Figure 1 ( A ) Schematic of experimental setup depicting MECs grown as monolayers on a rigid rBM ( two - dimensional culture model , 2D ; top left ) or within a compliant rBM to generate multicellular spheroid structures with apical - basal polarity ( three - dimensional culture model , 3D ) top right ) . Heatmap of microarray analysis of gene expression in HMT - 3522 S - 1 MECs cultured either as a 2D monolayer or as spheroids ( Bottom ) . Expression of the top 200 differentially expressed genes between the two experimental conditions ( n = 3 independent biological replicates ; R = replicate ) . ( C ) Samples were stained with antibodies for β4 - integrin or laminin ( green ; yellow arrows ) . The actin and nucleus were counterstained with phalloidin ( red ) and DAPI ( blue ) , respectively . Scale bar , 10 μm . ( D ) HMT - 3522 S - 1 MECs plated as monolayers on a rigid rBM ( 2D ) or as spheroids within rBM ( 3D ) were treated with increasing doses of Paclitaxel ( Pac ) , TRAIL , Doxorubicin ( Doxo ) and irradiation ( IR ) . Percent cell death was quantified by immunofluorescence as percentage of cells stained positive for cleaved caspase - 3 at 48h post - treatment ( mean ± SEM ; n = 3 independent biological replicates ) . * * * * P < 0 . 0001 ( Student ' s t test ) . ( F ) Representative immunofluorescence microscopy images of MCF10A MECs plated in the indicated conditions for 18h and stained with phalloidin to reveal F - actin organization . Cells were plated as single cells on rigid glass coverslips ( 2D glass ; top left ) or fully embedded within rBM ( 3D fully embedded ; bottom left ) . Cell spreading was inhibited by plating cells on either laminin - 111 conjugated , 10 - μm micropatterned glass ( 2D / micropatterned , rigid substrate ; top middle ) or on compliant 75 Pa rBM - laminated polyacrylamide ( PA ) gels ( 2D / PA , soft substrate ; top right ) . The single 2D cells were overlaid with either purified laminin - 111 ( bottom middle ) or rBM ( bottom right ) to create a 3D ECM microenvironment . Images show maximum intensity z - projections of confocal stacks for F - actin phalloidin staining . Scale bar , 10 μ ( G ) Representative immunofluorescence microscopy images of MCF10A MECs stably expressing recombinant mCherry - tagged golgi marker ( mCherry - GalT ; red ; yellow arrows ) ligated with rBM in either 2D or 3D for 18h . The actin cortex and nucleus were counterstained with phalloidin ( green ) and DAPI ( blue ) , respectively . Images show the cross - sectional view of each cell compartment ( dashed lines ; xy plane ) and side view of confocal stacks ( xz plane ) in individual MCF10A MECs . Scale bar , 10 μm . ( H ) Golgi staining was assessed within non - spread MCF10A MECs ligated with rBM in 2D and 3D for 18h and values were plotted as a function of subcellular localization ( 2D , n = 8 ; 3D , n = 9 cells from two independent experiments ) . ( I ) Heatmap of RNA - seq experiment from MCF10A MECs ligated with rBM in 2D and 3D 12h post - plating . Data show the expression of the top 200 genes that are differentially expressed between the 2D and 3D rBM conditions . ( n = 3 independent biological replicates ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Schematic of experimental setup depicting MECs grown as monolayers on a rigid rBM (two-dimensional culture model, 2D; top left) or within a compliant rBM to generate multicellular spheroid structures with apical-basal polarity (three-dimensional culture model, 3D) top right). Heatmap of microarray analysis of gene expression in HMT-3522 S-1 MECs cultured either as a 2D monolayer or as spheroids (Bottom). Expression of the top 200 differentially expressed genes between the two experimental conditions (n=3 independent biological replicates; R=replicate).(C) Samples were stained with antibodies for β4-integrin or laminin (green; yellow arrows). The actin and nucleus were counterstained with phalloidin (red) and DAPI (blue), respectively. Scale bar, 10 μm.(D) HMT-3522 S-1 MECs plated as monolayers on a rigid rBM (2D) or as spheroids within rBM (3D) were treated with increasing doses of Paclitaxel (Pac), TRAIL, Doxorubicin (Doxo) and irradiation (IR). Percent cell death was quantified by immunofluorescence as percentage of cells stained positive for cleaved caspase-3 at 48h post-treatment (mean ± SEM; n=3 independent biological replicates). ****P < 0.0001 (Student's t test).(F) Representative immunofluorescence microscopy images of MCF10A MECs plated in the indicated conditions for 18h and stained with phalloidin to reveal F-actin organization. Cells were plated as single cells on rigid glass coverslips (2D glass; top left) or fully embedded within rBM (3D fully embedded; bottom left). Cell spreading was inhibited by plating cells on either laminin-111 conjugated, 10-μm micropatterned glass (2D/micropatterned, rigid substrate; top middle) or on compliant 75 Pa rBM-laminated polyacrylamide (PA) gels (2D/PA, soft substrate; top right). The single 2D cells were overlaid with either purified laminin-111 (bottom middle) or rBM (bottom right) to create a 3D ECM microenvironment. Images show maximum intensity z-projections of confocal stacks for F-actin phalloidin staining. Scale bar, 10 μ(G) Representative immunofluorescence microscopy images of MCF10A MECs stably expressing recombinant mCherry-tagged golgi marker (mCherry-GalT; red; yellow arrows) ligated with rBM in either 2D or 3D for 18h. The actin cortex and nucleus were counterstained with phalloidin (green) and DAPI (blue), respectively. Images show the cross-sectional view of each cell compartment (dashed lines; xy plane) and side view of confocal stacks (xz plane) in individual MCF10A MECs. Scale bar, 10 μm. (H) Golgi staining was assessed within non-spread MCF10A MECs ligated with rBM in 2D and 3D for 18h and values were plotted as a function of subcellular localization (2D, n=8; 3D, n=9 cells from two independent experiments).(I) Heatmap of RNA-seq experiment from MCF10A MECs ligated with rBM in 2D and 3D 12h post-plating. Data show the expression of the top 200 genes that are differentially expressed between the 2D and 3D rBM conditions. (n=3 independent biological replicates).


Figure 2 ( A ) Bar graph showing qPCR quantification of SEC61B level in MCF10A MECs ligated with rBM in 2D and 3D 12h post - plating ( mean ± SEM ; n = 3 independent biological replicates ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD , * * P = 0 . 0022 . ( B ) MCF10A MECs expressing VSVGts045 - EGFP were ligated to rBM in 2D or 3D and incubated at 40 ° C for 16h to trap VSVGts045 - EGFP protein in the ER . Cells were shifted to 32 ° C for 2h to release VSVGts045 - EGFP into the secretory pathway . Representative fluorescence microscopy images of MECs expressing VSVGts045 - EGFP 2h post incubation at 32 ° C . Scale bar ( whole cell ) , 10 μm ; Scale bar ( magnified ) , 2 μm . ( C ) Scatter plot of the secretory trafficking efficiency of VSVGts045 - EGFP based on the fluorescence of VSVGts045 at the plasma membrane versus total fluorescence measured in cells from Panel B ( mean ± SEM ; 2D , n = 45 ; 3D , n = 49 cells from three independent experiments ) . * * * * P < 0 . 0001 ( Student ' s t test ) . ( D ) Representative line graph showing the changes in cytosolic [ Ca2 + ] levels of MCF10A MECs under different treatment . Fura - 2 - loaded MECs ligated to 2D ( blue ) or 3D rBM ( red ) were pre - incubated with EGTA to chelate extracellular Ca2 + in the bath solution , challenged with 2μM thapsigargin to induce ER Ca2 + release , and replenished with 4 mM Ca2 + in bath solution at the indicated time . F340 / F380 values for each cell were quantified over the course of imaging ( mean ± SD ; 2D , n = 7 ; 3D , n = 7 cells from one experiment ) . ( E ) Quantification of the amplitude of the Ca2 + response induced by thapsigargin ( ∆ F = F - F0 ) , where F0 is the basal fluorescence before thapsigargin treatment ( mean ± SEM ( 2D , n = 20 ; 3D , n = 21 cells from three independent experiments ) . * * * P = 0 . 0007 ( Student ' s t test ) . ( F ) Bar graph showing qPCR of the relative level of ATF3 mRNA in MCF10A MECs ligated with rBM in 2D and 3D 12h post - plating ( mean ± SEM ; n = 3 independent biological replicates ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD , * * P = 0 . 0037 . ( G ) Representative immunoblots of phosphorylated EIF2a ( pEIF2a ) , total EIF2a and alpha - tubulin in cell lysate from MCF10A MECs ligated to a rBM in 2D or 3D and corresponding quantification data ( mean ± SEM ; n = 6 independent biological replicates ) . Statistical analysis by Student ' s t test , * * P = 0 . 0049 . ( H ) Bar graph of qPCR data measuring the relative levels of CRACR2B mRNA in MCF10A MECs ligated with rBM in 2D and 3D 12h post - plating ( mean ± SEM ; n = 4 independent biological replicates ) . * P = 0 . 0371 ( Student ' s t test ) . ( I ) Representative fluorescence microscopy images of the ER - plasma membrane contact site reporter GFP - MAPPER in MCF10A MECs ligated with rBM in 2D or 3D 18h post plating . Scale bar ( whole cell ) , 10 μm ; Scale bar ( magnified ) , 1 μm . ( J ) Graph of the levels of GFP - MAPPER at the plasma membrane relative total cellular GFP - MAPPER fluorescence in MCF10A MECs ligated with rBM in 2D or 3D in Panel I ( mean ± SEM ; 2D , n = 52 ; 3D , n = 57 cells from three independent experiments ) . * * P = 0 . 0032 ( Student ' s t test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Bar graph showing qPCR quantification of SEC61B level in MCF10A MECs ligated with rBM in 2D and 3D 12h post-plating (mean ± SEM; n=3 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD, **P = 0.0022.(B) MCF10A MECs expressing VSVGts045-EGFP were ligated to rBM in 2D or 3D and incubated at 40°C for 16h to trap VSVGts045-EGFP protein in the ER. Cells were shifted to 32°C for 2h to release VSVGts045-EGFP into the secretory pathway. Representative fluorescence microscopy images of MECs expressing VSVGts045-EGFP 2h post incubation at 32°C. Scale bar (whole cell), 10 μm; Scale bar (magnified), 2 μm. (C) Scatter plot of the secretory trafficking efficiency of VSVGts045-EGFP based on the fluorescence of VSVGts045 at the plasma membrane versus total fluorescence measured in cells from Panel B (mean ± SEM; 2D, n=45; 3D, n=49 cells from three independent experiments). ****P < 0.0001 (Student's t test).(D) Representative line graph showing the changes in cytosolic [Ca2+] levels of MCF10A MECs under different treatment. Fura-2-loaded MECs ligated to 2D (blue) or 3D rBM (red) were pre-incubated with EGTA to chelate extracellular Ca2+ in the bath solution, challenged with 2μM thapsigargin to induce ER Ca2+ release, and replenished with 4 mM Ca2+ in bath solution at the indicated time. F340/F380 values for each cell were quantified over the course of imaging (mean ± SD; 2D, n=7; 3D, n=7 cells from one experiment). (E) Quantification of the amplitude of the Ca2+ response induced by thapsigargin (∆F=F-F0), where F0 is the basal fluorescence before thapsigargin treatment (mean ± SEM (2D, n=20; 3D, n=21 cells from three independent experiments). ***P = 0.0007 (Student's t test).(F) Bar graph showing qPCR of the relative level of ATF3 mRNA in MCF10A MECs ligated with rBM in 2D and 3D 12h post-plating (mean ± SEM; n=3 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD, **P = 0.0037.(G) Representative immunoblots of phosphorylated EIF2a (pEIF2a), total EIF2a and alpha-tubulin in cell lysate from MCF10A MECs ligated to a rBM in 2D or 3D and corresponding quantification data (mean ± SEM; n=6 independent biological replicates). Statistical analysis by Student's t test, **P = 0.0049.(H) Bar graph of qPCR data measuring the relative levels of CRACR2B mRNA in MCF10A MECs ligated with rBM in 2D and 3D 12h post-plating (mean ± SEM; n=4 independent biological replicates). *P = 0.0371 (Student's t test).(I) Representative fluorescence microscopy images of the ER-plasma membrane contact site reporter GFP-MAPPER in MCF10A MECs ligated with rBM in 2D or 3D 18h post plating. Scale bar (whole cell), 10 μm; Scale bar (magnified), 1 μm. (J) Graph of the levels of GFP-MAPPER at the plasma membrane relative total cellular GFP-MAPPER fluorescence in MCF10A MECs ligated with rBM in 2D or 3D in Panel I (mean ± SEM; 2D, n=52; 3D, n=57 cells from three independent experiments). **P = 0.0032 (Student's t test).


Figure 3 ( A ) Representative immunofluorescence microscopy images of MCF10A MECs ligated with either a rBM in 2D or 3D for 18h and stained with antibody targeting filamin ( green ) . F - actin was counterstained with phalloidin ( red ) . Images show maximum intensity z - projections of xy confocal stacks ( left ) and middle focal plane of confocal stacks from MECs ( right ) . Yellow arrows indicate filamin aggregates in individual MECs ligated to rBM in 2D . Yellow blunt - end lines highlight the extent of filamin distribution away from the cell edge . Scale bar , 10 μm . ( B ) The expression level of filamin in MCF10A MECs ligated to rBM in 2D and 3D for 18h was assessed via immunoblot and quantified relative to actin loading control ( mean ± SEM ; n = 5 independent biological replicates ) . * * * P = 0 . 0008 ( Student ' s t test ) . ( C ) Representative fluorescence microscopy images of MCF10A MECs co - expressing the ER - plasma membrane contact site reporter GFP - MAPPER and shRNA targeting filamin ( FLMN ) or luciferase ( Luc ) ligated to rBM in 2D for 18h . Scale bar ( whole cell ) , 10 μm ; Scale bar ( magnified ) , 2 μm . ( D ) Quantification of the levels of GFP - MAPPER at the plasma membrane versus total cellular fluorescence in MCF10A MECs stably expressing an shRNA targeting filamin ( FLMN ) or luciferase ( Luc ) ligated to rBM in 2D ( mean ± SEM ; 2D + shLuc , n = 38 ; 2D + shFLMN , n = 39 cells from three independent experiments ) . * * P < 0 . 0021 ( Student ' s t test ) . ( E ) Representative immunoblots of filamin , phosphorylated EIF2a ( pEIF2a ) , total EIF2a and alpha - tubulin in cell lysate from MCF10A MECs that express shRNA targeting filamin ( shFLMN ) or luciferase ( shLuc ) and ligated to a rBM in 2D or 3D for 18h and corresponding quantification data ( mean ± SEM ; n = 4 independent biological replicates ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD . 2D - shLuc versus 3D - shLuc , * * * P = 0 . 0003 . 2D - shLuc versus 2D - shFLMN , * * * P = 0 . 0004 . ( F ) Bar graph of the relative levels of ATF3 mRNA as measured by qPCR in MCF10A MECs expressing shRNA targeting filamin ( shFLMN ) or luciferase ( shLuc ) and ligated with rBM in 2D or 3D for 18h ( mean ± SEM ; n = 4 independent biological replicates ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD . 2D - shLuc versus 3D - shLuc , * * * P = 0 . 0005 . 2D - shLuc versus 2D - shFLMN , * P = 0 . 0359 . ( G ) Representative immunoblots of filamin , phosphorylated EIF2a ( pEIF2a ) , total EIF2a and alpha - tubulin in cell lysate from MCF10A MECs ligated to rBM in 3D and treated with ethanol ( mock ) or doxycycline ( FLMN ) for 18h to induce filamin expression and corresponding quantification data ( mean ± SEM ; n = 5 independent biological replicates ) . * * P = 0 . 0018 ( Student ' s t test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Representative immunofluorescence microscopy images of MCF10A MECs ligated with either a rBM in 2D or 3D for 18h and stained with antibody targeting filamin (green). F-actin was counterstained with phalloidin (red). Images show maximum intensity z-projections of xy confocal stacks (left) and middle focal plane of confocal stacks from MECs (right). Yellow arrows indicate filamin aggregates in individual MECs ligated to rBM in 2D. Yellow blunt-end lines highlight the extent of filamin distribution away from the cell edge. Scale bar, 10 μm.(B) The expression level of filamin in MCF10A MECs ligated to rBM in 2D and 3D for 18h was assessed via immunoblot and quantified relative to actin loading control (mean ± SEM; n=5 independent biological replicates). ***P = 0.0008 (Student's t test).(C) Representative fluorescence microscopy images of MCF10A MECs co-expressing the ER-plasma membrane contact site reporter GFP-MAPPER and shRNA targeting filamin (FLMN) or luciferase (Luc) ligated to rBM in 2D for 18h. Scale bar (whole cell), 10 μm; Scale bar (magnified), 2 μm.(D) Quantification of the levels of GFP-MAPPER at the plasma membrane versus total cellular fluorescence in MCF10A MECs stably expressing an shRNA targeting filamin (FLMN) or luciferase (Luc) ligated to rBM in 2D (mean ± SEM; 2D + shLuc, n=38; 2D + shFLMN , n=39 cells from three independent experiments). **P < 0.0021 (Student's t test).(E) Representative immunoblots of filamin, phosphorylated EIF2a (pEIF2a), total EIF2a and alpha-tubulin in cell lysate from MCF10A MECs that express shRNA targeting filamin (shFLMN) or luciferase (shLuc) and ligated to a rBM in 2D or 3D for 18h and corresponding quantification data (mean ± SEM; n=4 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. 2D-shLuc versus 3D-shLuc, ***P = 0.0003. 2D-shLuc versus 2D-shFLMN, ***P = 0.0004.(F) Bar graph of the relative levels of ATF3 mRNA as measured by qPCR in MCF10A MECs expressing shRNA targeting filamin (shFLMN) or luciferase (shLuc) and ligated with rBM in 2D or 3D for 18h (mean ± SEM; n=4 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. 2D-shLuc versus 3D-shLuc, ***P = 0.0005. 2D-shLuc versus 2D-shFLMN, *P = 0.0359.(G) Representative immunoblots of filamin, phosphorylated EIF2a (pEIF2a), total EIF2a and alpha-tubulin in cell lysate from MCF10A MECs ligated to rBM in 3D and treated with ethanol (mock) or doxycycline (FLMN) for 18h to induce filamin expression and corresponding quantification data (mean ± SEM; n=5 independent biological replicates). **P = 0.0018 (Student's t test).


Figure 4 ( A ) Schematic showing the principles behind Atomic Force Microscopy ( AFM ) ( left ) . A cantilever at the end of the microscope probe is deflected when it is in contact with the cell surface . Cell cortex - mediated resistance to indentation alters the path of the laser beam focused on the cantilever that is then reflected onto a photodetector to enable measurement of cellular cortical actin tension . AFM was used to measure the cortical actin tension in MCF10A MECs ligated to a laminin - 111 substrate in 2D or 3D ( right ) and treated with blebbistatin ( Bleb ) to reduce cortical actin tension , induced to overexpress filamin expression ( FLMN ) , or activated ROCK ( ROCK ) to increase cortical actin tension . MECs were indented using a 2 - µm beaded tip on the AFM cantilever and the Hertz model was used to fit each indentation curve to extract the Young ' s modulus of the cell cortex ( mean ± SEM ; 2D , n = 123 ; 3D , n = 184 ; 3D + FLMN , n = 83 ; 2D + Bleb , n = 66 ; 3D + Bleb , n = 65 ; 3D + ROCK , n = 212 ; n = AFM indentation from > 30 cells from three independent experiments ) . Statistical analysis by one - way ANOVA followed by Tukey ' s multiple comparisons test . 2D versus 3D , * * * * P < 0 . 0001 ; 3D versus 3D + FLMN , * * * * P < 0 . 0001 ; 3D versus 3D + ROCK , * * * * P < 0 . 0001 ; 2D versus 2D + Bleb , * * * P = 0 . 0004 . ( B ) Schematic of the principles behind traction force microscopy ( left ) . MECs are plated on compliant polyacrylamide gels containing 100 nm fluorescent beads ( close to the cell - polyacrylamide gel interface ) . Traction stresses are calculated based on the bead displacement induced by substrate deformation and relaxation . Quantification of the traction stresses in individual MCF10A MECs ligated with rBM in 2D and 3D and treated in the absence and presence of blebbistatin ( Bleb ) for 18h ( right ; mean ± SEM ; 2D , n = 139 ; 3D , n = 124 ; 2D + Bleb , n = 42 ; 3D + Bleb , n = 43 cells from three independent experiments ) . The background bead displacement was measured from gel areas that lacked ligated cells ( n = 8 fields from one experiment ) . Statistical analysis by one - way ANOVA followed by Tukey ' s multiple comparisons test . * * * * P < 0 . 0001 . ( C ) Schematic of the principle of active microrheology ( left ) . MCF10A MECs endocytosed 0 . 5 μm polystyrene particles , which were trapped and oscillated using laser optical tweezers to measure the cytoplasmic modulus . The cytoplasmic modulus was measured for MECs ligated to a rBM in 2D or 3D and treated in the absence or presence of blebbistatin ( Bleb ) to reduce cortical actin tension ( right ) ; individual modulus values were calculated based on the slope in the linear range of the normalized force - displacement curve ( mean ± SEM ; 2D , n = 66 ; 3D , n = 69 ; 2D + Bleb , n = 46 ; 3D + Bleb , n = 60 cells from three independent experiments ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD . 2D versus 3D , * * P = 0 . 0028 . 2D versus 2D + Bleb , * P = 0 . 0182 . ( D ) Schematic depicting strategy used to measure cortical tension using laser ablation . ( Top ) Cells with high cortical tension exhibit plasma membrane blebbing when cortical actin is severed by a pulsed laser , whereas cells with lower cortical tension do not . ( Bottom left ) Bar graph of the laser ablation response of MCF10A MECs ligated to a rBM in 2D or 3D ( mean ± SD ; 2D , n = 35 ; 3D , n = 40 cells from three independent experiments ) . ( Bottom right ) Bar graph showing the laser ablation response of MCF10A MECs ligated to a rBM in 2D or 3D and treated in the absence or presence of blebbistatin ( 2D + Bleb and 3D + Bleb ) or expressing constitutively active ROCK ( 3D + ROCK ) ( mean ; 2D , n = 14 ; 3D , n = 15 ; 2D + Bleb , n = 17 ; 3D + Bleb , n = 14 ; 3D + ROCK , n = 10 cells from one experiment ) . ( E ) Representative fluorescence and brightfield images of bleb formation induced by laser ablation in MECs stably expressing LifeAct - RFP . Arrowhead : the site of laser ablation . Scale bar , 10 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Schematic showing the principles behind Atomic Force Microscopy (AFM) (left). A cantilever at the end of the microscope probe is deflected when it is in contact with the cell surface. Cell cortex-mediated resistance to indentation alters the path of the laser beam focused on the cantilever that is then reflected onto a photodetector to enable measurement of cellular cortical actin tension. AFM was used to measure the cortical actin tension in MCF10A MECs ligated to a laminin-111 substrate in 2D or 3D (right) and treated with blebbistatin (Bleb) to reduce cortical actin tension, induced to overexpress filamin expression (FLMN), or activated ROCK (ROCK) to increase cortical actin tension. MECs were indented using a 2-µm beaded tip on the AFM cantilever and the Hertz model was used to fit each indentation curve to extract the Young's modulus of the cell cortex (mean ± SEM; 2D, n=123; 3D, n=184; 3D+FLMN, n=83; 2D+Bleb, n=66; 3D + Bleb, n= 65; 3D+ROCK, n=212; n=AFM indentation from >30 cells from three independent experiments). Statistical analysis by one-way ANOVA followed by Tukey's multiple comparisons test. 2D versus 3D, ****P < 0.0001; 3D versus 3D + FLMN, ****P < 0.0001; 3D versus 3D + ROCK, ****P < 0.0001; 2D versus 2D + Bleb, ***P=0.0004.(B) Schematic of the principles behind traction force microscopy (left). MECs are plated on compliant polyacrylamide gels containing 100 nm fluorescent beads (close to the cell-polyacrylamide gel interface). Traction stresses are calculated based on the bead displacement induced by substrate deformation and relaxation. Quantification of the traction stresses in individual MCF10A MECs ligated with rBM in 2D and 3D and treated in the absence and presence of blebbistatin (Bleb) for 18h (right; mean ± SEM; 2D, n=139; 3D, n=124; 2D+Bleb, n=42; 3D + Bleb, n= 43 cells from three independent experiments). The background bead displacement was measured from gel areas that lacked ligated cells (n=8 fields from one experiment). Statistical analysis by one-way ANOVA followed by Tukey's multiple comparisons test. ****P < 0.0001.(C) Schematic of the principle of active microrheology (left). MCF10A MECs endocytosed 0.5 μm polystyrene particles, which were trapped and oscillated using laser optical tweezers to measure the cytoplasmic modulus. The cytoplasmic modulus was measured for MECs ligated to a rBM in 2D or 3D and treated in the absence or presence of blebbistatin (Bleb) to reduce cortical actin tension (right); individual modulus values were calculated based on the slope in the linear range of the normalized force-displacement curve (mean ± SEM; 2D, n= 66; 3D, n=69; 2D+Bleb, n=46; 3D+Bleb, n=60 cells from three independent experiments). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. 2D versus 3D, **P = 0.0028. 2D versus 2D + Bleb, *P = 0.0182.(D) Schematic depicting strategy used to measure cortical tension using laser ablation. (Top) Cells with high cortical tension exhibit plasma membrane blebbing when cortical actin is severed by a pulsed laser, whereas cells with lower cortical tension do not. (Bottom left) Bar graph of the laser ablation response of MCF10A MECs ligated to a rBM in 2D or 3D (mean ± SD; 2D, n= 35; 3D, n=40 cells from three independent experiments). (Bottom right) Bar graph showing the laser ablation response of MCF10A MECs ligated to a rBM in 2D or 3D and treated in the absence or presence of blebbistatin (2D+Bleb and 3D+Bleb) or expressing constitutively active ROCK (3D+ROCK) (mean; 2D, n= 14; 3D, n=15; 2D+Bleb, n=17; 3D+Bleb, n=14; 3D+ ROCK, n=10 cells from one experiment).(E) Representative fluorescence and brightfield images of bleb formation induced by laser ablation in MECs stably expressing LifeAct-RFP. Arrowhead: the site of laser ablation. Scale bar, 10 μm.


Figure 5 ( A ) Representative line graph showing the changes in cytosolic [ Ca2 + ] levels of MCF10A MECs in response to treatment . Fura - 2 - loaded MECs ligated with rBM in 2D were treated with DMSO ( 2D ; blue ) or with blebbistatin ( 2D + Bleb ; red ) and preincubated with EGTA to chelate extracellular Ca2 + in the bath solution , challenged with 2 μM thapsigargin to induce ER Ca2 + release , and replenished with 4 mM Ca2 + in bath solution at the indicated times . F340 / F380 values for each cell were quantified over the course of imaging and plotted ( mean ± SD ; 2D , n = 7 ; 2D + Bleb , n = 7 cells from one experiment ) . ( B ) Quantification of the amplitude of the Ca2 + response ( ∆ F = F - F0 ) induced by thapsigargin , where F0 is the basal fluorescence before thapsigargin treatment . The data shown indicated mean ± SEM ( 2D , n = 13 ; 2D + Bleb , n = 12 cells from two independent experiments ) . * * * * P < 0 . 0001 ( Student ' s t test ) . ( C ) Representative immunoblots of phosphorylated EIF2a ( pEIF2a ) , total EIF2a and alpha - tubulin in cell lysate from MCF10A MECs ligated to a rBM in 2D or 3D and treated in the absence or presence of blebbistatin ( Bleb ) . Corresponding quantification data are shown at bottom ( mean ± SEM ; n = 4 independent biological replicates ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD . 2D versus 3D , * * P = 0 . 0022 . 2D versus 2D + Bleb , * P = 0 . 0248 . ( D ) Bar graph of qPCR data measuring the relative levels of ATF3 mRNA in MECs ligated with rBM in 2D and treated in the absence or presence of blebbistatin ( Bleb ) ( mean ± SEM ; n = 4 independent biological replicates ) . * P = 0 . 0158 ( Student ' s t test ) . ( E ) Graph of the levels of GFP - MAPPER at the plasma membrane relative total cellular GFP - MAPPER fluorescence in MCF10A MECs ligated with rBM in 2D and treated in the absence or presence of blebbistatin ( Bleb ) ( mean ± SEM ; 2D , n = 39 ; 2D + Bleb , n = 39 cells from three independent experiments ) . * * * P = 0 . 0002 ( Student ' s t test ) . ( G ) Scatter plot of the number of proximity ligation assay ( PLA ) puncta within individual cells expressing myc - PERK that were ligated to rBM in 2D or 3D , treated in the absence or presence of blebbistatin ( Bleb ) for 25h and stained with PLA antibody probes specific for endogenous filamin and myc - PERK . Representative images of PLA staining from these experiments can be found in Panel H . Background PLA signal was measured from cells stained in the absence of primary antibodies ( mean ± SEM ; 2D , n = 47 ; 3D , n = 32 cells ; 2D + Bleb , n = 39 ; background , n = 14 cells from three independent experiments ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD . * * * * P < 0 . 0001 . ( H ) Representative fluorescence microscopy images of cells expressing myc - PERK that were ligated to rBM in 2D or 3D , treated in the absence or presence of blebbistatin ( Bleb ) for 25h and stained with PLA antibody probes specific for endogenous filamin and myc - PERK ( red puncta ) . F - actin was stained with phalloidin ( I ) Representative line graph showing the changes in cytosolic [ Ca2 + ] levels of MCF10A MECs in response to treatment . Fura - 2 - loaded MCF10A MECs ligated with rBM in 2D and expressing filamin repeats 21 - 23 ( 2D + FLMNIg21 - Ig23 ) or vector control ( 2D ) were pre - incubated with EGTA to chelate extracellular Ca2 + in the bath solution , challenged with 2 μM thapsigargin to induce ER Ca2 + release , and replenished with 4 mM Ca2 + in bath solution at the indicated time . F340 / F380 values of individual cells were quantified over the course of imaging ( mean ± SD ( 2D , n = 8 ; 2D + FLMNIg21 - Ig23 , n = 7 cells from one experiment ) . ( J ) Quantification of the amplitude of the Ca2 + response ( ∆ F = F - F0 ) induced by thapsigargin , where F0 is the basal fluorescence before thapsigargin treatment . The data shown indicated mean ± SEM ( 2D , n = 21 ; 2D + FLMNIg21 - Ig23 , n = 19 cells from three independent experiments ) . * * * * P < 0 . 0001 ( Student ' s t test ) . ( K ) Bar graph of qPCR data measuring the relative levels of ATF3 mRNA in MCF10A MECs expressing luciferase control or filamin repeats 21 - 23 ( FLMNIg21 - Ig23 ) ligated with rBM in 2D for 18h ( mean ± SEM ; n = 3 independent biological replicates ) . * P = 0 . 032 ( Student ' s t test ) . ( L ) Representative immunoblots of phosphorylated EIF2a ( pEIF2a ) , total EIF2a and alpha - tubulin in cell lysate from MCF10A MECs expressing luciferase ( control ) or filamin repeats 21 - 23 ( FLMNIg21 - Ig23 - myc ) that were ligated with rBM in 2D . Corresponding quantification data are shown at bottom ( mean ± SEM ; n = 5 independent biological replicates ) . * P = 0 . 0112 ( Student ' s t test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Representative line graph showing the changes in cytosolic [Ca2+] levels of MCF10A MECs in response to treatment. Fura-2-loaded MECs ligated with rBM in 2D were treated with DMSO (2D; blue) or with blebbistatin (2D+Bleb; red) and preincubated with EGTA to chelate extracellular Ca2+ in the bath solution, challenged with 2 μM thapsigargin to induce ER Ca2+ release, and replenished with 4 mM Ca2+ in bath solution at the indicated times. F340/F380 values for each cell were quantified over the course of imaging and plotted (mean ± SD; 2D, n=7; 2D + Bleb, n=7 cells from one experiment). (B) Quantification of the amplitude of the Ca2+ response (∆F=F-F0) induced by thapsigargin, where F0 is the basal fluorescence before thapsigargin treatment. The data shown indicated mean ± SEM (2D, n=13; 2D + Bleb, n=12 cells from two independent experiments). ****P < 0.0001 (Student's t test).(C) Representative immunoblots of phosphorylated EIF2a (pEIF2a), total EIF2a and alpha-tubulin in cell lysate from MCF10A MECs ligated to a rBM in 2D or 3D and treated in the absence or presence of blebbistatin (Bleb). Corresponding quantification data are shown at bottom (mean ± SEM; n=4 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. 2D versus 3D, **P = 0.0022. 2D versus 2D + Bleb, *P = 0.0248.(D) Bar graph of qPCR data measuring the relative levels of ATF3 mRNA in MECs ligated with rBM in 2D and treated in the absence or presence of blebbistatin (Bleb) (mean ± SEM; n=4 independent biological replicates). *P = 0.0158 (Student's t test).(E) Graph of the levels of GFP-MAPPER at the plasma membrane relative total cellular GFP-MAPPER fluorescence in MCF10A MECs ligated with rBM in 2D and treated in the absence or presence of blebbistatin (Bleb) (mean ± SEM; 2D, n=39; 2D + Bleb, n=39 cells from three independent experiments). ***P = 0.0002 (Student's t test).(G) Scatter plot of the number of proximity ligation assay (PLA) puncta within individual cells expressing myc-PERK that were ligated to rBM in 2D or 3D, treated in the absence or presence of blebbistatin (Bleb) for 25h and stained with PLA antibody probes specific for endogenous filamin and myc-PERK. Representative images of PLA staining from these experiments can be found in Panel H. Background PLA signal was measured from cells stained in the absence of primary antibodies (mean ± SEM; 2D, n=47; 3D, n=32 cells; 2D + Bleb, n=39; background, n=14 cells from three independent experiments). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. ****P < 0.0001.(H) Representative fluorescence microscopy images of cells expressing myc-PERK that were ligated to rBM in 2D or 3D, treated in the absence or presence of blebbistatin (Bleb) for 25h and stained with PLA antibody probes specific for endogenous filamin and myc-PERK (red puncta). F-actin was stained with phalloidin(I) Representative line graph showing the changes in cytosolic [Ca2+] levels of MCF10A MECs in response to treatment. Fura-2-loaded MCF10A MECs ligated with rBM in 2D and expressing filamin repeats 21-23 (2D+FLMNIg21-Ig23) or vector control (2D) were pre-incubated with EGTA to chelate extracellular Ca2+ in the bath solution, challenged with 2 μM thapsigargin to induce ER Ca2+ release, and replenished with 4 mM Ca2+ in bath solution at the indicated time. F340/F380 values of individual cells were quantified over the course of imaging (mean ± SD (2D, n=8; 2D + FLMNIg21-Ig23, n=7 cells from one experiment). (J) Quantification of the amplitude of the Ca2+ response (∆F=F-F0) induced by thapsigargin, where F0 is the basal fluorescence before thapsigargin treatment. The data shown indicated mean ± SEM (2D, n=21; 2D + FLMNIg21-Ig23, n=19 cells from three independent experiments). ****P < 0.0001 (Student's t test).(K) Bar graph of qPCR data measuring the relative levels of ATF3 mRNA in MCF10A MECs expressing luciferase control or filamin repeats 21-23 (FLMNIg21-Ig23) ligated with rBM in 2D for 18h (mean ± SEM; n=3 independent biological replicates). *P = 0.032 (Student's t test).(L) Representative immunoblots of phosphorylated EIF2a (pEIF2a), total EIF2a and alpha-tubulin in cell lysate from MCF10A MECs expressing luciferase (control) or filamin repeats 21-23 (FLMNIg21-Ig23-myc) that were ligated with rBM in 2D. Corresponding quantification data are shown at bottom (mean ± SEM; n=5 independent biological replicates). *P = 0.0112 (Student's t test).


Figure 6 ( A ) Bar graph of qPCR data measuring the levels of SEC61B mRNA in MECs ligated with rBM in 2D and treated in the absence and presence of blebbistatin ( Bleb ) ( mean ± SEM ; n = 3 independent biological replicates ) . * * P = 0 . 0046 ( Student ' s t test ) . ( B ) Change in the Helmholtz free energy of membrane protrusion assembly ( ∆ F ) as a function of cell membrane excess area ( A / Ap ) , which is the variable conjugate to the cortical actin tension . ( D ) Representative time - lapse confocal microscopy images of MECs stably expressing LifeAct - RFP that were ligated to a rBM in 2D ( 2D ) or 3D ( 3D ) . Time in seconds ( s ) is indicated in each inset ; scale bar , 10 μm . ( E ) Bar graph of protrusion length measurements in MECs ligated to rBM in 2D , 3D , or in 2D treated with blebbistatin ( 2D + Bleb ) ( mean ± SEM ; 2D , n = 139 ; 3D , n = 106 ; 2D + Bleb , n = 88 protrusions from three independent experiments ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD . * * * * P < 0 . 0001 . ( F ) Bar graph of protrusion residence time measurements in MECs ligated to rBM in 2D ( 2D ) , 3D ( 3D ) , or in 2D treated with blebbistatin ( 2D + Bleb ) ( mean ± SEM ; 2D , n = 30 ; 3D , n = 30 ; 2D + Bleb , n = 38 protrusions from three independent experiments ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD . * * * * P < 0 . 0001 . ( G ) 30 - nm thick slice through a cellular tomogram focusing on protrusions emanating from a vitrified no - spread MECs cultured on a rBM in the absence ( left , 2D ) or in the presence of blebbistatin ( right , 2D + Bleb ) . The position of the cell body is towards the upper right - hand corner ( marked with white asterisk ) . Scale bars , 200 nm . Note : under conditions of low cortical actin tension ( blebbistatin treatment ) the protrusions visualized in these MECs appeared to be highly interdigitated with sharp kinks , consistent with a compliant phenotype . By contrast , the protrusions observed in the 2D samples ( higher cortical actin tension ) were predominantly straight and outwardly projected , suggesting they were stiffer than the protrusions formed in the MECs with lower cortical actin tension ( e . g . blebbistatin treated ) . ( H ) Quantification of membrane protrusion width in tomograms from non - spread MECs interacting with rBM treated with ( 2D + Bleb ) and without blebbistatin ( 2D ) . n > 2000 protrusions . Statistical significance of differences between the distributions was assessed using Mann - Whitney rank tests . * * * * P < 0 . 0001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Bar graph of qPCR data measuring the levels of SEC61B mRNA in MECs ligated with rBM in 2D and treated in the absence and presence of blebbistatin (Bleb) (mean ± SEM; n=3 independent biological replicates). **P = 0.0046 (Student's t test).(B) Change in the Helmholtz free energy of membrane protrusion assembly (∆F) as a function of cell membrane excess area (A/Ap), which is the variable conjugate to the cortical actin tension.(D) Representative time-lapse confocal microscopy images of MECs stably expressing LifeAct-RFP that were ligated to a rBM in 2D (2D) or 3D (3D). Time in seconds (s) is indicated in each inset; scale bar, 10 μm.(E) Bar graph of protrusion length measurements in MECs ligated to rBM in 2D, 3D, or in 2D treated with blebbistatin (2D + Bleb) (mean ± SEM; 2D, n= 139; 3D, n=106; 2D + Bleb, n=88 protrusions from three independent experiments). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. ****P < 0.0001. (F) Bar graph of protrusion residence time measurements in MECs ligated to rBM in 2D (2D), 3D (3D), or in 2D treated with blebbistatin (2D+ Bleb) (mean ± SEM; 2D, n= 30; 3D, n=30; 2D + Bleb, n=38 protrusions from three independent experiments). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. ****P < 0.0001.(G) 30-nm thick slice through a cellular tomogram focusing on protrusions emanating from a vitrified no-spread MECs cultured on a rBM in the absence (left, 2D) or in the presence of blebbistatin (right, 2D+ Bleb). The position of the cell body is towards the upper right-hand corner (marked with white asterisk). Scale bars, 200 nm. Note: under conditions of low cortical actin tension (blebbistatin treatment) the protrusions visualized in these MECs appeared to be highly interdigitated with sharp kinks, consistent with a compliant phenotype. By contrast, the protrusions observed in the 2D samples (higher cortical actin tension) were predominantly straight and outwardly projected, suggesting they were stiffer than the protrusions formed in the MECs with lower cortical actin tension (e.g. blebbistatin treated). (H) Quantification of membrane protrusion width in tomograms from non-spread MECs interacting with rBM treated with (2D+Bleb) and without blebbistatin (2D). n > 2000 protrusions. Statistical significance of differences between the distributions was assessed using Mann-Whitney rank tests. ****P < 0.0001.


Figure 7 ( C ) ( Top ) Model of how exocyst components ( including Exo70 ) tether vesicles during exocytosis . Representative fluorescence microscopy images of MECs stably co - expressing EGFP - Exo70 ( Exo70 ) and the plasma membrane reporter farnesylated mCherry ( PM ) ligated to rBM in 2D , 3D , and treated with blebbistatin ( 2D + Bleb ) . Fluorescent images of whole cells are shown ( left ) and cell - edge protrusions are magnified within inset ( right ) . Colocalization of Exo70 and membrane protrusions are highlighted with yellow arrows . Scale bar ( whole cell ) , 5 μm ; Scale bar ( magnified inset ) , 1 μm . ( 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 . ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(C) (Top) Model of how exocyst components (including Exo70) tether vesicles during exocytosis. Representative fluorescence microscopy images of MECs stably co-expressing EGFP-Exo70 (Exo70) and the plasma membrane reporter farnesylated mCherry (PM) ligated to rBM in 2D, 3D, and treated with blebbistatin (2D+Bleb). Fluorescent images of whole cells are shown (left) and cell-edge protrusions are magnified within inset (right). Colocalization of Exo70 and membrane protrusions are highlighted with yellow arrows. Scale bar (whole cell), 5 μm; Scale bar (magnified inset), 1 μm.(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.(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).(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.


Figure 8 ( 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 ) . ( 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 . ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(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).(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.(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.(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.(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).


Figure 1A - 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A-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.


Figure 2One - 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 ) . 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2One-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).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).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).


Figure 3A 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 ) . 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 . 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 . 05Mosquitoes 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A 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).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. 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 > 0.05Mosquitoes 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).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).


Figure 4B 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 ) . 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 .

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Figure 4B 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). 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.


Figure 5B 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 . 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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Figure 5B 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.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.