state: n = 8; 4 experiments, IMQ: n = 11-12; 4 experiments.


Representative indicating ear skin sections stained for H & E before after IMQ - induced psoriasis ( scale bar = 100μm ) . of epidermal thickening from H & E stained sections . Steady - state : n = 8 ; 4 experiments , IMQ : n = 11 - 12 ; 4 experiments . Each symbol is average thickening from 5 measured in μm .

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

Representative micrographs indicating ear skin sections stained for H&E before and after IMQ-induced psoriasis (scale bar = 100μm). Quantification of epidermal thickening from H&E stained sections. Steady-state: n = 8; 4 experiments, IMQ: n = 11-12; 4 experiments. Each symbol is the average thickening from 5 micrographs measured in μm.


Flow cytometric of γδ T in STAT4 − / − ( − / − ) littermate control mice ( + / − ) . In graphs , each symbol represents a mouse median . Numbers of γδT17 in LN skin ( as in Fig EV1 ) before ( steady - state ) after ( IMQ ) IMQ - induced psoriasis . Steady - state : n = 6 ; 3 experiments , IMQ : n = 11 ; 4 experiments .

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

Flow cytometric analysis of γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). In graphs, each symbol represents a mouse and line the median. Numbers of γδT17 cells in the LN and skin (staining as in Fig EV1) before (steady-state) and after (IMQ) IMQ-induced psoriasis. Steady-state: n = 6; 3 experiments, IMQ: n = 11; 4 experiments.


Representative indicating ear skin sections stained for H & E before after IMQ - induced psoriasis ( scale bar = 100μm ) . of epidermal thickening from H & E stained sections . Steady - state : n = 6 ; 3 experiments , IMQ : n = 8 ; 3 experiments . Each symbol is average thickening from 5 measured in μm .

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

Representative micrographs indicating ear skin sections stained for H&E before and after IMQ-induced psoriasis (scale bar = 100μm). Quantification of epidermal thickening from H&E stained sections. Steady-state: n = 6; 3 experiments, IMQ: n = 8; 3 experiments. Each symbol is the average thickening from 5 micrographs measured in μm.


Flow cytometric of lymph node γδ T in RORγtCRE - STAT3F / F ( Cre + ) littermate control mice ( Cre − ) . In graphs , each symbol represents a mouse or experiment median . Representative dot plots showing IL - 17A ( A ) or IL - 17F ( D ) IFNγ production in γδ T before ( steady - state ) after IMQ - induced psoriasis . Numbers in gate indicate % positive ; numbers outside gate indicate mean of IL - 17A or IL - 17F . ( B , E ) Frequency of IL - 17A + ( B ) IL - 17F + ( E ) γδ T before ( steady - state ) after ( IMQ ) IMQ - induced psoriasis . ( C , F ) of mean ( MFI ) of IL - 17A ( C ) IL - 17F ( F ) in γδ T after IMQ - induced psoriasis ( each color represents a different experiment ) .

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

Flow cytometric analysis of lymph node γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse or experiment and line the median. Representative dot plots showing IL-17A (A) or IL-17F (D) and IFNγ production in γδ T cells before (steady-state) and after IMQ-induced psoriasis. Numbers in gate indicate % positive cells; numbers outside the gate indicate mean fluorescence intensity of IL-17A or IL-17F. (B, E) Frequency of IL-17A+ (B) and IL-17F+ (E) γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis. (C, F) Quantification of mean fluorescence intensity (MFI) of IL-17A (C) and IL-17F (F) staining in γδ T cells after IMQ-induced psoriasis (each color represents a different experiment).


Frequency of IL - 17A + IL - 17F + γδ T or IL - 17A ( G ) IL - 17F ( H ) MFI following culture with IL - 1β , IL - 23 or nothing . Each symbol represents one experiment .

Frequency of IL-17A+ and IL-17F+ γδ T cells or IL-17A (G) and IL-17F (H) MFI following culture with IL-1β, IL-23 or nothing. Each symbol represents one experiment.


Flow cytometric of lymph node γδ T in RORγtCRE - STAT3F / F ( Cre + ) littermate control mice ( Cre − ) . In graphs , each symbol represents a mouse or experiment median . Representative dot plots showing IL - 22 IL - 17A production in γδ T ( A ) frequency of IL - 22 + γδ T ( B ) before ( steady - state ) after ( IMQ ) IMQ - induced psoriasis . Steady - state : n = 6 ; 3 experiments , IMQ : n = 8 - 9 ; 3 experiments .

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

Flow cytometric analysis of lymph node γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse or experiment and line the median. Representative dot plots showing IL-22 and IL-17A production in γδ T cells (A) and frequency of IL-22+ γδ T cells (B) before (steady-state) and after (IMQ) IMQ-induced psoriasis. Steady-state: n = 6; 3 experiments, IMQ: n = 8-9; 3 experiments.


Flow cytometric of lymph node γδ T in RORγtCRE - STAT3F / F ( Cre + ) littermate control mice ( Cre − ) . In graphs , each symbol represents a mouse or experiment median . Representative dot plots showing IL - 22 IL - 17A production in γδ T ( C ) frequency of IL - 22 + γδ T ( D ) following culture without ( − ) or with 40ng / ml recombinant IL - 23 ( n = 9 - 10 , 3 experiments ) .

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

Flow cytometric analysis of lymph node γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse or experiment and line the median. Representative dot plots showing IL-22 and IL-17A production in γδ T cells (C) and frequency of IL-22+ γδ T cells (D) following culture without (−) or with 40ng/ml recombinant IL-23 (n = 9-10, 3 experiments).


Frequency of IL - 22 + γδ T following culture with IL - 1β , IL - 23 or nothing . Each symbol represents one experiment . Open circles = Cre + ( each color represents a different culture condition ) .

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 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

Frequency of IL-22+ γδ T cells following culture with IL-1β, IL-23 or nothing. Each symbol represents one experiment. Open circles = Cre+ (each color represents a different culture condition).


Flow cytometric of lymph node γδ T in STAT4 − / − ( − / − ) littermate control mice ( + / − ) . ( A , ) Frequency of IL - 17A + ( A ) γδ T before ( steady - state ) after ( IMQ ) IMQ - induced psoriasis . ( B ) Frequency of IL - 17F + γδ T before ( steady - state ) after ( IMQ ) IMQ - induced psoriasis . ( C ) of mean ( MFI ) of IL - 17A in γδ T after IMQ - induced psoriasis ( D ) of mean ( MFI ) of IL - 17F in γδ T after IMQ - induced psoriasis .

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

Flow cytometric analysis of lymph node γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). (A, ) Frequency of IL-17A+ (A) γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis. (B) Frequency of IL-17F+ γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis. (C) Quantification of mean fluorescence intensity (MFI) of IL-17A staining in γδ T cells after IMQ-induced psoriasis (D) Quantification of mean fluorescence intensity (MFI) of IL-17F staining in γδ T cells after IMQ-induced psoriasis.


Flow cytometric of lymph node γδ T in STAT4 − / − ( − / − ) littermate control mice ( + / − ) . Frequency of IL - 22 + γδ T before ( steady - state ) after ( IMQ ) IMQ - induced psoriasis .

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

Flow cytometric analysis of lymph node γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). Frequency of IL-22+ γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis.


Flow cytometric of lymph node γδ T in STAT4 − / − ( − / − ) littermate control mice ( + / − ) . Frequency of IL - 22 + γδ T following culture without ( − ) or with 40ng / ml recombinant IL -

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

Flow cytometric analysis of lymph node γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). Frequency of IL-22+ γδ T cells following culture without (−) or with 40ng/ml recombinant IL-23


( A ) Homer1c - GFP signal ( magenta ) immunostaining for surface AMPARs ( cyan ) SP ( green ) in neurons treated for 48h with TTX , or untreated ( UT ) . Scale bars : 10 µm ( low magnification ) , 5 µm ( insets ) . Dotted lines indicate outline of dendrites . Arrowheads indicate SP + synapses . ( B ) of SP + synapses for untreated ( UT ) TTX - treated neurons ( dot plots represent different ; UT : n = 26 ; TTX : n = 33 , n indicates number of , from 2 cultures ) . % SP + spines : * * P = 0 . 019 ( Mann Whitney ) . Data information : Data are represented as mean ± .

O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O 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) Homer1c-GFP signal (magenta) and immunostaining for surface AMPARs (cyan) and SP (green) in neurons treated for 48h with TTX, or untreated (UT). Scale bars: 10 µm (low magnification), 5 µm (insets). Dotted lines indicate the outline of dendrites. Arrowheads indicate SP+ synapses. (B) Percentage of SP+ synapses for untreated (UT) and TTX-treated neurons (dot plots represent different cells; UT: n = 26; TTX: n = 33, n indicates the number of cells, from 2 cultures). % SP+ spines: **P = 0.019 (Mann Whitney test). Data information: Data are represented as mean ± SEM.


( C ) AMPAR synaptic for SP + versus SP - synapses in UT TTX - treated neurons . AMPAR synaptic was normalized to SP - or small synapses , respectively , from UT condition . ( UT : SP - , n = 1455 , SP + , n = 516 ; TTX : SP - , n = 1529 , SP + , n = 724 , n indicates number of synapses , from 2 cultures ) . * * P < 0 . 01 , * * * * P < 0 . 0001 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis followed by Dunn ' s multiple comparison ) . Data information : Data are represented as mean ± .

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 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

(C) AMPAR synaptic fluorescence intensity for SP+ versus SP- synapses in UT and TTX-treated neurons. AMPAR synaptic fluorescence intensity was normalized to SP- or small synapses, respectively, from UT condition. (UT: SP-, n = 1455, SP +, n = 516; TTX: SP-, n = 1529, SP+, n = 724, n indicates the number of synapses, from 2 cultures). **P < 0.01, ****P < 0.0001, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data are represented as mean ± SEM.


( A ) Example of a FRAP experiment : SP - SP + spines from same cultured hippocampal neuron transfected with SEP - GluA2 ( gray color - coded ) + RFP - SP ( red ) 10s before , 0 , 370 , 750 s after photobleaching . Scale bars : 1 µm . Dotted lines represent outline of dendrites . Arrowheads indicate dendritic spines which were targeted for photobleaching . ( B ) SEP - GluA2 at SP - vs SP + spines . Each pair of dot plots represents average for SP - SP + spines from a same neuron ( 4 cultures ) . * * P = 0 . 0061 ( two - paired Student ' s t ) . ( C ) of FRAP dynamics for SP - vs SP + spines . Recovery curves represent SEP - GluA2 average per ( n = 14 , from 4 cultures ) . two traces were fitted using double exponential components equations convergence of traces to a common fit was tested using extra sum of squares F . F indicates that traces are best fitted by two divergent models ( P < 0 . 0001 ) . ( D ) of recovery fraction 750 s after photobleaching . Each pair of dot plots represents average recovery for SP - SP + spines from a same neuron ( 4 cultures ) . * * P = 0 . 0067 ( Wilcoxon matched - pairs signed rank ) . Data information : Data are represented as mean ± .

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

(A) Example images of a FRAP experiment: SP- and SP+ spines from the same cultured hippocampal neuron transfected with SEP-GluA2 (gray and color-coded) + RFP-SP (red) 10s before, and 0, 370, 750 s after the photobleaching. Scale bars: 1 µm. Dotted lines represent the outline of dendrites. Arrowheads indicate dendritic spines which were targeted for photobleaching. (B) SEP-GluA2 fluorescence intensity at SP- vs SP+ spines. Each pair of dot plots represents average fluorescence for SP- and SP+ spines from a same neuron (4 cultures). **P = 0.0061 (two-tailed paired Student's t test). (C) Quantification of FRAP dynamics for SP- vs SP+ spines. Recovery curves represent SEP-GluA2 fluorescence average per cell (n = 14 cells, from 4 cultures). The two traces were fitted using double exponential components equations and the convergence of the traces to a common fit was tested using the extra sum of squares F test. The F test indicates that the traces are best fitted by two divergent models (P < 0.0001). (D) Quantification of the recovery fraction 750 s after the photobleaching. Each pair of dot plots represents average recovery for SP- and SP+ spines from a same neuron (4 cultures). **P = 0.0067 (Wilcoxon matched-pairs signed rank test). Data information: Data are represented as mean ± SEM.


( A ) Immunoblot of SP in lysates from COS - 7 which were untransfected , transfected with RFP - SP + empty vector or co - transfected with SP - shRNA - GFP + RFP - SP ( knock - down ) or shRNA - resistant RFP - SP ( rescue ) . β - actin GFP signals illustrate equal loading SP - shRNA expression level , respectively . ( B ) of SP expression from 6 different experiments , normalized to β - actin signal . * P < 0 . 05 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis followed by Dunn ' s multiple comparison ) . Data information : Data represent mean ± .

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

(A) Immunoblot analysis of SP in lysates from COS-7 cells which were untransfected, transfected with RFP-SP + empty vector or co-transfected with SP-shRNA-GFP + RFP-SP (knock-down) or shRNA-resistant RFP-SP (rescue). β-actin and GFP signals illustrate equal protein loading and SP-shRNA expression level, respectively. (B) Quantification of SP expression from 6 different experiments, normalized to the β-actin signal. *P < 0.05, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data represent mean ± SEM.


( C ) Homer - DsRed ( green ) immunostained endogenous SP ( red ) in dendrites from neurons transfected with either empty vector , scramble shRNA or SP - shRNA with GFP reporter ( blue ) . Scale bar : 10 µm . ( D ) Average SP for same conditions ( EV : n = 47 ; Scr - shRNA : n = 53 ; SP - shRNA : n = 62 , n indicates number of , from 3 cultures ; normalized to empty vector condition ) . * * P < 0 . 01 , * * * * P < 0 . 0001 ( Kruskal - Wallis followed by Dunn ' s multiple comparison ) . ( E ) Fraction of SP + synapses for same conditions . ( EV : n = 47 ; Scr - shRNA : n = 53 ; SP - shRNA : n = 62 , n indicates number of from 3 cultures ) . * P < 0 . 05 ( Kruskal - Wallis followed by Dunn ' s multiple comparison ) . Data information : Data represent mean ± .

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

(C) Homer-DsRed (green) and immunostained endogenous SP (red) in dendrites from neurons transfected with either empty vector, scramble shRNA or SP-shRNA with GFP reporter (blue). Scale bar: 10 µm. (D) Average SP intensity for same conditions (EV: n = 47; Scr-shRNA: n = 53; SP-shRNA: n = 62, n indicates the number of cells, from 3 cultures; normalized to empty vector condition). **P < 0.01, ****P < 0.0001 (Kruskal-Wallis test followed by Dunn's multiple comparison test). (E) Fraction of SP+ synapses for same conditions. (EV: n = 47; Scr-shRNA: n = 53; SP-shRNA: n = 62, n indicates the number of cells from 3 cultures). *P < 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data represent mean ± SEM.


( F ) Homer1c - DsRed ( red ) immunostained surface AMPARs ( green ) in dendrites from neurons transfected with either SP - shRNA - GFP or empty vector ( EV ) with GFP reporter ( blue ) treated with TTX or left untreated ( UT ) . Scale bar : 5 µm . ( G ) AMPAR synaptic normalized to untreated empty vector ( EV ) condition ( EV : UT , n = 42 , TTX , n = 33 ; SP - shRNA : UT , n = 30 , TTX , n = 31 , n indicates number of , from 3 cultures ) . * P < 0 . 05 , * * P < 0 . 01 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis followed by Dunn ' s multiple comparison ) . Data information : Data represent mean ± .

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

(F) Homer1c-DsRed (red) and immunostained surface AMPARs (green) in dendrites from neurons transfected with either SP-shRNA-GFP or empty vector (EV) with GFP reporter (blue) and treated with TTX or left untreated (UT). Scale bar: 5 µm. (G) AMPAR synaptic fluorescence intensity normalized to untreated empty vector (EV) condition (EV: UT, n = 42, TTX, n = 33; SP-shRNA: UT, n = 30, TTX, n = 31, n indicates the number of cells, from 3 cultures). *P < 0.05, **P < 0.01, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data represent mean ± SEM.


( A ) showing Homer1c - GFP ( green ) immunostaining for surface AMPARs ( blue ) endogenous SP ( red ) in neurons transfected with miR - 124 or control miR - 67 ( miR - Ctrl ) , treated with TTX , or left untreated ( UT ) . Scale bar : 5 µ ( B ) of SP + synapses for same conditions as in ( A ) ( miR - Ctrl : UT , n = 25 , TTX , n = 26 ; miR - 124 : UT , n = 30 , TTX , n = 30 , n indicates number of , from 3 cultures ) . * * * * P < 0 . 0001 , ns , not significant , P > 0 . 05 ( two - way ANOVA followed by Tukey ' s multiple comparison ) . ( C ) Homer1c - GFP for same condition as in ( A ) , normalized to untreated miR - Ctrl ( miR - Ctrl : UT , n = 25 , TTX , n = 26 ; miR - 124 : UT , n = 30 , TTX , n = 30 , n indicates number of , from 3 cultures ) . ns , not significant , P > 0 . 05 ( two - way ANOVA followed by Tukey ' s multiple comparison ) . ( D ) AMPAR synaptic for same condition as in ( A ) , normalized to untreated miR - Ctrl ( miR - Ctrl : UT , n = 25 , TTX , n = 26 , miR - 124 : UT , n = 30 , TTX , n = 30 , n indicates number of , from 3 cultures ) . * * * P < 0 . 001 , * P < 0 . 01 , ns , not significant , P > 0 . 05 ( two - way ANOVA followed by Tukey ' s multiple comparison ) . Data information : Data are represented as mean ± .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O 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) Micrographs showing Homer1c-GFP (green) and immunostaining for surface AMPARs (blue) and endogenous SP (red) in neurons transfected with miR-124 or control miR-67 (miR-Ctrl), and treated with TTX, or left untreated (UT). Scale bar: 5 µ (B) Percentage of SP+ synapses for same conditions as in (A) (miR-Ctrl: UT, n = 25, TTX, n = 26; miR-124: UT, n = 30, TTX, n = 30, n indicates the number of cells, from 3 cultures). ****P < 0.0001, ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test). (C) Homer1c-GFP intensity for same condition as in (A), normalized to untreated miR-Ctrl (miR-Ctrl: UT, n = 25, TTX, n = 26; miR-124: UT, n = 30, TTX, n = 30, n indicates the number of cells, from 3 cultures). ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test). (D) AMPAR synaptic fluorescence intensity for same condition as in (A), normalized to untreated miR-Ctrl (miR-Ctrl: UT, n = 25, TTX, n = 26, miR-124: UT, n = 30, TTX, n = 30, n indicates the number of cells, from 3 cultures). ***P < 0.001, *P < 0.01, ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test). Data information: Data are represented as mean ± SEM.


( A ) showing dendrites from neurons transfected with Homer1c - DsRed ( magenta ) SEP - GluA2 constructs containing wild - type ( WT ) or mutated ( MUT ) 3 ' UTR ( green ) treated with TTX for 48 h , or left untreated ( UT ) . Scale bar : 10 µm . ( B ) SEP - GluA2 synaptic for each condition , normalized to GluA2 - 3 ' UTR - WT untreated neurons ( 3 ' UTR - WT : UT , n = 29 , TTX , n = 37 ; 3 ' UTR - MUT : UT , n = 18 , TTX , n = 14 ; n indicates number of , from 3 cultures ) . * P < 0 . 05 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis followed by Dunn ' s multiple comparison ) . Data information : Data are represented as mean ± .

O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Micrographs showing dendrites from neurons transfected with Homer1c-DsRed (magenta) and SEP-GluA2 constructs containing wild-type (WT) or mutated (MUT) 3'UTR (green) and treated with TTX for 48 h, or left untreated (UT). Scale bar: 10 µm. (B) SEP-GluA2 synaptic fluorescence intensity for each condition, normalized to GluA2-3'UTR-WT untreated neurons (3'UTR-WT: UT, n = 29, TTX, n = 37; 3'UTR-MUT: UT, n = 18, TTX, n = 14; n indicates the number of cells, from 3 cultures). *P < 0.05, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data are represented as mean ± SEM.


( C ) showing dendrites from neurons transfected with Homer1c - BFP ( magenta ) , GFP - SP - shRNA ( gray ) a rescue RFP - SP construct containing wild - type ( WT ) or mutated ( MUT ) 3 ' UTR ( geen ) treated with TTX , or left untreated ( UT ) . Scale bar : 10 µm . ( D ) of SP + synapses for each condition ( 3 ' UTR - WT : UT , n = 26 , TTX , n = 28 ; 3 ' UTR - MUT : UT , n = 26 , TTX , n = 24 ; n indicates number of , from 3 cultures ) . * * P < 0 . 01 , * * * P < 0 . 001 , ns , not significant , P > 0 . 05 ( two - way ANOVA followed by Tukey ' s multiple comparison ) . Data information : Data are represented as mean ± .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Micrographs showing dendrites from neurons transfected with Homer1c-BFP (magenta), GFP-SP-shRNA (gray) and a rescue RFP-SP construct containing wild-type (WT) or mutated (MUT) 3'UTR (geen) and treated with TTX, or left untreated (UT). Scale bar: 10 µm. (D) Percentage of SP+ synapses for each condition (3'UTR-WT: UT, n = 26, TTX, n = 28; 3'UTR-MUT: UT, n = 26, TTX, n = 24; n indicates the number of cells, from 3 cultures). **P < 0.01, ***P < 0.001, ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test). Data information: Data are represented as mean ± SEM.


( E ) showing Homer1c - GFP ( magenta ) immunostaining for surface AMPARs ( cyan ) endogenous SP ( green ) in neurons transfected with or without 50 nM SP TSB - LNA treated with TTX , or left untreated ( UT ) . Scale bar : 10 µm . ( F ) of SP + synapses for each condition ( Control : UT , n = 36 , TTX , n = 52 ; SP TSB - LNA : UT , n = 62 , TTX , n = 29 ; n indicates number of from 3 cultures ) . * P < 0 . 05 , * * P < 0 . 01 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis followed by Dunn ' s multiple comparison ) . ( G ) AMPAR synaptic for each condition , normalized to control untreated neurons ( Control : UT , n = 30 , TTX , n = 36 ; SP TSB - LNA : UT , n = 37 , TTX , n = 20 ; n indicates number of from 2 cultures ) . * P < 0 . 05 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis followed by Dunn ' s multiple comparison ) . Data information : Data are represented as mean ± .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O 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) Micrographs showing Homer1c-GFP (magenta) and immunostaining for surface AMPARs (cyan) and endogenous SP (green) in neurons transfected with or without 50 nM SP TSB-LNA and treated with TTX, or left untreated (UT). Scale bar: 10 µm. (F) Percentage of SP+ synapses for each condition (Control: UT, n = 36, TTX, n = 52; SP TSB-LNA: UT, n = 62, TTX, n = 29; n indicates the number of cells from 3 cultures). *P < 0.05, **P < 0.01, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). (G) AMPAR synaptic fluorescence intensity for each condition, normalized to control untreated neurons (Control: UT, n = 30, TTX, n = 36; SP TSB-LNA: UT, n = 37, TTX, n = 20; n indicates the number of cells from 2 cultures). *P < 0.05, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data are represented as mean ± SEM.


( A ) showing puro - PLA of newly synthesized SP ( green ) in neurons immunostained for MAP - 2 ( magenta ) DAPI ( blue ) treated with TTX for 24 h or left untreated . on bottom panels show no in absence of puromycin treatment or when omitting SP antibody . Scale bars : 30 µm ( large view ) , 5 µm ( insets ) .

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

(A) Micrographs showing puro-PLA staining of newly synthesized SP (green) in neurons immunostained for MAP-2 (magenta) and DAPI (blue) and treated with TTX for 24 h or left untreated. The images on bottom panels show no staining in the absence of puromycin treatment or when omitting SP primary antibody. Scale bars: 30 µm (large view), 5 µm (insets).


( C ) Density of SP puro - PLA , normalized to untreated condition ( UT ) ( UT : n = 62 ; TTX : n = 57 , n represents number of , from 3 cultures ) . * P < 0 . 05 ( Mann - Whitney ) . ( D ) SP puro - PLA cluster normalized to untreated condition ( UT ) ( UT : n = 62 ; TTX : n = 57 , n indicates number of , from 3 cultures ) . * * * * P < 0 . 0001 ( Mann - Whitney ) . Data information : Data are represented as mean ± .

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

(C) Density of SP puro-PLA puncta, normalized to the untreated condition (UT) (UT: n = 62; TTX: n = 57, n represents the number of cells, from 3 cultures). *P < 0.05 (Mann-Whitney test). (D) SP puro-PLA cluster fluorescence intensity normalized to the untreated condition (UT) (UT: n = 62; TTX: n = 57, n indicates the number of cells, from 3 cultures). ****P < 0.0001 (Mann-Whitney test). Data information: Data are represented as mean ± SEM.


( E ) showing puro - PLA of newly synthesized SP ( green ) in neurons transfected with Homer1c - GFP ( magenta ) treated with TTX for 24 h or left untreated . Scale bar : 5 µm . Arrowheads indicate puro - PLA + spines . ( F ) of SP puro - PLA + synapses ( UT , n = 26 , TTX , n = 33 ; n indicates number of , from 2 cultures ) . * * P < 0 . 01 , ns , not significant ( Mann - Whitney ) . ( G ) Homer1c - GFP integrated at SP puro - PLA - vs SP puro - PLA + synapses ( n = 32 , from 2 cultures ) . * * * * P < 0 . 0001 , ns , not significant ( Wilcoxon matched - pairs signed rank ) . Data information : Data are represented as mean ± .

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

(E) Micrographs showing puro-PLA staining of newly synthesized SP (green) in neurons transfected with Homer1c-GFP (magenta) and treated with TTX for 24 h or left untreated. Scale bar: 5 µm. Arrowheads indicate puro-PLA+ spines. (F) Percentage of SP puro-PLA+ synapses (UT, n = 26, TTX, n = 33; n indicates the number of cells, from 2 cultures). **P < 0.01, ns, not significant (Mann-Whitney test). (G) Homer1c-GFP integrated fluorescence intensity at SP puro-PLA- vs SP puro-PLA+ synapses (n = 32 cells, from 2 cultures). ****P < 0.0001, ns, not significant (Wilcoxon matched-pairs signed rank test). Data information: Data are represented as mean ± SEM.


( A ) Confocal showing dendrites from CA3 pyramidal transfected with RFP - SP ( green ) containing wild - type ( WT ) or mutated ( MUT ) 3 ' UTR + SP - shRNA - BFP ( magenta ) . Arrowheads indicate SP + spines . Scale bar : 10 µm . ( B ) Graph plot showing spine head volume vs SP integrated ( n = 5 ) . Each plot represents a single spine for which SP integrated has been normalized to average SP of SP + spines in same neuron . Plots for SP - SP + spines appear in grey magenta , respectively . black dotted represents linear regression ( R2 = 0 . 25 , P = 0 . 0004 ) . ( C ) Paired data showing spine head volume for SP - vs SP + spines . Each pair of plot represents average values for SP - vs SP + spines for a given neuron . * P = 0 . 0031 ( two - Student t ) . Data information : Data are represented as mean ± .

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

(A) Confocal images showing dendrites from CA3 pyramidal cells transfected with RFP-SP (green) containing wild-type (WT) or mutated (MUT) 3'UTR + SP-shRNA-BFP (magenta). Arrowheads indicate SP+ spines. Scale bar: 10 µm. (B) Graph plot showing spine head volume vs SP integrated intensity (n = 5 cells). Each plot represents a single spine for which SP integrated intensity has been normalized to average SP intensity of SP+ spines in the same neuron. Plots for SP- and SP+ spines appear in grey and magenta, respectively. The black dotted line represents linear regression (R2 = 0.25, P = 0.0004). (C) Paired data showing spine head volume for SP- vs SP+ spines. Each pair of plot represents average values for SP- vs SP+ spines for a given neuron. *P = 0.0031 (two-tailed Student t test). Data information: Data are represented as mean ± SEM.


( F ) Higher magnification of dendritic spines from neurons transfected with rescue RFP - SP ( magenta ) containing wild - type ( WT ) or mutated ( MUT ) 3 ' UTR BFP - shRNA - SP ( grey ) contacted by GFP - Syp + terminals ( green ) . Arrowheads indicate putative synaptic contacts . Scale bar : 5 μm . ( G ) of SP + spines in neurons expressing RFP - SP - 3 ' UTR - WT vs - MUT depending on apposition or not to a GFP - Syp + terminal ( SP - 3 ' UTR - WT : TetTx - , n = 220 , TetTx + , n = 26 ; SP - 3 ' UTR - MUT : TetTx - , n = 616 , TetTx + , n = 25 ; n indicates number of spines from 5 - 7 pairs ) . ( H ) Spine head volume in same conditions as in ( G ) ( SP - 3 ' UTR - WT : TetTx - , n = 26 , TetTx + , n = 26 spines from 5 pairs , 4 slices ; SP - 3 ' UTR - MUT : TetTx - , n = 25 , TetTx + , n = 25 ; n indicates number of spines from 7 pairs , 7 slices ) . * * * P < 0 . 001 , * P < 0 . 05 , ns , not significant ( Kruskall - Wallis followed by Dunn ' s multiple ) . ( I ) Cumulative probability distributions of spine head volumes for TetTx - TetTx + spines from neurons expressing RFP - SP - 3 ' UTR - WT ( SP - WT , gray green ) or RFP - SP - 3 ' UTR - MUT ( SP - MUT , gray green ) . Data information : Data are represented as mean ± .

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

(F) Higher magnification of dendritic spines from neurons transfected with rescue RFP-SP (magenta) containing wild-type (WT) or mutated (MUT) 3'UTR and BFP-shRNA-SP (grey) and contacted by GFP-Syp+ terminals (green). Arrowheads indicate putative synaptic contacts. Scale bar: 5 μm. (G) Percentage of SP+ spines in neurons expressing RFP-SP-3'UTR-WT vs -MUT and depending on the apposition or not to a GFP-Syp+ terminal (SP-3'UTR-WT: TetTx-, n = 220, TetTx+, n = 26; SP-3'UTR-MUT: TetTx-, n = 616, TetTx+, n = 25; n indicates the number of spines from 5-7 pairs). (H) Spine head volume in same conditions as in (G) (SP-3'UTR-WT: TetTx-, n = 26, TetTx+, n = 26 spines from 5 pairs, 4 slices; SP-3'UTR-MUT: TetTx-, n = 25, TetTx+, n = 25; n indicates the number of spines from 7 pairs, 7 slices). ***P < 0.001, *P < 0.05, ns, not significant (Kruskall-Wallis test followed by Dunn's multiple test). (I) Cumulative probability distributions of spine head volumes for TetTx- and TetTx+ spines from neurons expressing RFP-SP-3'UTR-WT (SP-WT, light gray and light green) or RFP-SP-3'UTR-MUT (SP-MUT, dark gray and dark green). Data information: Data are represented as mean ± SEM.


( c ) Intravascular endothelial interactions transmigration of neutrophils ( Ly - 6G + CX3CR - 1 - ; red ) cMOs ( Ly - 6G - CX3CR - 1low ; green ) to perivascular tissue as assessed 6 h after intrascrotal stimulation with recombinant murine uPA , PAI - 1 , or uPA - PAI - 1 in postcapillary venules of cremaster of CX3CR - 1GFP / + mice by multi - channel in vivo microscopy . Representative still ( scale bar : 50 : µm ) quantitative data are shown ( mean ± for n = 4 mice per group ; # p < 0 . 05 vs . saline ; * p < 0 . 05 vs . uPA ; § p < 0 . 05 vs . PAI - 1 ; One - way ANOVA ) .

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

(c) Intravascular endothelial cell interactions and transmigration of neutrophils (Ly-6G+ CX3CR-1-; red) and cMOs (Ly-6G- CX3CR-1low; green) to the perivascular tissue as assessed 6 h after intrascrotal stimulation with recombinant murine uPA, PAI-1, or uPA-PAI-1 in postcapillary venules of the cremaster of CX3CR-1GFP/+ mice by multi-channel in vivo microscopy. Representative still images (scale bar: 50: µm) and quantitative data are shown (mean±SEM for n=4 mice per group; #p<0.05 vs. saline; *p<0.05 vs. uPA; §p<0.05 vs. PAI-1; One-way ANOVA).


( b ) Expression of PECAM - 1 / CD31 ( red ) ICAM - 1 / CD54 or VCAM - 1 / CD106 ( green ) in cremaster muscle of WT mice as assessed ex vivo by confocal scanning microscopy 6 h after intrascrotal injection of recombinant murine uPA - PAI - 1 or saline , representative ( scale bar : 50 µm ) quantitative data are shown ( mean ± for n = 4 mice per group ; # p < 0 . 05 vs . saline ; t ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O 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) Expression of PECAM-1/CD31 (red) and ICAM-1/CD54 or VCAM-1/CD106 (green) in the cremaster muscle of WT mice as assessed ex vivo by confocal laser scanning microscopy 6 h after intrascrotal injection of recombinant murine uPA-PAI-1 or saline, representative images (scale bar: 50 µm) and quantitative data are shown (mean±SEM for n=4 mice per group; #p<0.05 vs. saline; t test).


( g ) Quantitative of LFA - 1 / CD11a clustering on surface of mouse neutrophils under increasing flow conditions in vitro in presence of either E - selectin / CD62E + ICAM - 1 / CD54 , E - selectin / CD62E + ICAM - 1 / CD54 + KC , or E - selectin / CD62E + ICAM - 1 / CD54 + KC + uPA - PAI - 1 as assessed by spinning disk confocal microscopy , representative quantitative data are shown ( scale bar : 10 μm ; mean ± SD ; n = 3 mice ; n = 10 - 13 flow chambers , n = 122 - 222 ) ; * p < 0 . 05 vs . E - selectin / CD62E + ICAM - 1 / CD54 ; One - way ANOVA ) .

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

(g) Quantitative analysis of LFA-1/CD11a clustering on the surface of mouse neutrophils under increasing flow conditions in vitro in the presence of either E-selectin/CD62E + ICAM-1/CD54, E-selectin/CD62E + ICAM-1/CD54 + KC, or E-selectin/CD62E + ICAM-1/CD54 + KC + uPA-PAI-1 as assessed by spinning disk confocal microscopy, representative images and quantitative data are shown (scale bar: 10 μm; mean±SD; n=3 mice; n=10-13 flow chambers, n =122-222 cells); *p<0.05 vs. E-selectin/CD62E + ICAM-1/CD54; One-way ANOVA).


( b ) Correlation analyses of expression of uPA ( PLAU gene ) , PAI - 1 ( SERPINE1 gene ) , neutrophil marker gene FPR1 , overall survival of breast cancer ( stages 0 1 ) with high low PLAU or SERPINE1 gene expression ( cut - off z ≥ 2 . 0 ) in METABRIC breast cancer cohort .

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(b) Correlation analyses of RNA expression levels of uPA (PLAU gene), PAI-1 (SERPINE1 gene), and the neutrophil marker gene FPR1, and overall survival of breast cancer patients (stages 0 and 1) with high and low PLAU or SERPINE1 gene expression levels (cut-off z≥ 2.0) in the METABRIC breast cancer cohort.


( c ) Surface expression of MMP - 9 , VEGF , NE as assessed on circulating neutrophils isolated from peripheral blood of WT mice ( saline ) or from peritoneal cavity of WT mice 6 h after intraperitoneal stimulation with uPA - PAI - 1 ( uPA - PAI - 1 ) by multi - channel flow cytometry , quantitative data are shown ( mean ± for n = 4 - 6 mice per group ; # p < 0 . 05 vs . saline ; t ) .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O 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) Surface expression of MMP-9, VEGF, and NE as assessed on circulating neutrophils isolated from the peripheral blood of WT mice (saline) or from the peritoneal cavity of WT mice 6 h after intraperitoneal stimulation with uPA-PAI-1 (uPA-PAI-1) by multi-channel flow cytometry, quantitative data are shown (mean±SEM for n=4-6 mice per group; #p<0.05 vs. saline; t test).


( f ) Formation of NETs ( histone H3 + ; green ) as assessed ex vivo by confocal microscopy in cremaster muscle of WT mice 6 h after intra - scrotal injection of uPA - PAI - 1 , TNF , or saline , PECAM - 1 / CD31 + postcapillary venules ( blue ) Ly - 6G + neutrophils ( red ) are depicted . Representative are shown ( scale bar : 50 µm ) .

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(f) Formation of NETs (histone H3+; green) as assessed ex vivo by confocal microscopy in the cremaster muscle of WT mice 6 h after intra-scrotal injection of uPA-PAI-1, TNF, or saline, PECAM-1/CD31+ postcapillary venules (blue) and Ly-6G+ neutrophils (red) are depicted. Representative images are shown (scale bar: 50 µm).


( a ) Effect of compound WX - 340 on binding of recombinant murine uPA to PAI - 1 as assessed by ELISA , quantitative data are shown ( mean ± for n = 3 experiments per group ; # p < 0 . 05 vs . drug vehicle ; One - way ANOVA ) .

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(a) Effect of compound WX-340 on binding of recombinant murine uPA to PAI-1 protein as assessed by ELISA, quantitative data are shown (mean±SEM for n=3 experiments per group; #p<0.05 vs. drug vehicle; One-way ANOVA).


( d ) Relative development rates in treated with WX - 340 a priori or therapeutically after 1 week after injection on a daily basis as assessed in an orthotopic model of 4T1 breast cancer in WT mice ( mean ± for n = 4 - 6 mice per group ; * p < 0 . 05 vs . drug vehicle ; One - way ANOVA ) .

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(d) Relative development rates and tumor weight in animals treated with WX-340 a priori or therapeutically after 1 week after tumor cell injection on a daily basis as assessed in an orthotopic model of 4T1 breast cancer in WT mice (mean±SEM for n=4-6 mice per group; *p<0.05 vs. drug vehicle; One-way ANOVA).