( D ) Bar graph of the relative level of EGFP - Exo70 at the plasma membrane versus cytoplasm in MECs ligated to rBM in 2D or 3D in the absence and presence of blebbistatin ( Bleb ) . Membrane localization of EGFP - Exo70 was quantified as a ratio of plasma membrane to cytoplasmic fluorescence ( mean ± SEM ; 2D , n = 59 ; 3D , n = 60 ; 2D + Bleb , n = 57 ; 3D + Bleb , n = 54 cells from three independent experiments ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD . * * * * P < 0 . 0001 .

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

(D) Bar graph of the relative level of EGFP-Exo70 at the plasma membrane versus cytoplasm in MECs ligated to rBM in 2D or 3D in the absence and presence of blebbistatin (Bleb). Membrane localization of EGFP-Exo70 was quantified as a ratio of plasma membrane to cytoplasmic fluorescence (mean ± SEM; 2D, n= 59; 3D, n=60; 2D + Bleb, n=57; 3D + Bleb, n=54 cells from three independent experiments). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. ****P < 0.0001.


( E ) 30 - nm thick slices through a cellular tomogram focusing on subplasmalemmal regions enriched with membrane - encased compartments of vitrified MECs cultured on rBM treated with ( 2D , right panel ) or without ( 2D + Bleb , left panel ) blebbistatin . The position of the cell body is towards the upper right - hand corners ( marked with white asterisk ) . Scale bars , 200 nm .

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

(E) 30-nm thick slices through a cellular tomogram focusing on subplasmalemmal regions enriched with membrane-encased compartments of vitrified MECs cultured on rBM treated with (2D, right panel) or without (2D+Bleb, left panel) blebbistatin. The position of the cell body is towards the upper right-hand corners (marked with white asterisk). Scale bars, 200 nm.


( F ) Quantification of the number of membrane - enclosed compartments ( vesicles ) proximal to the plasma membrane that were classified as either ' filled ' ( black ) or ' empty ' ( white ) , based on the absence or presence of encapsulated macromolecules . Membrane - enclosed compartments were independently classified by three cryo - EM experts using 28 tomograms per condition . The resulting standard deviation for assigning ' empty ' versus ' filled ' was 10 . 3 % ( n = 3 independent expert classifications ) . Statistical significance of differences between the distributions was assessed using Mann - Whitney rank tests . * * * * P < 0 . 0001 .

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

(F) Quantification of the number of membrane-enclosed compartments (vesicles) proximal to the plasma membrane that were classified as either 'filled' (black) or 'empty' (white), based on the absence or presence of encapsulated macromolecules. Membrane-enclosed compartments were independently classified by three cryo-EM experts using 28 tomograms per condition. The resulting standard deviation for assigning 'empty' versus 'filled' was 10.3% (n=3 independent expert classifications). Statistical significance of differences between the distributions was assessed using Mann-Whitney rank tests. ****P < 0.0001.


( H ) Volcano plot showing differentially associated proteins in MECs ligated to rBM in 2D treated with vehicle ( DMSO ; blue ; log 2 > 0 . 5 ; high cortical actin tension ) versus blebbistatin ( Bleb ; red ; log 2 > 0 . 5 ; low cortical actin tension ) . Differentially expressed genes with an adjusted p - value < 0 . 1 and log2 ( fold change ) > 0 . 5 are shown ( n = 2 biological replicates ) .

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

(H) Volcano plot showing differentially associated proteins in MECs ligated to rBM in 2D treated with vehicle (DMSO; blue; log 2> 0.5; high cortical actin tension) versus blebbistatin (Bleb; red; log 2> 0.5; low cortical actin tension). Differentially expressed genes with an adjusted p-value<0.1 and log2 (fold change) > 0.5 are shown (n=2 biological replicates).


( J ) Representative immunofluorescence microscopy images of MECs ligated to rBM in 2D , 3D , or 2D treated with blebbistatin ( 2D + Bleb ) and immunostained with antibodies targeting ANXA2 or S100A14 ( green ) . Cellular F - actin was counterstained using phalloidin ( red ) and actin - rich protrusions are highlighted with yellow arrows . Fluorescent images of whole cells ( left ) and actin - rich protrusions containing ANXA2 or S100A14 are magnified in the inset ( right ) . The edge of the actin cortex in MECs ligated to rBM in 2D was marked with a yellow dashed line . Scale bar ( whole cell ) , 5 μm ; Scale bar ( magnified insets ) , 1 μm .

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 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

(J) Representative immunofluorescence microscopy images of MECs ligated to rBM in 2D, 3D, or 2D treated with blebbistatin (2D+ Bleb) and immunostained with antibodies targeting ANXA2 or S100A14 (green). Cellular F-actin was counterstained using phalloidin (red) and actin-rich protrusions are highlighted with yellow arrows. Fluorescent images of whole cells (left) and actin-rich protrusions containing ANXA2 or S100A14 are magnified in the inset (right). The edge of the actin cortex in MECs ligated to rBM in 2D was marked with a yellow dashed line. Scale bar (whole cell), 5 μm; Scale bar (magnified insets), 1 μm.


( A ) Graph of the percent cell death measured in MCF10A MECs ligated to rBM in 2D and 3D ( left ) and in 2D treated with blebbistatin ( 2D + Bleb ) 48h post - plating ( right ) that was quantified using calcein AM and ethidium homodimer staining ( mean ± SEM ; 2D , n = 3 ; 3D , n = 3 independent experiments ; 2D + DMSO , n = 5 ; 2D + Bleb , n = 5 independent experiments ) . 2D versus 3D , * * * P = 0 . 0010 , 2D versus 2D + Bleb , * * P = 0 . 0248 ( Student ' s t test ) .

O O O O 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 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 O O 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

(A) Graph of the percent cell death measured in MCF10A MECs ligated to rBM in 2D and 3D (left) and in 2D treated with blebbistatin (2D+Bleb) 48h post-plating (right) that was quantified using calcein AM and ethidium homodimer staining (mean ± SEM; 2D, n= 3; 3D, n=3 independent experiments; 2D + DMSO, n = 5; 2D + Bleb, n=5 independent experiments). 2D versus 3D, ***P = 0.0010, 2D versus 2D + Bleb, **P = 0.0248 (Student's t test).


( B ) Bar graph of the percent cell death measured in MCF10A MECs stably expressing shRNAs targeting Luciferase ( Luc ) , filamin ( FLMN ) or actinin ( ACTN ) and ligated to rBM in 2D . Cell death was assessed at 48h post - plating via calcein AM and ethidium homodimer staining ( mean ± SEM ; n = 3 independent biological replicates ) . * * P = 0 . 0034 ; ns = not significant ( Student ' s t test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bar graph of the percent cell death measured in MCF10A MECs stably expressing shRNAs targeting Luciferase (Luc), filamin (FLMN) or actinin (ACTN) and ligated to rBM in 2D. Cell death was assessed at 48h post-plating via calcein AM and ethidium homodimer staining (mean ± SEM; n =3 independent biological replicates). **P = 0.0034; ns= not significant (Student's t test).


( C ) Bar graph of the percent cell death in MCF10A MECs expressing luciferase control or filamin repeats 21 - 23 ( FLMNIg21 - Ig23 ) that were ligated with rBM in 2D . Cell death was assessed at 48h post - plating via calcein AM and ethidium homodimer staining ( mean ± SEM ; n = 3 independent biological replicates ) . * P = 0 . 0489 ( Student ' s t test ) .

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

(C) Bar graph of the percent cell death in MCF10A MECs expressing luciferase control or filamin repeats 21-23 (FLMNIg21-Ig23) that were ligated with rBM in 2D. Cell death was assessed at 48h post-plating via calcein AM and ethidium homodimer staining (mean ± SEM; n =3 independent biological replicates). *P = 0.0489 (Student's t test).


( D ) Bar graph of the levels of endogenous Exo70 in MECs stably expressing shRNA against Exo70 or luciferase ( Luc ) ( mean ± SEM ; n = 3 independent biological replicates ) . * * p = 0 . 0086 .

O O O O O O O O O O O O O O O O O O O O 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) Bar graph of the levels of endogenous Exo70 in MECs stably expressing shRNA against Exo70 or luciferase (Luc) (mean ± SEM; n =3 independent biological replicates). ** p = 0.0086.


( E ) Bar graph of the percent cell death measured in MECs expressing shRNA targeting Exo70 ( shExo70 ) or luciferase ( control ) and ligated with rBM in 2D or 3D . Cell death was assessed at 48h post - plating via calcein AM and ethidium homodimer staining ( mean ± SEM ; n = 3 independent biological replicates ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD . 2D versus 3D , * * * P = 0 . 0004 . 3D + shLuc versus 3D + shExo70 , * * P = 0 . 0091 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bar graph of the percent cell death measured in MECs expressing shRNA targeting Exo70 (shExo70) or luciferase (control) and ligated with rBM in 2D or 3D. Cell death was assessed at 48h post-plating via calcein AM and ethidium homodimer staining (mean ± SEM; n=3 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. 2D versus 3D, ***P = 0.0004. 3D + shLuc versus 3D + shExo70, **P = 0.0091.


( F ) Spheroids were induced to express constitutively active ROCK or left uninduced ( control ) and co - stained with antibodies for laminin or S100A14 ( green ) , phalloidin ( red ) and DAPI ( blue ) . Scale bar , 10 μm .

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

(F) Spheroids were induced to express constitutively active ROCK or left uninduced (control) and co-stained with antibodies for laminin or S100A14 (green), phalloidin (red) and DAPI (blue). Scale bar, 10 μm.


( G ) Representative immunoblots of phosphorylated EIF2a ( pEIF2a ) , total EIF2a and alpha - tubulin in cell lysate from MCF10A MEC spheroids induced to express constitutively active ROCK or left uninduced ( control ) . Corresponding quantification data are plotted at bottom ( mean ± SEM ; n = 3 independent biological replicates ) . * P = 0 . 0442 ( Student ' s t test ) .

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

(G) Representative immunoblots of phosphorylated EIF2a (pEIF2a), total EIF2a and alpha-tubulin in cell lysate from MCF10A MEC spheroids induced to express constitutively active ROCK or left uninduced (control). Corresponding quantification data are plotted at bottom (mean ± SEM; n=3 independent biological replicates). *P = 0.0442 (Student's t test).


( H ) Spheroids were induced to express constitutively active ROCK or left uninduced ( control ) , treated with Paclitaxel ( 2nM ) , lysed and immunoblotted for cleaved caspase - 3 and alpha - tubulin ( top ) . In parallel , spheroids were stained with antibody and phalloidin to evaluate the levels of caspase - 3 ( green ) and actin organization ( red ) , respectively . Scale bar , 10 μm .

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

(H) Spheroids were induced to express constitutively active ROCK or left uninduced (control), treated with Paclitaxel (2nM), lysed and immunoblotted for cleaved caspase-3 and alpha-tubulin (top). In parallel, spheroids were stained with antibody and phalloidin to evaluate the levels of caspase-3 (green) and actin organization (red), respectively. Scale bar, 10 μm.


( I ) Bar graph of the relative mRNA levels of ER stress response genes including ATF3 , STC2 , PPP1R15A , and PMAIP1 in microarray analyses of TRAIL treated HMT - 3522 S - 1 MECs plated as monolayers on a rigid rBM ( 2D ) or as spheroids ( 3D ) ( mean ± SEM ; n = 3 independent experiments ) . ATF3 , * * * P = 0 . 0006 ; STC2 , * P = 0 . 0353 ; PPP1R15A , * P = 0 . 0254 ; PMAIP1 , * P = 0 . 0127 ( Student ' s t test ) .

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

(I) Bar graph of the relative mRNA levels of ER stress response genes including ATF3, STC2, PPP1R15A, and PMAIP1 in microarray analyses of TRAIL treated HMT-3522 S-1 MECs plated as monolayers on a rigid rBM (2D) or as spheroids (3D) (mean ± SEM; n=3 independent experiments). ATF3, ***P = 0.0006; STC2, *P = 0.0353; PPP1R15A, *P = 0.0254; PMAIP1, *P = 0.0127 (Student's t test).


A - D Heat maps of the occurrence frequency of effective variants within each genotype in DENV1 ( A ) , DENV2 ( B ) , DENV3 ( C ) , and DENV4 ( D ) . Residues with the occurrence frequency < 20 % before 1995 were shown in the heat maps . Red square frames represent ten stable substitutions , including 171T in DENV1 - I ( A ) , 131Q , 170T , 340T , and 380V in DENV2 - Asian American ( B ) , 226K and 228E in DENV2 - Asian I ( B ) , 301S and 377I in DENV3 - I ( C ) , and 265A in DENV4 - I ( D ) . Gray boxes represent the data that were not available .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Heat maps of the occurrence frequency of effective variants within each genotype in DENV1 (A), DENV2 (B), DENV3 (C), and DENV4 (D). Residues with the occurrence frequency <20% before 1995 were shown in the heat maps. Red square frames represent ten stable substitutions, including 171T in DENV1-I (A), 131Q, 170T, 340T, and 380V in DENV2-Asian American (B), 226K and 228E in DENV2-Asian I (B), 301S and 377I in DENV3-I (C), and 265A in DENV4-I (D). Gray boxes represent the data that were not available.


E - H Growth kinetics and plaque morphology DENV2 - Asian I 16681 strain ( 16681 , U87411 ) ( E ) , DENV2 - Asian American DENV2 / US / BID - V1164 / 1986 strain ( BID , EU482568 ) ( F ) , DENV3 - I PF89 / 27643 strain ( PF89 , AY744677 ) ( G ) , and DENV4 - I H241 strain ( H241 , AY947539 ) ( H ) . Vero cells were infected with each mutant at a multiplicity of infection ( MOI ) of 0 . 01 , and the cell supernatant was collected at Days 1 , 3 , 5 , 7 , and 9 for the plaque assay . Data information : In ( E - H ) , data are presented as mean ± SEM of n = 4 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H Growth kinetics and plaque morphology DENV2-Asian I 16681 strain (16681, U87411) (E), DENV2-Asian American DENV2/US/BID-V1164/1986 strain (BID, EU482568) (F), DENV3-I PF89/27643 strain (PF89, AY744677) (G), and DENV4-I H241 strain (H241, AY947539) (H). Vero cells were infected with each mutant at a multiplicity of infection (MOI) of 0.01, and the cell supernatant was collected at Days 1, 3, 5, 7, and 9 for the plaque assay. Data information: In (E-H), data are presented as mean ± SEM of n = 4 biological replicates.


One - week - old mosquitoes were thoracically infected with DENV2 - Asian I ( A ) , DENV2 - Asian American ( B ) , DENV3 - I mutants , respectively . A . aegypti mosquitoes were infected intrathoracically with 10 p . f . u . of DENV . The DENV viral loads were assessed at 3 days postinfection via qRT - PCR and were normalized against A . aegypti actin . Data information : one dot represents one mosquito , and the horizontal lines represent the median of the results . data are presented as mean ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . , not significant ( p > 0 . 05 ) .

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

One-week-old mosquitoes were thoracically infected with DENV2-Asian I (A), DENV2-Asian American (B), DENV3-I mutants, respectively. A. aegypti mosquitoes were infected intrathoracically with 10 p.f.u. of DENV. The DENV viral loads were assessed at 3 days postinfection via qRT-PCR and were normalized against A. aegypti actin. Data information: one dot represents one mosquito, and the horizontal lines represent the median of the results. data are presented as mean ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).


One - week - old mosquitoes were thoracically infected with DENV4 - I ( D ) mutants , respectively . A . aegypti mosquitoes were infected intrathoracically with 10 p . f . u . of DENV . The DENV viral loads were assessed at 3 days postinfection via qRT - PCR and were normalized against A . aegypti actin . Data information : one dot represents one mosquito , and the horizontal lines represent the median of the results . data are presented as mean ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . , not significant ( p > 0 . 05 ) .

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

One-week-old mosquitoes were thoracically infected with DENV4-I (D) mutants, respectively. A. aegypti mosquitoes were infected intrathoracically with 10 p.f.u. of DENV. The DENV viral loads were assessed at 3 days postinfection via qRT-PCR and were normalized against A. aegypti actin. Data information: one dot represents one mosquito, and the horizontal lines represent the median of the results. data are presented as mean ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).


Four - week - old AG6 mice were infected with DENV ( 2 , 500 p . f . u . ) by footpad inoculation . Blood samples were collected for DENV viremia detection from Day 1 to Day 7 . The viral loads from AG6 mice infected with DENV2 - Asian I ( E ) , were measured by qRT - PCR and were normalized against mouse GAPDH . Data information : n = 6 biological replicates . data are presented as mean ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . , not significant ( p > 0 . 05 ) .

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

Four-week-old AG6 mice were infected with DENV (2,500 p.f.u.) by footpad inoculation. Blood samples were collected for DENV viremia detection from Day 1 to Day 7. The viral loads from AG6 mice infected with DENV2-Asian I (E), were measured by qRT-PCR and were normalized against mouse GAPDH. Data information: n = 6 biological replicates. data are presented as mean ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).


Four - week - old AG6 mice were infected with DENV ( 2 , 500 p . f . u . ) by footpad inoculation . Blood samples were collected for DENV viremia detection from Day 1 to Day 7 . The viral loads from AG6 mice infected with DENV2 - Asian American ( F ) , DENV3 - I ( G ) were measured by qRT - PCR and were normalized against mouse GAPDH . Data information : , n = 6 biological replicates . In ( F ) , n = 5 biological replicates . data are presented as mean ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . , not significant ( p > 0 . 05 ) .

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

Four-week-old AG6 mice were infected with DENV (2,500 p.f.u.) by footpad inoculation. Blood samples were collected for DENV viremia detection from Day 1 to Day 7. The viral loads from AG6 mice infected with DENV2-Asian American (F), DENV3-I (G) were measured by qRT-PCR and were normalized against mouse GAPDH. Data information: , n = 6 biological replicates. In (F), n = 5 biological replicates. data are presented as mean ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).


Four - week - old AG6 mice were infected with DENV ( 2 , 500 p . f . u . ) by footpad inoculation . Blood samples were collected for DENV viremia detection from Day 1 to Day 7 . The viral loads from AG6 mice infected with DENV4 - I ( H ) were measured by qRT - PCR and were normalized against mouse GAPDH . Data information : n = 6 biological replicates . data are presented as mean ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . , not significant ( p > 0 . 05 ) .

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

Four-week-old AG6 mice were infected with DENV (2,500 p.f.u.) by footpad inoculation. Blood samples were collected for DENV viremia detection from Day 1 to Day 7. The viral loads from AG6 mice infected with DENV4-I (H) were measured by qRT-PCR and were normalized against mouse GAPDH. Data information: n = 6 biological replicates. data are presented as mean ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).


I , J Infection of DENV2 - Asian I mutants on hu - moDC ( I ) and hu - moMØ ( J ) . The cells were infected with 0 . 01 MOI of the indicated viruses . The infected cells were detected at 48 h postinfection by qRT - PCR , and DENV viral loads were normalized against human GAPDH . Data information : n = 6 biological replicates . data are presented as mean ± SEM . The data were analyzed statistically using the unpaired t - test ( I , J ) . The P value represents a comparison between the mutants and corresponding WT within genotypes . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . , not significant ( p > 0 . 05 ) .

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

I, J Infection of DENV2-Asian I mutants on hu-moDC (I) and hu-moMØ (J). The cells were infected with 0.01 MOI of the indicated viruses. The infected cells were detected at 48 h postinfection by qRT-PCR, and DENV viral loads were normalized against human GAPDH. Data information: n = 6 biological replicates. data are presented as mean ± SEM. The data were analyzed statistically using the unpaired t-test (I, J). The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).


A The five - year occurrence frequency of residues in 226th and 228th sites from < 1995 to 2016 - 2020 .

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A The five-year occurrence frequency of residues in 226th and 228th sites from <1995 to 2016-2020.


Four - week - old AG6 mice were infected with 2 , 500 p . f . u . of viruses by footpad inoculation . The viremia of infected AG6 mice from Day 1 to Day 7 was measured using qRT - PCR . Data information : , n = 5 biological replicates . the data are presented as means ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

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Four-week-old AG6 mice were infected with 2,500 p.f.u. of viruses by footpad inoculation. The viremia of infected AG6 mice from Day 1 to Day 7 was measured using qRT-PCR. Data information: , n = 5 biological replicates. the data are presented as means ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).


Four - week - old AG6 mice were infected with 2 , 500 p . f . u . of viruses by footpad inoculation . Two days postinfection , infected mice were euthanized , and the viral loads in the bone marrow ( C ) were measured by qRT - PCR . Data information : n = 5 biological replicates . the data are presented as means ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

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

Four-week-old AG6 mice were infected with 2,500 p.f.u. of viruses by footpad inoculation. Two days postinfection, infected mice were euthanized, and the viral loads in the bone marrow (C) were measured by qRT-PCR. Data information: n = 5 biological replicates. the data are presented as means ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).


Four - week - old AG6 mice were infected with 2 , 500 p . f . u . of viruses by footpad inoculation . Two days postinfection , infected mice were euthanized , and the viral loads in the spleen ( D ) , and footpad ( E ) were measured by qRT - PCR . Data information : n = 5 biological replicates . the data are presented as means ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

Four-week-old AG6 mice were infected with 2,500 p.f.u. of viruses by footpad inoculation. Two days postinfection, infected mice were euthanized, and the viral loads in the spleen (D), and footpad (E) were measured by qRT-PCR. Data information: n = 5 biological replicates. the data are presented as means ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).


Four - week - old AG6 mice were infected with 2 , 500 p . f . u . of viruses by footpad inoculation . The survival rates of mice ( F ) were recorded daily . Data information : In ( F ) , n = 10 biological replicates . In ( F ) , the data were analyzed statistically using the log - rank ( Mantel - Cox ) test . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

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

Four-week-old AG6 mice were infected with 2,500 p.f.u. of viruses by footpad inoculation. The survival rates of mice (F) were recorded daily. Data information: In (F), n = 10 biological replicates. In (F), the data were analyzed statistically using the log-rank (Mantel-Cox) test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).


Mosquito infectivity was determined by qRT - PCR at 8 days after the blood meal . The number at the top of each column represents the infected number / total number ( H ) . Data information : In ( H one dot represents one mosquito , and the horizontal lines represent the median of the results . In ( H , the data were analyzed statistically using the Fisher ' s exact test . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 .

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

Mosquito infectivity was determined by qRT-PCR at 8 days after the blood meal. The number at the top of each column represents the infected number/total number (H). Data information: In (H one dot represents one mosquito, and the horizontal lines represent the median of the results. In (H, the data were analyzed statistically using the Fisher's exact test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05


Mosquitoes were thoracically infected with 10 p . f . u . of viruses . Ten days postinfection , five infected mosquitoes were allowed to bite one four - week - old AG6 mouse . From Day 1 to Day 8 , infected mice were subjected to daily biting by naive A . aegypti , and blood samples were collected for DENV2 viremia detection by qRT - PCR ( J ) . Data information : In ( J ) , n = 4 biological replicates . In J ) , the data are presented as means ± SEM . The data were analyzed statistically using the Mann - Whitney test J ) . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

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

Mosquitoes were thoracically infected with 10 p.f.u. of viruses. Ten days postinfection, five infected mosquitoes were allowed to bite one four-week-old AG6 mouse. From Day 1 to Day 8, infected mice were subjected to daily biting by naive A. aegypti, and blood samples were collected for DENV2 viremia detection by qRT-PCR (J). Data information: In (J), n = 4 biological replicates. In J), the data are presented as means ± SEM. The data were analyzed statistically using the Mann-Whitney test J). The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).


Mosquitoes were thoracically infected with 10 p . f . u . of viruses . Ten days postinfection , five infected mosquitoes were allowed to bite one four - week - old AG6 mouse . From Day 1 to Day 8 , infected mice were subjected to daily biting by naive A . aegypti , and Mosquitoes were collected at 8 days after the blood meal for qRT - PCR to compare the infection rate of DENV2 ( K Data information : In K , the data were analyzed statistically using the Fisher ' s exact test . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

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

Mosquitoes were thoracically infected with 10 p.f.u. of viruses. Ten days postinfection, five infected mosquitoes were allowed to bite one four-week-old AG6 mouse. From Day 1 to Day 8, infected mice were subjected to daily biting by naive A. aegypti, and Mosquitoes were collected at 8 days after the blood meal for qRT-PCR to compare the infection rate of DENV2 (K Data information: In K, the data were analyzed statistically using the Fisher's exact test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).


Mosquitoes were thoracically infected with 10 p . f . u . of viruses . Ten days postinfection , five infected mosquitoes were allowed to bite one four - week - old AG6 mouse . From Day 1 to Day 8 , infected mice were subjected to daily biting by naive A . aegypti , and Mosquitoes were collected at 8 days after the blood meal for qRT - PCR to compare the infection rate of DENV2 L ) . The number at the top of each column represents the infected number / total number ( L ) . Data information : In L ) , one dot represents one mosquito , and the horizontal lines represent the median of the results . In , L ) , the data were analyzed statistically using the Fisher ' s exact test . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

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

Mosquitoes were thoracically infected with 10 p.f.u. of viruses. Ten days postinfection, five infected mosquitoes were allowed to bite one four-week-old AG6 mouse. From Day 1 to Day 8, infected mice were subjected to daily biting by naive A. aegypti, and Mosquitoes were collected at 8 days after the blood meal for qRT-PCR to compare the infection rate of DENV2 L). The number at the top of each column represents the infected number/total number (L). Data information: In L), one dot represents one mosquito, and the horizontal lines represent the median of the results. In , L), the data were analyzed statistically using the Fisher's exact test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).


B The 293T cells were transfected with a pcDNA3 . 1 recombinant plasmid with a human DC - SIGN gene . The cells transfected by an empty pcDNA3 . 1 plasmid served as negative controls . Thirty - six hours after transfection , the cells were infected with DENV2 ( 0 . 01 MOI ) . The DENV2 viral loads were assessed at 48 h postinfection via qRT - PCR and were normalized against human GAPDH . Data information : In ( B ) , n = 6 biological replicates . In ( B , the data are presented as means ± SEM and were analyzed statistically using an unpaired t - test . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

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B The 293T cells were transfected with a pcDNA3.1 recombinant plasmid with a human DC-SIGN gene. The cells transfected by an empty pcDNA3.1 plasmid served as negative controls. Thirty-six hours after transfection, the cells were infected with DENV2 (0.01 MOI). The DENV2 viral loads were assessed at 48 h postinfection via qRT-PCR and were normalized against human GAPDH. Data information: In (B), n = 6 biological replicates. In (B, the data are presented as means ± SEM and were analyzed statistically using an unpaired t-test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).


C hu - moDCs were infected with 0 . 01 MOI of DENV2 . The infected cells were detected at 48 h postinfection by qRT - PCR . Data information : In ( C ) , n = 5 biological replicates . In C ) , the data are presented as means ± SEM and were analyzed statistically using an unpaired t - test . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

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C hu-moDCs were infected with 0.01 MOI of DENV2. The infected cells were detected at 48 h postinfection by qRT-PCR. Data information: In (C), n = 5 biological replicates. In C), the data are presented as means ± SEM and were analyzed statistically using an unpaired t-test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).


D The binding affinity between DC - SIGN and purified DENV2 virions measured by SPR . E The binding affinity between DC - SIGN ( R309A / R312A ) and purified DENV2 virions measured by SPR .

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D The binding affinity between DC-SIGN and purified DENV2 virions measured by SPR. E The binding affinity between DC-SIGN (R309A/R312A) and purified DENV2 virions measured by SPR.


G The binding affinity between mosGCTL - AAEL011408 and purified DENV2 virions measured by SPR .

G The binding affinity between mosGCTL-AAEL011408 and purified DENV2 virions measured by SPR.


B The AOF prediction of the T226K substitution ( left panel ) and the G228E substitution ( right panel ) in the DENV2 Asian I genotype .

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B The AOF prediction of the T226K substitution (left panel) and the G228E substitution (right panel) in the DENV2 Asian I genotype.


C The AOF prediction of the T226K substitution ( left panel ) and the G228E substitution ( right panel ) in the DENV2 Cosmopolitan genotype .

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C The AOF prediction of the T226K substitution (left panel) and the G228E substitution (right panel) in the DENV2 Cosmopolitan genotype.


The performance of the GPR - 2 in modeling the prevalence of the DENV2 Asian I genotype .

The performance of the GPR-2 in modeling the prevalence of the DENV2 Asian I genotype.


E The prevalence of the DENV2 Cosmopolitan genotype predicted by the GPR - 2 model ( orange dashed line + squares ) . The orange shadow denotes the predicted prevalence range of the DENV2 Cosmopolitan genotype , with the AOF of T226K / G228E substitutions predicted by the numerical kinetics , GPR - 1 , and support vector machine ( SVR ) models serving as the GPR - 2 input data .

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E The prevalence of the DENV2 Cosmopolitan genotype predicted by the GPR-2 model (orange dashed line + squares). The orange shadow denotes the predicted prevalence range of the DENV2 Cosmopolitan genotype, with the AOF of T226K/G228E substitutions predicted by the numerical kinetics, GPR-1, and support vector machine (SVR) models serving as the GPR-2 input data.


Schematic of single ‐ plane acquisition FRAP on endogenously tagged Swi6 ‐ EGFP . Representative still images of a FRAP experiment are shown . See also Movie EV1 . Green sphere : yeast nucleus containing Swi6 ‐ EGFP ; green dots : heterochromatic Swi6 ‐ EGFP ; red circle : bleached area .

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Schematic of single‐plane acquisition FRAP on endogenously tagged Swi6‐EGFP. Representative still images of a FRAP experiment are shown. See also Movie EV1. Green sphere: yeast nucleus containing Swi6‐EGFP; green dots: heterochromatic Swi6‐EGFP; red circle: bleached area.


Normalized average intensities ( black dots ) of FRAP on Swi6 ‐ EGFP heterochromatic loci ( n = 31 ) fitted to a one ‐ component model ( blue line ) . The dashed lines indicate the final relative intensity that is set to 1 and the fluorescence half ‐ recovery time ( t 1 / 2 ) .

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Normalized average intensities (black dots) of FRAP on Swi6‐EGFP heterochromatic loci (n = 31) fitted to a one‐component model (blue line). The dashed lines indicate the final relative intensity that is set to 1 and the fluorescence half‐recovery time (t 1/2).


Fluorescence t 1 / 2 values of FRAP on Swi6 ‐ EGFP heterochromatic loci . The box bounds the interquartile range ( IQR ) divided by the median , and whiskers extend to a maximum of 1 . 5 × IQR beyond the box .

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Fluorescence t 1/2 values of FRAP on Swi6‐EGFP heterochromatic loci. The box bounds the interquartile range (IQR) divided by the median, and whiskers extend to a maximum of 1.5 × IQR beyond the box.


Normalized fluorescence recovery values of FRAP on Swi6 ‐ EGFP heterochromatic loci . The box bounds the interquartile range ( IQR ) divided by the median , and whiskers extend to a maximum of 1 . 5 × IQR beyond the box .

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Normalized fluorescence recovery values of FRAP on Swi6‐EGFP heterochromatic loci. The box bounds the interquartile range (IQR) divided by the median, and whiskers extend to a maximum of 1.5 × IQR beyond the box.


FLIP experiment showing a bleached ( FRAP , red circle ) and unbleached , adjacent locus ( FLIP , blue circle ) in the same nucleus . The graph displays average relative fluorescence of FLIP ( blue ) , FRAP ( red ) , and control loci ( no bleach , green ) in other non ‐ bleached cells .

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FLIP experiment showing a bleached (FRAP, red circle) and unbleached, adjacent locus (FLIP, blue circle) in the same nucleus. The graph displays average relative fluorescence of FLIP (blue), FRAP (red), and control loci (no bleach, green) in other non‐bleached cells.


Schematic of a line ‐ scan FRAP experiment . Fluorescence intensities are measured along a line of a non ‐ bleached nucleus ( Ctrl nucleus ) and a bleached nucleus ( FRAP nucleus ) in Swi6 ‐ EGFP clr4 ∆ cells . Red box : bleached region . Right : raw data example .

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Schematic of a line‐scan FRAP experiment. Fluorescence intensities are measured along a line of a non‐bleached nucleus (Ctrl nucleus) and a bleached nucleus (FRAP nucleus) in Swi6‐EGFP clr4∆ cells. Red box: bleached region. Right: raw data example.


Average relative intensities of a line ‐ scan FRAP experiment on Swi6 ‐ EGFP in heterochromatin ( blue line ) , euchromatin ( green line ) , and clr4 ∆ ( red line ) . The curves have a gliding average of 40 .

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Average relative intensities of a line‐scan FRAP experiment on Swi6‐EGFP in heterochromatin (blue line), euchromatin (green line), and clr4∆ (red line). The curves have a gliding average of 40.


A , BAverage relative intensities over time and corresponding fluorescence t 1 / 2 values of heterochromatic Swi6 obtained from FRAP experiments performed with cells expressing NLS ‐ Swi6 ‐ EGFP ( blue ) or NLS ‐ Swi6 ‐ KR25A ‐ EGFP ( red ) .

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A, BAverage relative intensities over time and corresponding fluorescence t 1/2 values of heterochromatic Swi6 obtained from FRAP experiments performed with cells expressing NLS‐Swi6‐EGFP (blue) or NLS‐Swi6‐KR25A‐EGFP (red).


C , DAverage relative intensities over time and corresponding fluorescence t 1 / 2 values of heterochromatic Swi6 obtained from FRAP experiments performed with wild ‐ type ( brown ) or cid14 ∆ ( dark green ) cells expressing Swi6 ‐ EGFP .

C, DAverage relative intensities over time and corresponding fluorescence t 1/2 values of heterochromatic Swi6 obtained from FRAP experiments performed with wild‐type (brown) or cid14∆ (dark green) cells expressing Swi6‐EGFP.


E , FAverage relative intensities over time and corresponding fluorescence t 1 / 2 values of heterochromatic Swi6 obtained from FRAP experiments performed with wild ‐ type cells expressing heterochromatic NLS ‐ Swi6 ‐ EGFP ( blue ) or NLS ‐ Swi6 KR25A ‐ EGFP ( red ) , or cid14 ∆ cells expressing NLS ‐ Swi6 ‐ EGFP ( green ) or NLS ‐ Swi6 ‐ KR25A ‐ EGFP ( yellow ) . Data information : In ( B ) , ( D ) and ( F ) , the box bounds the interquartile range ( IQR ) divided by the median , and whiskers extend to a maximum of 1 . 5 × IQR beyond the box .

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E, FAverage relative intensities over time and corresponding fluorescence t 1/2 values of heterochromatic Swi6 obtained from FRAP experiments performed with wild‐type cells expressing heterochromatic NLS‐Swi6‐EGFP (blue) or NLS‐Swi6 KR25A‐EGFP (red), or cid14∆ cells expressing NLS‐Swi6‐EGFP (green) or NLS‐Swi6‐KR25A‐EGFP (yellow).Data information: In (B), (D) and (F), the box bounds the interquartile range (IQR) divided by the median, and whiskers extend to a maximum of 1.5 × IQR beyond the box.


Schematic of a chromosome indicating Swi6 ‐ EGFP heterochromatic domains ( green ) . Cnp1 ‐ mCherry and Taz1 ‐ mCherry ( red ) mark centromeres and telomeres , respectively . Representative images demonstrating Swi6 ‐ EGFP co ‐ localization with centromeres ( Cnp1 ‐ mCherry ) and telomeres ( Taz1 ‐ mCherry ) are shown .

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Schematic of a chromosome indicating Swi6‐EGFP heterochromatic domains (green). Cnp1‐mCherry and Taz1‐mCherry (red) mark centromeres and telomeres, respectively. Representative images demonstrating Swi6‐EGFP co‐localization with centromeres (Cnp1‐mCherry) and telomeres (Taz1‐mCherry) are shown.


Fluorescence t 1 / 2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing NLS ‐ Swi6 ‐ EGFP .

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Fluorescence t 1/2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing NLS‐Swi6‐EGFP.


Fluorescence t 1 / 2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing the RNA ‐ binding mutant NLS ‐ Swi6 ‐ KR25A ‐ EGFP . Fluorescence t 1 / 2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing Swi6 ‐ EGFP .

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Fluorescence t 1/2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing the RNA‐binding mutant NLS‐Swi6‐KR25A‐EGFP.Fluorescence t 1/2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing Swi6‐EGFP.


Fluorescence t 1 / 2 values of centromeric Swi6 obtained from FRAP experiments performed after release of cells expressing NLS ‐ Swi6 ‐ EGFP ( black dots ) or the RNA ‐ binding mutant NLS ‐ Swi6 ‐ KR25A ‐ EGFP ( red dots ) from G1 / S cell cycle arrest . Each dot represents one FRAP experiment at the respective time after release from cell cycle arrest . Swi6 is dispersed in M phase due to H3S10 phosphorylation , and M phase was therefore not included in the FRAP analysis .

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Fluorescence t 1/2 values of centromeric Swi6 obtained from FRAP experiments performed after release of cells expressing NLS‐Swi6‐EGFP (black dots) or the RNA‐binding mutant NLS‐Swi6‐KR25A‐EGFP (red dots) from G1/S cell cycle arrest. Each dot represents one FRAP experiment at the respective time after release from cell cycle arrest. Swi6 is dispersed in M phase due to H3S10 phosphorylation, and M phase was therefore not included in the FRAP analysis.


Fluorescence t 1 / 2 values obtained from FRAP experiments performed on centromeric Swi6 . A total of 40 , 66 , and 104 individual cells residing in the G1 , S , and G2 phases of the cell cycle were analyzed , respectively . Representative bright ‐ field images of cells in G1 , S , or G2 are shown . Scale bars = 2 μm .

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Fluorescence t 1/2 values obtained from FRAP experiments performed on centromeric Swi6. A total of 40, 66, and 104 individual cells residing in the G1, S, and G2 phases of the cell cycle were analyzed, respectively. Representative bright‐field images of cells in G1, S, or G2 are shown. Scale bars = 2 μm.


Fluorescence t 1 / 2 values obtained from FRAP experiments performed on centromeric Swi6 of cells expressing NLS ‐ Swi6 ‐ EGFP and NLS ‐ Swi6 ‐ KR25A ‐ EGFP that reside in the G2 of the cell cycle .

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Fluorescence t 1/2 values obtained from FRAP experiments performed on centromeric Swi6 of cells expressing NLS‐Swi6‐EGFP and NLS‐Swi6‐KR25A‐EGFP that reside in the G2 of the cell cycle.


Fluorescence t 1 / 2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing the RNA ‐ binding mutant NLS ‐ Swi6 ‐ KR25A ‐ EGFP .

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Fluorescence t 1/2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing the RNA‐binding mutant NLS‐Swi6‐KR25A‐EGFP.


H3K9me2 ChIP ‐ seq enrichment profiles for swi6 + and swi6 ∆ cells on centromere 1 . Centromeric repeat elements ( dg / dh ) are indicated . Inverted repeats ( IRC1 ) constitute heterochromatin boundaries in wild ‐ type cells ( Cam et al , 2005 ) . The y ‐ axes represent log2 ChIP ‐ seq enrichments in 200 ‐ bp genomically tiled windows calculated over clr4Δ cells . Two independent biological replicates for each genotype were performed ( replicates 1 and 2 ) . Positions of genomic elements on the plus and minus strands are indicated . Blue , protein ‐ coding genes ; white , repeats ; green , long terminal repeat ( LTR ) .

H3K9me2 ChIP‐seq enrichment profiles for swi6+ and swi6∆ cells on centromere 1. Centromeric repeat elements (dg/dh) are indicated. Inverted repeats (IRC1) constitute heterochromatin boundaries in wild‐type cells (Cam et al, 2005). The y‐axes represent log2 ChIP‐seq enrichments in 200‐bp genomically tiled windows calculated over clr4Δ cells. Two independent biological replicates for each genotype were performed (replicates 1 and 2). Positions of genomic elements on the plus and minus strands are indicated. Blue, protein‐coding genes; white, repeats; green, long terminal repeat (LTR).


H3K9me2 ChIP ‐ seq enrichment profiles for swi6 + and swi6 ∆ cells on the telomere of the right arm of chromosome 1 . Two independent biological replicates for each genotype were performed ( replicates 1 and 2 ) .

H3K9me2 ChIP‐seq enrichment profiles for swi6+ and swi6∆ cells on the telomere of the right arm of chromosome 1. Two independent biological replicates for each genotype were performed (replicates 1 and 2).


Differential expression of transcripts coming from centromere 1 assessed by tiling microarray gene expression analysis . Expression data for swi6 + and swi6 ∆ cells were taken from a previous publication ( Woolcock et al , 2012 ) .

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Differential expression of transcripts coming from centromere 1 assessed by tiling microarray gene expression analysis. Expression data for swi6+ and swi6∆ cells were taken from a previous publication (Woolcock et al, 2012).


C - EH3K9me2 levels on heterochromatin ‐ adjacent genes assessed by ChIP - PCR in swi6 + , swi6 ∆ , and swi6 ‐ L315E ( CSD dimerization mutant ) ( C ) , swi6 ‐ W104A ( H3K9me2 binding mutant ) ( D ) , and epe1 ∆ ( E ) cells . Enrichments over clr4 ∆ are normalized to adh1 + . Average fold enrichment with s . d . is shown for three ( C and E ) or four ( D ) independent experiments . P ‐ values were generated by the Student ' s t ‐ test ( two ‐ tailed distribution , two ‐ sample , unequal variance ) .

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C-EH3K9me2 levels on heterochromatin‐adjacent genes assessed by ChIP-PCR in swi6+, swi6∆, and swi6‐L315E (CSD dimerization mutant) (C), swi6‐W104A (H3K9me2 binding mutant) (D), and epe1∆ (E) cells. Enrichments over clr4∆ are normalized to adh1+. Average fold enrichment with s.d. is shown for three (C and E) or four (D) independent experiments. P‐values were generated by the Student's t‐test (two‐tailed distribution, two‐sample, unequal variance).


sRNA reads mapping to centromere 1 in swi6 + ( green ) and swi6 ∆ ( blue ) cells . The dashed red box highlights the loss of brdrRNAs at the IRC1R in the absence of Swi6 . Centromeric repeat elements and the central core are indicated . Counts were normalized to the library size . Asterisks denote tRNA fragments . Note that tRNA genes flank IRC3 ( Fig EV4 ) but not IRC1R .

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sRNA reads mapping to centromere 1 in swi6+ (green) and swi6∆ (blue) cells. The dashed red box highlights the loss of brdrRNAs at the IRC1R in the absence of Swi6. Centromeric repeat elements and the central core are indicated. Counts were normalized to the library size. Asterisks denote tRNA fragments. Note that tRNA genes flank IRC3 (Fig EV4) but not IRC1R.


H3K9me2 levels on heterochromatin ‐ adjacent genes assessed by ChIP - PCR in swi6 + , swi6 ∆ , tas3 L479E , and tas3 L479E swi6 ∆ cells . Enrichments over clr4 ∆ are normalized to adh1 + . Average fold enrichment with s . d . is shown for three independent experiments . P ‐ values were generated by Student ' s t ‐ test ( two ‐ tailed distribution , two ‐ sample , unequal variance ) .

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H3K9me2 levels on heterochromatin‐adjacent genes assessed by ChIP-PCR in swi6+, swi6∆, tas3 L479E, and tas3 L479E swi6∆ cells. Enrichments over clr4∆ are normalized to adh1+. Average fold enrichment with s.d. is shown for three independent experiments. P‐values were generated by Student's t‐test (two‐tailed distribution, two‐sample, unequal variance).


a , Northern blot analysis of lamp - 2 expression . Total RNA was hybridized using a lamp - 2 cDNA2 probe and a murine glyceraldehyde - 3 - phosphate dehydrogenase probe .

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a, Northern blot analysis of lamp-2 expression. Total RNA was hybridized using a lamp-2 cDNA2 probe and a murine glyceraldehyde-3-phosphate dehydrogenase probe.


b , Western blot analysis of LAMP - 2 expression . In control liver and kidney extracts the glycosylated LAMP - 2 molecules were detected . In LAMP - 2 - / - tissues no LAMP - 2 product was found .

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b, Western blot analysis of LAMP-2 expression. In control liver and kidney extracts the glycosylated LAMP-2 molecules were detected. In LAMP-2-/- tissues no LAMP-2 product was found.


c , Increased mortality in LAMP - 2 deficient outbred ( C57B6 / Jx129SV ) mice . Forty - two control ( closed circles ) and LAMP - 2 - deficient mice ( open circles ) were monitored over 300 days .

c, Increased mortality in LAMP-2 deficient outbred (C57B6/Jx129SV) mice. Forty-two control (closed circles) and LAMP-2-deficient mice (open circles) were monitored over 300 days.


d , Loss of weight in early dying ( n = 7 males ) and surviving LAMP - 2 - deficient mice ( n = 11 males ) in comparison with control mice ( n = 12 males ) monitored for 60 days after birth .

d, Loss of weight in early dying (n = 7 males) and surviving LAMP-2-deficient mice (n = 11 males) in comparison with control mice ( n = 12 males) monitored for 60 days after birth.


a , Ultrastructure of autophagic vacuoles ( arrowheads ) in pancreatic acinar gland cells from a 6 . 5 - month - old LAMP - 2 - deficient mouse .

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a, Ultrastructure of autophagic vacuoles (arrowheads) in pancreatic acinar gland cells from a 6.5-month-old LAMP-2-deficient mouse.


b , Autophagic vacuoles containing glycogen ( g ) , cytosol and cellular constituents in a hepatocyte of an 8 - day - old LAMP - 2 - / - mouse . m , mitochrondria ; bc , bile canaliculus . c , Accumulation of numerous early autophagic vacuoles in cultured LAMP - 2 - / - hepatocytes . Arrow , large late autophagic vacuole containing partially degraded material . Insert , magnification of the region indicated by an asterisk with typical early autophagic vacuoles containing undigested cellular constituents . Scale bars : a , 3 . 5 µm ; b , 0 . 2 µm ; c , 1 . 3 µm ; insert , 0 . 9 µm .

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b, Autophagic vacuoles containing glycogen (g), cytosol and cellular constituents in a hepatocyte of an 8-day-old LAMP-2-/- mouse. m, mitochrondria; bc, bile canaliculus. c, Accumulation of numerous early autophagic vacuoles in cultured LAMP-2-/- hepatocytes. Arrow, large late autophagic vacuole containing partially degraded material. Insert, magnification of the region indicated by an asterisk with typical early autophagic vacuoles containing undigested cellular constituents. Scale bars: a, 3.5 µm; b, 0.2 µm; c, 1.3 µm; insert, 0.9 µm.


a , Vacuole in a fibre of the soleus muscle . b , Ultrastructure of a vacuole in a cardiomyocyte . Scale bars : a , 1 µm ; b , 1 . 5 µm .

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a, Vacuole in a fibre of the soleus muscle. b, Ultrastructure of a vacuole in a cardiomyocyte. Scale bars: a, 1 µm; b, 1.5 µm.


c , Typical original twitch recordings of developed force of heart muscles from control ( thin line ) and LAMP - 2 - deficient ( thick line ) mice . d , Average values of developed force . Preparations of LAMP - 2 deficient mice had a significantly lower baseline developed force at a stimulation frequency of 4 Hz and 10 Hz ; P 0 . 01 .

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c, Typical original twitch recordings of developed force of heart muscles from control (thin line) and LAMP-2-deficient (thick line) mice. d, Average values of developed force. Preparations of LAMP-2 deficient mice had a significantly lower baseline developed force at a stimulation frequency of 4 Hz and 10 Hz; P  0.01.


a , Volume density of early ( Avi ) and late ( Avd ) autophagic vacuoles from four independent cultures . Examples of Avi and Avd are shown in b and c , respectively . Bars represent 0 . 4 µm .

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a, Volume density of early (Avi) and late (Avd) autophagic vacuoles from four independent cultures. Examples of Avi and Avd are shown in b and c, respectively. Bars represent 0.4 µm.


d , Labelling density of cathepsin - D as estimated in two independent experiments . e , Labelling density of LAMP - 1 .

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d, Labelling density of cathepsin-D as estimated in two independent experiments. e, Labelling density of LAMP-1.


f , Degradation of long - lived proteins followed over 24 h in amino acid and fetal - calf - serum - containing medium . g , Effect of amino - acid ( AA ) deprivation and 3 - methyladenine ( MA ) on protein degradation during 4 h of chase . The error bars in a , d , e and g represent s . e . m .

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f, Degradation of long-lived proteins followed over 24 h in amino acid and fetal-calf-serum-containing medium. g, Effect of amino-acid (AA) deprivation and 3-methyladenine (MA) on protein degradation during 4 h of chase. The error bars in a, d, e and g represent s.e.m.


Localization of R - LPS ( green ) and S - LPS ( red ) in exponential phase bacteria . Scale bars : 2 μm . Distribution of R - LPS and S - LPS in exponential phase culture analyzed by flow cytometry ( one representative example among 3 biological replicates , n = 20 , 000 events ) . Numbers in each corner corresponds to % of relative frequencies .

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Localization of R-LPS (green) and S-LPS (red) in exponential phase bacteria. Scale bars: 2 μm. Distribution of R-LPS and S-LPS in exponential phase culture analyzed by flow cytometry (one representative example among 3 biological replicates, n = 20,000 events). Numbers in each corner corresponds to % of relative frequencies.


Roughness measurements on R - LPS labeled ( green ) WT and Δgmd cells . AFM images of whole bacteria and of the separated areas ( coloured squares in first images , 0 . 4 x 0 . 4 μm2 ) are shown . The arithmetic roughness Ra is indicated below each area . Scale bars : 1 μm . Quantitative roughness measurement of R - LPS labeled WT and Δgmd cells . The areas with the lower roughness ( more regular surfaces ) of Δgmd cells were assigned as areas 1 . mAb : monoclonal antibody . Ab : antibody . nWT = 13 bacteria . nΔgmd = 7 bacteria . Differences were statistically analyzed by t - test . * * P < 0 . 01 . ns : not significant .

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Roughness measurements on R-LPS labeled (green) WT and Δgmd cells. AFM images of whole bacteria and of the separated areas (coloured squares in first images, 0.4 x 0.4 μm2) are shown. The arithmetic roughness Ra is indicated below each area. Scale bars: 1 μm. Quantitative roughness measurement of R-LPS labeled WT and Δgmd cells. The areas with the lower roughness (more regular surfaces) of Δgmd cells were assigned as areas 1. mAb: monoclonal antibody. Ab: antibody. nWT = 13 bacteria. nΔgmd = 7 bacteria. Differences were statistically analyzed by t-test. **P < 0.01. ns: not significant.


Detection of forces between AFM tip functionalized with mAb against R - LPS and unlabeled WT cells . Height image and the corresponding adhesion image are shown on top and zooms of depicted areas ( black squares , 0 . 4 x 0 . 4 μm2 ) below . The arithmetic roughness Ra and percentage of adhesion Padh are indicated below each area . Scale bar : 1 μm . Correlation between roughness and detection of R - LPS on unlabeled WT cells . The areas with the lower roughness ( more regular surfaces ) were assigned as areas 1 . The boxplot represents the mean values ( squares ) , the median ( horizontal line ) , the 25 % and 75 % quartiles ( box limits ) and the standard deviations ( whiskers ) . n = 9 bacteria . Error bars : s . d . Statistical differences were analyzed by t - test . * * P < 0 . 01 . * P < 0 . 05 .

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Detection of forces between AFM tip functionalized with mAb against R-LPS and unlabeled WT cells. Height image and the corresponding adhesion image are shown on top and zooms of depicted areas (black squares, 0.4 x 0.4 μm2) below. The arithmetic roughness Ra and percentage of adhesion Padh are indicated below each area. Scale bar: 1 μm. Correlation between roughness and detection of R-LPS on unlabeled WT cells. The areas with the lower roughness (more regular surfaces) were assigned as areas 1. The boxplot represents the mean values (squares), the median (horizontal line), the 25% and 75% quartiles (box limits) and the standard deviations (whiskers). n = 9 bacteria. Error bars: s.d. Statistical differences were analyzed by t-test. **P < 0.01. *P < 0.05.


Co - localization of Omp2b ( red ) and R - LPS ( green ) in exponential phase bacteria . Scale bars : 2 μm .

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Co-localization of Omp2b (red) and R-LPS (green) in exponential phase bacteria. Scale bars: 2 μm.


Co - detection of Omp2b ( red ) and R - LPS by AFM . R - LPS was detected using AFM tips functionalized with mAb against R - LPS . AFM images of whole bacteria and of the separated areas ( red squares in first images , 0 . 4 x 0 . 4 μm2 ) are shown . The percentages of adhesion Padh are indicated below each area . Scale bars : 1 μm .

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Co-detection of Omp2b (red) and R-LPS by AFM. R-LPS was detected using AFM tips functionalized with mAb against R-LPS. AFM images of whole bacteria and of the separated areas (red squares in first images, 0.4 x 0.4 μm2) are shown. The percentages of adhesion Padh are indicated below each area. Scale bars: 1 μm.


Adhesion ratio between the Omp2b positive area compared to the Omp2b negative area . Grey dashed line shows ratio = 1 corresponding to homogeneity in surface structure . nWT = 13 bacteria , nΔgmd = 13 bacteria . Statistical analysis with Mann - Whitney U test showed * * P < 0 . 01 .

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Adhesion ratio between the Omp2b positive area compared to the Omp2b negative area. Grey dashed line shows ratio = 1 corresponding to homogeneity in surface structure. nWT = 13 bacteria, nΔgmd = 13 bacteria. Statistical analysis with Mann-Whitney U test showed **P < 0.01.


Localization of initially labeled Omp25 ( green ) on TRSE - labeled ( red ) bacteria before ( 0 h ) and after 2 h of growth in the absence of both labelings ( 2 h ) . As a control , Omp25 localization after pulse - chase labeling with TRSE is shown at the bottom panel . Scale bars : 2 μm .

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Localization of initially labeled Omp25 (green) on TRSE-labeled (red) bacteria before (0 h) and after 2 h of growth in the absence of both labelings (2 h). As a control, Omp25 localization after pulse-chase labeling with TRSE is shown at the bottom panel. Scale bars: 2 μm.


Localization of initially labeled Omp2b ( green ) on TRSE - labeled ( red ) bacteria before ( 0 h ) and after 2 h of growth in the absence of both labelings ( 2 h ) . Scale bars : 2 μm .

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Localization of initially labeled Omp2b (green) on TRSE-labeled (red) bacteria before (0 h) and after 2 h of growth in the absence of both labelings (2 h). Scale bars: 2 μm.


Co - localization of Omp25 and Omp2b positive pixels with TRSE positive or negative pixels , respectively . Error bars correspond to s . d . from independent experiments . Differences were statistically analyzed by t - test . * * * P < 0 . 001 . nOmp25 = 279 bacteria , 85666 pixels ( 5 biological replicates ) . nOmp2b = 252 bacteria , 74630 pixels ( 3 biological replicates ) .

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Co-localization of Omp25 and Omp2b positive pixels with TRSE positive or negative pixels, respectively. Error bars correspond to s.d. from independent experiments. Differences were statistically analyzed by t-test. ***P < 0.001. nOmp25 = 279 bacteria, 85666 pixels (5 biological replicates). nOmp2b = 252 bacteria, 74630 pixels (3 biological replicates).


Short pulse labeling of exponential phase bacteria with the fluorescent D - amino acid HADA . Merge is showing old pole marker PdhS - mCherry ( red ) and PG insertion sites ( white ) . Scale bars : 2 μm .

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Short pulse labeling of exponential phase bacteria with the fluorescent D-amino acid HADA. Merge is showing old pole marker PdhS-mCherry (red) and PG insertion sites (white). Scale bars: 2 μm.


Demographic representation of HADA labeling . Bacteria were sorted according to their cell length and oriented with the old pole at the bottom of the demograph by PdhS - mCherry fluorescence signal . NFI : normalized fluorescence intensity . n = 393 bacteria ( 3 biological replicates ) .

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Demographic representation of HADA labeling. Bacteria were sorted according to their cell length and oriented with the old pole at the bottom of the demograph by PdhS-mCherry fluorescence signal. NFI: normalized fluorescence intensity. n = 393 bacteria (3 biological replicates).


Localization of S - LPS ( green ) in the eFluor - labeled ( magenta ) inducible rough strain Δgmd plac - gmd possessing the old pole marker PdhS - mCherry ( red ) . The strains Δgmd and Δgmd plac - gmd - IPTG were used as negative controls . Scale bars : 2 μm .

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Localization of S-LPS (green) in the eFluor-labeled (magenta) inducible rough strain Δgmd plac-gmd possessing the old pole marker PdhS-mCherry (red). The strains Δgmd and Δgmd plac-gmd -IPTG were used as negative controls. Scale bars: 2 μm.


Demograph represents fluorescence profile of S - LPS labeling from ( A ) . Cells were oriented with the old pole at the bottom of the graph by PdhS - mCherry and sorted by cell length . NFI : normalized fluorescence intensity . n = 468 bacteria ( 3 biological replicates ) .

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Demograph represents fluorescence profile of S-LPS labeling from (A). Cells were oriented with the old pole at the bottom of the graph by PdhS-mCherry and sorted by cell length. NFI: normalized fluorescence intensity. n = 468 bacteria (3 biological replicates).


Short pulse labeling of LPS ( 2 h ) by Kdo - N3 ( green ) on eFluor - labeled ( magenta ) bacteria expressing the old pole marker PdhS - mCherry ( red ) . Scale bars : 2 μm .

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Short pulse labeling of LPS (2 h) by Kdo-N3 (green) on eFluor-labeled (magenta) bacteria expressing the old pole marker PdhS-mCherry (red). Scale bars: 2 μm.


Demographs represent fluorescence intensities of eFluor ( OM , left ) and Kdo - N3 ( LPS , right ) labeling . Cells were sorted by cell length and aligned with the old pole at the bottom of the graph by PdhS - mCherry localization . NFI : normalized fluorescence intensity . n = 566 bacteria ( 3 biological replicates ) .

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Demographs represent fluorescence intensities of eFluor (OM, left) and Kdo-N3 (LPS, right) labeling. Cells were sorted by cell length and aligned with the old pole at the bottom of the graph by PdhS-mCherry localization. NFI: normalized fluorescence intensity. n = 566 bacteria (3 biological replicates).


Distribution of R - LPS and S - LPS in Δgmd plac - gmd after 3 , 6 , 9 and 24 h post - induction analyzed by flow cytometry ( one representative example among 3 biological replicates , n = 20 , 000 events ) .

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Distribution of R-LPS and S-LPS in Δgmd plac-gmd after 3, 6, 9 and 24 h post-induction analyzed by flow cytometry (one representative example among 3 biological replicates, n = 20,000 events).


Quantification of S - LPS labeling from ( A ) as relative frequencies . Error bars correspond to s . d . from 3 independent experiments . n0h = 77 bacteria , n3h = 254 bacteria , n6h = 616 bacteria , n9h = 937 bacteria , n24h = 1059 bacteria .

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Quantification of S-LPS labeling from (A) as relative frequencies. Error bars correspond to s.d. from 3 independent experiments. n0h = 77 bacteria, n3h = 254 bacteria, n6h = 616 bacteria, n9h = 937 bacteria, n24h = 1059 bacteria.


( A ) Binding assay for Hsc70 binding in the presence of WT DNAJB1 : control of DNAJB1 / Hsc70 , αSyn fibres + DNAJB1 / Hsc70 , αSyn fibres + DNAJB1 / Hsc70 / Apg2 , control of DNAJB1 / Hsc70 / Apg2 . The ' * ' highlights the condition where an increase of Hsc70 in the bound fraction is observed . P , pellet ; S , supernatant . The amount of Hsc70 recruited to the fibres is shown in the pellet fractions . ( B ) Binding assay for Hsc70 binding in the presence of the truncated ΔJ - DNAJB1 , lacking the J domain . The gel layout is similar to the previous one with ΔJ - DNAJB1 replacing WT DNAJB1 . Both Apg2 and J domain are required for enhancement of Hsc70 binding . ( C ) Histogram of Hsc70 bound fraction in each sample ( N = 3 independent experiments , WT : WT DNAJB1 , ΔJ : ΔJ - DNAJB1 mutant , - Apg2 : condition with fibres + DNAJB1 / Hsc70 , + Apg2 : condition with fibres + DNAJB1 / Hsc70 / Apg2 ) . A 2 - way ANOVA was performed with Tukey ' s multiple comparisons test ( * * : P = 0 . 003 ; * * * * : P < 0 . 0001 ) . Mean Hsc70 binding values with SD are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Binding assay for Hsc70 binding in the presence of WT DNAJB1: control of DNAJB1/Hsc70, αSyn fibres + DNAJB1/Hsc70, αSyn fibres + DNAJB1/Hsc70/Apg2, control of DNAJB1/Hsc70/Apg2. The '*' highlights the condition where an increase of Hsc70 in the bound fraction is observed. P, pellet; S, supernatant. The amount of Hsc70 recruited to the fibres is shown in the pellet fractions. (B) Binding assay for Hsc70 binding in the presence of the truncated ΔJ-DNAJB1, lacking the J domain. The gel layout is similar to the previous one with ΔJ-DNAJB1 replacing WT DNAJB1. Both Apg2 and J domain are required for enhancement of Hsc70 binding. (C) Histogram of Hsc70 bound fraction in each sample (N = 3 independent experiments, WT: WT DNAJB1, ΔJ: ΔJ-DNAJB1 mutant, -Apg2: condition with fibres + DNAJB1/Hsc70, +Apg2: condition with fibres + DNAJB1/Hsc70/Apg2). A 2-way ANOVA was performed with Tukey's multiple comparisons test (**: P=0.003; ****: P<0.0001). Mean Hsc70 binding values with SD are shown.


( D ) TIRF microscopy images of the labelled αSyn fibres and Hsc70 in the absence or in the presence of Apg2 . Scale bar , 1 µm . ( E , F ) Quantitation of fluorescence intensity of Hsc70 ( E ) and αSyn ( F ) in the two conditions ( + / - Apg2 , N = 3 independent experiments , n = 65 fibres ) . Mean Hsc70 and αSyn fluorescence intensities with SD are shown . ( G ) Quantitation of differences in Hsc70 binding between the two ends of the fibres , normalised to the local αSyn intensity , in the absence and presence of Apg2 ( N = 3 independent experiments , n = 54 fibres ) . Mean Hsc70 fluorescence intensities with SD are shown . Data information : ( E - G ) For each plot , data were tested for normality by performing a Shapiro - Wilk test . As the data did not display a normal distribution , they were analysed using a two - tailed Mann - Whitney test ( * * * * : P < 0 . 0001 , ns : not significant ) .

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(D) TIRF microscopy images of the labelled αSyn fibres and Hsc70 in the absence or in the presence of Apg2. Scale bar, 1 µm. (E, F) Quantitation of fluorescence intensity of Hsc70 (E) and αSyn (F) in the two conditions (+/- Apg2, N = 3 independent experiments, n = 65 fibres). Mean Hsc70 and αSyn fluorescence intensities with SD are shown. (G) Quantitation of differences in Hsc70 binding between the two ends of the fibres, normalised to the local αSyn intensity, in the absence and presence of Apg2 (N = 3 independent experiments, n = 54 fibres). Mean Hsc70 fluorescence intensities with SD are shown. Data information: (E-G) For each plot, data were tested for normality by performing a Shapiro-Wilk test. As the data did not display a normal distribution, they were analysed using a two-tailed Mann-Whitney test (****: P<0.0001, ns: not significant).


Binding assay with different dilutions of Apg2 incubated with DNAJB1 / Hsc70 and ( A ) with αSyn fibres or ( B ) without αSyn fibres ( control ) . 5 mM ATP was present in the buffer throughout .

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Binding assay with different dilutions of Apg2 incubated with DNAJB1/Hsc70 and (A) with αSyn fibres or (B) without αSyn fibres (control). 5 mM ATP was present in the buffer throughout.


A - L ) Serum starved HUVECs were stimulated with designed scaffolds normalized to 18 nM of F - domains for 15 minutes and analyzed by immunoblotting for pAKT ( S473 ) , pERK1 / 2 ( T202 / Y204 ) , and pFAK ( Y397 ) phosphorylation . A , E , I ) pAKT , B , F , J ) pERK1 / 2 , and C , G , K ) pFAK phosphorylation levels are quantified and normalized to Icos - cage signaling levels . Appendix Figure S1B shows the detection of two bands expected for pERK1 / 2 . Representative immunoblot gels of cells stimulated with or without scaffolds at 18 nM of F - domain : D ) helical bundles , H ) trimeric and tetrameric homo - oligomers and tetrahedral nanocages , L ) Icosahedral nanocages and trimeric subunit .

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A-L) Serum starved HUVECs were stimulated with designed scaffolds normalized to 18 nM of F-domains for 15 minutes and analyzed by immunoblotting for pAKT (S473), pERK1/2 (T202/Y204), and pFAK (Y397) phosphorylation. A, E, I) pAKT, B, F, J) pERK1/2, and C, G, K) pFAK phosphorylation levels are quantified and normalized to Icos-cage signaling levels. Appendix Figure S1B shows the detection of two bands expected for pERK1/2. Representative immunoblot gels of cells stimulated with or without scaffolds at 18 nM of F-domain: D) helical bundles, H) trimeric and tetrameric homo-oligomers and tetrahedral nanocages, L) Icosahedral nanocages and trimeric subunit.


M - P ) Dose - response curves , EC50 , and EMAX for pAKT and pERK1 / 2 activation . The curves , EC50 and EMAX values are calculated using Prism , GraphPad . N , P ) Darker red color indicate lower EC50 and higher EMAX values .

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M-P) Dose-response curves, EC50, and EMAX for pAKT and pERK1/2 activation. The curves, EC50 and EMAX values are calculated using Prism, GraphPad. N, P) Darker red color indicate lower EC50 and higher EMAX values.


C ) Comparison of cell migration induced by Ang1 , Ang2 , and designed F - domain scaffolds after 12 hours of treatment . The change in wound area is normalized to vehicle control . All experiments have at least 3 biological replicates . ( * ) is the comparison between F - domain scaffolds vs Ang1 , and ( # ) is the comparison between F - domain scaffolds vs Vehicle .

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C) Comparison of cell migration induced by Ang1, Ang2, and designed F-domain scaffolds after 12 hours of treatment. The change in wound area is normalized to vehicle control. All experiments have at least 3 biological replicates. (*) is the comparison between F-domain scaffolds vs Ang1, and (#) is the comparison between F-domain scaffolds vs Vehicle.


E ) Representative increase in vascular stability by Icos1 . The number of nodes ( blue ) , meshes ( red ) , and tubes ( green ) were quantified using angiogenesis analyzer plugin in ImageJ . Scale bar is100 µm .

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E) Representative increase in vascular stability by Icos1. The number of nodes (blue), meshes (red), and tubes (green) were quantified using angiogenesis analyzer plugin in ImageJ. Scale bar is100 µm.


A - C ) Representative images of FOXO1 localization upon scaffolds administration . Scale bars are 10 µm D ) Quantifications of nuclear FOXO1 shown as percent of cells with nuclear FOXO1 stain , 100 cells were counted per sample .

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A-C) Representative images of FOXO1 localization upon scaffolds administration. Scale bars are 10 µm D) Quantifications of nuclear FOXO1 shown as percent of cells with nuclear FOXO1 stain, 100 cells were counted per sample.


E ) Cells were stimulated with scaffolds or angiopoietins and stained with Tie2 antibodies for analyzing plasma membrane Tie2 clusters . Dark gray bars : Quantification shows the percent % of total cells that exhibit large Tie2 clusters on the plasma membrane .

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E) Cells were stimulated with scaffolds or angiopoietins and stained with Tie2 antibodies for analyzing plasma membrane Tie2 clusters. Dark gray bars: Quantification shows the percent % of total cells that exhibit large Tie2 clusters on the plasma membrane.


K , L ) Representatives immunoblot gel ( K ) and quantifications ( L ) for α5 integrin knockdown experiments using 20 nM of siRNA .

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K, L) Representatives immunoblot gel (K) and quantifications (L) for α5 integrin knockdown experiments using 20 nM of siRNA.


C , D ) CCI - TBI brain sections were stained for reactive astrocytes ( GFAP , blue ) , microglia ( Iba1 , green ) and serum albumin ( magenta ) to highlight tissue margins ( dotted white line ) and quantify secondary damage as a function of lesion area ( E ; * p = 0 . 04 , n = 4 , the mean ± SEM is plotted ) . Bar , 1 mm . E ) Cumulative blood extravasation into brain tissue ( white outline ) was imaged by albumin immunofluorescence to establish an unbiased intensity - threshold mask ( red ) and quantify the area of blood serum extravasation ( mm2 ) in Icos1ø vs . Icos1 treated animals ( * * p = 0 . 01 , n = 4 , the mean ± SEM is plotted ) . Scale bar is 500 µm .

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C, D) CCI-TBI brain sections were stained for reactive astrocytes (GFAP, blue), microglia (Iba1, green) and serum albumin (magenta) to highlight tissue margins (dotted white line) and quantify secondary damage as a function of lesion area (E; *p = 0.04, n = 4, the mean ± SEM is plotted ). Bar, 1 mm. E) Cumulative blood extravasation into brain tissue (white outline) was imaged by albumin immunofluorescence to establish an unbiased intensity-threshold mask (red) and quantify the area of blood serum extravasation (mm2) in Icos1ø vs. Icos1 treated animals (**p = 0.01, n = 4, the mean ± SEM is plotted). Scale bar is 500 µm.


F - H ) Brain tissues stained by GFAP - ( magenta ) and CD31 - immunofluorescence ( green ) were imaged by confocal microscopy to measure angiogenesis as a function of vessel diameter ( G ; * * p = 0 . 003 , n = 4 , the boxes show 25th to 75th percentiles and whiskers represent the min and max values . ) and quantify the number of CD31 - vessels ( H ; * * p = 0 . 007 , n = 4 , the mean ± SEM is plotted ) at the site of injury in Icos1ø and Icos1 treated animals 4 days post - injury . Scale bars in G and H is 100 µm .

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F-H) Brain tissues stained by GFAP- (magenta) and CD31-immunofluorescence (green) were imaged by confocal microscopy to measure angiogenesis as a function of vessel diameter (G; **p = 0.003, n = 4, the boxes show 25th to 75th percentiles and whiskers represent the min and max values.) and quantify the number of CD31-vessels (H; **p = 0.007, n = 4, the mean ± SEM is plotted) at the site of injury in Icos1ø and Icos1 treated animals 4 days post-injury. Scale bars in G and H is 100 µm.


I ) Activation of pAKT ( Magenta ) was examined in Icos1ø and Icos1 treated - animals within or around vascular ( CD31 + , green ) and pericytes ( NG2 + cells , yellow ) of the BBB . High - resolution confocal images ( insets ) reveal the prevalence of activated pAKT ( magenta ) co - stain ( cyan arrows ) in pericytes ( yellow ) of the BBB along vasculature ( green , or dotted outline ) versus reactive neuroglia ( NG2 + pAKT - ) in the parenchyma ( red arrows ) in Icos1ø vs . Icos1 treated animals . Scale bar is 10 µm .

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I) Activation of pAKT (Magenta) was examined in Icos1ø and Icos1 treated-animals within or around vascular (CD31+, green) and pericytes (NG2+ cells, yellow) of the BBB. High-resolution confocal images (insets) reveal the prevalence of activated pAKT (magenta) co-stain (cyan arrows) in pericytes (yellow) of the BBB along vasculature (green, or dotted outline) versus reactive neuroglia (NG2+pAKT-) in the parenchyma (red arrows) in Icos1ø vs. Icos1 treated animals. Scale bar is 10 µm.


A Age , gender and BMI information of the Ashkenazi Jewish Centenarian cohort . Table showing the age , gender and BMI information in control and centenarian groups . Pie chart showing the percentage of male / female individuals in control and centenarian groups . Right panel showing the age and BMI distribution of all individuals recruited in targeted capture - seq . Data were presented as mean and SD , Students t - test , two tailed , * * * p < 0 . 001 .

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A Age, gender and BMI information of the Ashkenazi Jewish Centenarian cohort. Table showing the age, gender and BMI information in control and centenarian groups. Pie chart showing the percentage of male/female individuals in control and centenarian groups. Right panel showing the age and BMI distribution of all individuals recruited in targeted capture-seq. Data were presented as mean and SD, Students t-test, two tailed, *** p<0.001.


B , C Histograms of normalized allele counts in individuals 75 + years old compared to all age groups ( N = 125 , 748 ) across ( B ) all chromosome 19 SNPs and small insertions / deletions , and ( C ) missense SNPs and indels only . Dotted vertical lines show centSIRT6 .

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B, C Histograms of normalized allele counts in individuals 75+ years old compared to all age groups (N=125,748) across (B) all chromosome 19 SNPs and small insertions/deletions, and (C) missense SNPs and indels only. Dotted vertical lines show centSIRT6.


E Michaelis - Menten kinetic parameters as calculated by saturation curves using differential concentrations of myristoylated peptide or NAD + . n = 3 technical replicates ( TR ) ; error bars = SD .

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E Michaelis-Menten kinetic parameters as calculated by saturation curves using differential concentrations of myristoylated peptide or NAD+. n=3 technical replicates (TR); error bars=SD.


A , B Deacetylase activity on H3K9 ( A ) and H3K18 ( B ) residues shows reduced activity in centSIRT6 allele . Designer histones were incubated with purified SIRT6 and 5 mM NAD + , then resolved by SDS - PAGE and analyzed by immunoblotting with acetyl - specific histone antibodies . n = 3 TR ; error bars = SD . Students t - test , two tailed . Asterisks indicate p < 0 . 05 compared to WT , color corresponds to allele . H3K9ac , H3K18ac and H3 are blots , SIRT6 loading Coomassie stain HeLa histone preps were also

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

A, B Deacetylase activity on H3K9 (A) and H3K18 (B) residues shows reduced activity in centSIRT6 allele. Designer histones were incubated with purified SIRT6 and 5 mM NAD+, then resolved by SDS-PAGE and analyzed by immunoblotting with acetyl-specific histone antibodies. n=3 TR; error bars=SD. Students t-test, two tailed. Asterisks indicate p<0.05 compared to WT, color corresponds to allele. H3K9ac, H3K18ac and H3 are blots, SIRT6 loading Coomassie stain HeLa histone preps were also probed


C Quantitative mass spec of histone H3 peptide purified from human cells expressing different SIRT6 alleles did not reveal a difference in acetylation levels . Relative fraction represents the portion of the peptide encompassing H3K9 - 17 compared to the summed total of all of the peptide quantitation values for the same region . n = 3 biological replicates ( BR ) ; error bars = SD . Students t - test , two

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C Quantitative mass spec of histone H3 peptide purified from human cells expressing different SIRT6 alleles did not reveal a difference in acetylation levels. Relative fraction represents the portion of the peptide encompassing H3K9-17 compared to the summed total of all of the peptide quantitation values for the same region. n=3 biological replicates (BR); error bars=SD. Students t-test, two tailed


D Whole cell histone H3 acetylation levels in cumate - inducible SIRT6 human fibroblasts , assessed by Western blot . Cu , cumate ; PQ , paraquat . Cumate dosage required for equivalent SIRT6 protein abundance was determined by Western blot , and administered accordingly to respective cell lines Cells were incubated with appropriate cumate dose for 48hrs prior to harvest , with PQ - treated cells receiving PQ 24hrs after initial cumate induction .

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D Whole cell histone H3 acetylation levels in cumate-inducible SIRT6 human fibroblasts, assessed by Western blot. Cu, cumate; PQ, paraquat. Cumate dosage required for equivalent SIRT6 protein abundance was determined by Western blot, and administered accordingly to respective cell lines Cells were incubated with appropriate cumate dose for 48hrs prior to harvest, with PQ-treated cells receiving PQ 24hrs after initial cumate induction.