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 1 ( C ) Patients suffering from major depressive disorder ( MDD ) show a blunted ACTH response to CRH ( black line , N = 10 ) , compared with control ( gray line , N = 10 ) - data from ( von Bardeleben et al , 1988 ) , shown are mean ± SEM . ( D ) Patients suffering from anorexia and admitted to treatment show a blunted ACTH response and hypercortisolemia , which resolves within 6 - 24 months after weight normalization - data from ( Gold et al , 1986a ) . However , 3 - 4 weeks after weight normalization , cortisol dynamics are normal whereas ACTH dynamics are blunted . Pregnancy is associated with elevated cortisol levels due to CRH secretion by the placenta . 3 weeks after delivery , cortisol levels and dynamics return to normal , whereas ACTH dynamics are blunted - data from ( Magiakou et al , 1996 ) . After 12 weeks , ACTH dynamics normalize as well , while cortisol dynamics remain normal . Individuals recovering from alcohol abuse show hypercortisolemia and blunted ACTH response after admission - data from ( von Bardeleben et al , 1989 ) . After 2 - 6 weeks , these individuals show normal cortisol dynamics , but blunted ACTH responses persist . In all panels , control patient data are denoted by thin gray line ( Anorexia : N = 13 . Pregnancy : N was unspecified . Alcohol abuse disorder : N = 11 ) , and case data by a thicker black line ( Anorexia : left panel , N = 9 , center panel , N = 5 , right panel , N = 6 . Pregnancy : N = 17 . Alcohol abuse disorder : N = 20 ) . Shown are mean ± SEM for all panels .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(C) Patients suffering from major depressive disorder (MDD) show a blunted ACTH response to CRH (black line, N=10), compared with control (gray line, N=10) - data from (von Bardeleben et al, 1988), shown are mean ± SEM.(D) Patients suffering from anorexia and admitted to treatment show a blunted ACTH response and hypercortisolemia, which resolves within 6-24 months after weight normalization- data from (Gold et al, 1986a). However, 3-4 weeks after weight normalization, cortisol dynamics are normal whereas ACTH dynamics are blunted. Pregnancy is associated with elevated cortisol levels due to CRH secretion by the placenta. 3 weeks after delivery, cortisol levels and dynamics return to normal, whereas ACTH dynamics are blunted- data from (Magiakou et al, 1996). After 12 weeks, ACTH dynamics normalize as well, while cortisol dynamics remain normal. Individuals recovering from alcohol abuse show hypercortisolemia and blunted ACTH response after admission - data from (von Bardeleben et al, 1989). After 2-6 weeks, these individuals show normal cortisol dynamics, but blunted ACTH responses persist. In all panels, control patient data are denoted by thin gray line (Anorexia: N=13. Pregnancy: N was unspecified. Alcohol abuse disorder: N=11), and case data by a thicker black line (Anorexia: left panel, N=9, center panel, N=5, right panel, N=6. Pregnancy: N=17. Alcohol abuse disorder: N=20). Shown are mean ± SEM for all panels.


Figure 2A Structure of autoinhibited ScV1 . The catalytic A ( pink ) and non catalytic B ( green ) subunits are arranged as a heterohexamer around the central rotor subunits D ( red ) and F ( cyan ) . The three peripheral stalks ( EG heterodimers 1 - 3 , blue and orange , respectively ) are bound at the periphery of the B subunits . The inhibitory H subunit ( yellow ) is unique to the eukaryotic V - ATPase . Unless otherwise stated , the coloring scheme used here for the V1 subunits will be used from here on . B Interaction of the peripheral stalks with the catalytic hexamer . Each peripheral stalk crosses a non - catalytic A / B interface on its way from the top of the V1 to the base of the catalytic core . Representative view of the interaction is shown for EG3 . C Interaction of peripheral stalks EG1 and 2 with the catalytic hexamer . The peripheral stalks are bound to the top of the B subunits via their C - termini ( boxed ) . The electron density ( contoured at 1 . 2 σ ) for the C - terminal domains of EG1 and EG2 is shown in the panels to the left and right . D The structure of autoinhibited ScV1rotated 90 ° around the vertical from the view shown in panel A to highlight peripheral stalks EG2 and EG3 . Note the bent appearance of EG3 compared to EG2 along with the presence of a partially unstructured region known as the " bulge " in subunit G ( indicated with an arrow ) . The box shows electron density ( contoured at 1 . 2 σ ) for the EG3 N - termini contacting the C - terminal domain of the A subunit of a closed catalytic site ( see asterisk ) . The horizontal lines on the structure indicate the sections shown in panels E ( upper two lines ) and F ( lower two lines ) . E Section through the ScV1 electron density map showing interaction of the A3B3 catalytic hexamer with the peripheral stalks ( EG1 - 3 ) . View is rotated 90 ° from the view in A , looking up the central rotor . The A / B pairs forming the three catalytic sites are designated ( AB ) 1 - 3 according to which of the three peripheral stalks is bound to each B subunit . For example , ( AB ) 1 has peripheral stalk EG1 bound to its B subunit . The black arrowheads indicate the non - catalytic A / B interfaces . F Section through the ScV1 electron density map at the level of the N - termini of the peripheral stalks . Note the change in position of the N - termini of the peripheral stalks , which have each crossed a non - catalytic interface ( black arrowheads in E ) and are now in proximity to the C - termini of the catalytic A subunits ( ACT ) of the adjacent A / B pairs . While EG1 and EG2 are now near ACT from ( AB ) 3 and ( AB ) 1 , respectively ( see double headed arrows ) , only the bent EG3 peripheral stalk is close enough to contact the corresponding ACT from ( AB ) 2 ( see asterisk ) .

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 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 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 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

Figure 2A Structure of autoinhibited ScV1. The catalytic A (pink) and non catalytic B (green) subunits are arranged as a heterohexamer around the central rotor subunits D (red) and F (cyan). The three peripheral stalks (EG heterodimers 1-3, blue and orange, respectively) are bound at the periphery of the B subunits. The inhibitory H subunit (yellow) is unique to the eukaryotic V-ATPase. Unless otherwise stated, the coloring scheme used here for the V1 subunits will be used from here on.B Interaction of the peripheral stalks with the catalytic hexamer. Each peripheral stalk crosses a non-catalytic A/B interface on its way from the top of the V1 to the base of the catalytic core. Representative view of the interaction is shown for EG3.C Interaction of peripheral stalks EG1 and 2 with the catalytic hexamer. The peripheral stalks are bound to the top of the B subunits via their C-termini (boxed). The electron density (contoured at 1.2 σ) for the C-terminal domains of EG1 and EG2 is shown in the panels to the left and right.D The structure of autoinhibited ScV1rotated 90° around the vertical from the view shown in panel A to highlight peripheral stalks EG2 and EG3. Note the bent appearance of EG3 compared to EG2 along with the presence of a partially unstructured region known as the "bulge" in subunit G (indicated with an arrow). The box shows electron density (contoured at 1.2 σ) for the EG3 N-termini contacting the C-terminal domain of the A subunit of a closed catalytic site (see asterisk). The horizontal lines on the structure indicate the sections shown in panels E (upper two lines) and F (lower two lines).E Section through the ScV1 electron density map showing interaction of the A3B3 catalytic hexamer with the peripheral stalks (EG1-3). View is rotated 90° from the view in A, looking up the central rotor. The A/B pairs forming the three catalytic sites are designated (AB)1-3 according to which of the three peripheral stalks is bound to each B subunit. For example, (AB)1 has peripheral stalk EG1 bound to its B subunit. The black arrowheads indicate the non-catalytic A/B interfaces.F Section through the ScV1 electron density map at the level of the N-termini of the peripheral stalks. Note the change in position of the N-termini of the peripheral stalks, which have each crossed a non-catalytic interface (black arrowheads in E) and are now in proximity to the C-termini of the catalytic A subunits (ACT) of the adjacent A/B pairs. While EG1 and EG2 are now near ACT from (AB)3 and (AB)1, respectively (see double headed arrows), only the bent EG3 peripheral stalk is close enough to contact the corresponding ACT from (AB)2 (see asterisk).


Figure 3A The A3B3DF complex shown with the subunits unique to the eukaryotic V1 removed , highlighting the core conserved catalytic complex labeled as " Catalytic core " . The three peripheral stalks and inhibitory subunit H are found only in the eukaryotic V1 , whereas A3B3DF represents the full complement of subunits in the prokaryotic V1 complex . Notably , the prokaryotic V1 is an active ATP hydrolase ( Arai et al , 2009 ) . The orientation of the A and B subunits with respect to the membrane is indicated by the arrow on the left . B Conformational differences in the catalytic A subunits . The catalytic A subunits are shown aligned by their N - terminal β barrels , highlighting the change in conformation of C - terminal α helical bundles associated with the closed ( ( AB ) 2 and ( AB ) 3 ; dark and light gray , respectively ) and open ( ( AB ) 1 ; magenta ) catalytic sites . Movement of this domain in response to nucleotide occupancy is thought to drive rotation of the central rotor during catalysis . C ScV1 ' s catalytic hexamer contains one open and two closed catalytic sites . Side view of the three catalytic hexamer A / B pairs ( shown in surface representation ) . Note that the two closed pairs ( AB ) 2 and 3 are non - equivalently closed . D Alignment of the A3B3DF from ScV1 and nucleotide - free EhV1 by the catalytic hexamers . The A3B3DF from ScV1 is shown in the same color scheme as in Fig 2 , EhV1 is in blue . The alignment shows the difference in position of the central rotor subunit , D . Note that the C - terminus of subunit D ( DCT ) is longer in eukaryotic V - ATPase ( see electron density contoured at 1 . 2 σ in the box to the right ) . E ScV1 and EhV1are halted in different rotational positions . Alignment of the A3B3DF of ScV1 and nucleotide - free EhV1 ( blue , with dashed lines drawn through the A subunits ) by the central stalk subunit D , reveals that the two catalytic cores are halted in different rotational positions . The autoinhibited ScV1 requires 40 ° rotation to overlay its catalytic hexamer with that of the EhV1 . F Comparison of the D subunits of ScV1 ( red ) and nucleotide free EhV1 ( blue ) . HCT from ScV1 is shown in yellow and A subunit from EhV1 is shown in dark blue .

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 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 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 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 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 3A The A3B3DF complex shown with the subunits unique to the eukaryotic V1 removed, highlighting the core conserved catalytic complex labeled as "Catalytic core". The three peripheral stalks and inhibitory subunit H are found only in the eukaryotic V1, whereas A3B3DF represents the full complement of subunits in the prokaryotic V1 complex. Notably, the prokaryotic V1 is an active ATP hydrolase (Arai et al, 2009). The orientation of the A and B subunits with respect to the membrane is indicated by the arrow on the left.B Conformational differences in the catalytic A subunits. The catalytic A subunits are shown aligned by their N-terminal β barrels, highlighting the change in conformation of C-terminal α helical bundles associated with the closed ((AB)2 and (AB)3; dark and light gray, respectively) and open ((AB)1; magenta) catalytic sites. Movement of this domain in response to nucleotide occupancy is thought to drive rotation of the central rotor during catalysis.C ScV1's catalytic hexamer contains one open and two closed catalytic sites. Side view of the three catalytic hexamer A/B pairs (shown in surface representation). Note that the two closed pairs (AB)2 and 3 are non-equivalently closed.D Alignment of the A3B3DF from ScV1 and nucleotide-free EhV1 by the catalytic hexamers. The A3B3DF from ScV1 is shown in the same color scheme as in Fig 2, EhV1 is in blue. The alignment shows the difference in position of the central rotor subunit, D. Note that the C-terminus of subunit D (DCT) is longer in eukaryotic V-ATPase (see electron density contoured at 1.2 σ in the box to the right).E ScV1 and EhV1are halted in different rotational positions. Alignment of the A3B3DF of ScV1 and nucleotide-free EhV1 (blue, with dashed lines drawn through the A subunits) by the central stalk subunit D, reveals that the two catalytic cores are halted in different rotational positions. The autoinhibited ScV1 requires 40° rotation to overlay its catalytic hexamer with that of the EhV1.F Comparison of the D subunits of ScV1 (red) and nucleotide free EhV1 (blue). HCT from ScV1 is shown in yellow and A subunit from EhV1 is shown in dark blue.


Figure 4A ScV1 - H subunit interactions . Region of electron density ( contoured at 1 . 2 σ ) outlined by the box in the side view of ScV1 shown to the right . The H subunit N - terminal domain ( HNT ) is bound to the N - terminal α helices of peripheral stalk EG1 while its globular C - terminal domain ( HCT ) is bound at the base of the catalytic hexamer . B Interaction between HNT and peripheral stalk EG1 . View of HNT as in panel A rotated 110 ° along the horizontal , showing the α helical HEAT repeats of HNT forming a pocket for binding the N - termini of peripheral stalk EG1 . HNT ' s α helices forming the pocket are labeled as in ( Sagermann et al , 2001 ) . Pro197 on HNT ( red sphere ) has been previously shown to cross - link subunit E ( Jefferies Forgac , 2008 ) . C Interaction between HCT and the base of the catalytic hexamer . The H subunit inhibitory contacts at the base of the ScV1 are mediated by a loop in HCT ( highlighted in magenta ) that is seen to contact two short segments of the C - terminal domain of the subunit G from the open catalytic site ( AB ) 1 ( highlighted in purple ) and two turns of the N - terminal a helix of the central rotor subunit D ( highlighted in blue ) . D HCT binds to the open ( AB ) 1 pair . View of ScV1 looking along the central rotor subunit D towards the cytoplasm , showing the position of the H subunit binding at the interface of the open A / B pair ( AB ) 1 . The catalytic hexamer is shown in surface representation and the central rotor , peripheral stalks and subunit H are shown as ribbons .

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

Figure 4A ScV1-H subunit interactions. Region of electron density (contoured at 1.2 σ) outlined by the box in the side view of ScV1 shown to the right. The H subunit N-terminal domain (HNT) is bound to the N-terminal α helices of peripheral stalk EG1 while its globular C-terminal domain (HCT) is bound at the base of the catalytic hexamer.B Interaction between HNT and peripheral stalk EG1. View of HNT as in panel A rotated 110° along the horizontal, showing the α helical HEAT repeats of HNT forming a pocket for binding the N-termini of peripheral stalk EG1. HNT's α helices forming the pocket are labeled as in (Sagermann et al, 2001). Pro197 on HNT (red sphere) has been previously shown to cross-link subunit E (Jefferies Forgac, 2008).C Interaction between HCT and the base of the catalytic hexamer. The H subunit inhibitory contacts at the base of the ScV1 are mediated by a loop in HCT (highlighted in magenta) that is seen to contact two short segments of the C-terminal domain of the subunit G from the open catalytic site (AB)1 (highlighted in purple) and two turns of the N-terminal a helix of the central rotor subunit D (highlighted in blue).D HCT binds to the open (AB)1 pair. View of ScV1 looking along the central rotor subunit D towards the cytoplasm, showing the position of the H subunit binding at the interface of the open A/B pair (AB)1. The catalytic hexamer is shown in surface representation and the central rotor, peripheral stalks and subunit H are shown as ribbons.


Figure 5C Growth phenotype of H subunit mutants . Deletion of V - ATPase subunits from yeast results in a conditional lethal phenotype ( Vma - ) characterized by an inability to grow at pH 7 . 5 in the presence of 60 mM CaCl2 ( upper panel ) , but growth is observed at pH 5 ( lower panel ) . Subunit H mutations were introduced on a plasmid into a yeast strain deleted for the H subunit and their phenotypes assessed . Both humanH subunit isoforms ( HsH1 , HsH2 ) , a chimeric H subunit ( HChim ) containing ScHNT and HsHCT , mutations in yeast intended to mimic the length of the human H loop sequence and accessibility ( Hloop ) , and a point mutation of the conserved aspartic acid residue in the loop sequence ( HD410A ) are shown compared to the H subunit deletion ( ΔH ) and wild type H on a plasmid ( H WTpl ) . All grow on pH 5 medium , but the human isoforms failed to complement the deletion phenotype . D ATPase activity of H subunit mutants . The ScV1 sectors were purified from each strain that complemented the phenotype as well as from ScV1ΔH , for comparison of MgATPase activity . Average specific activities are shown plotted + / - s . e . m from two independent purifications . The ScV1 sector containing chromosomally encoded H subunit shows no detectable MgATPase activity ( Parra et al , 2000 ; Zhang et al , 2003 ) , whereas ScV1containing plasmid - borne wild type H ( H WTpl ) exhibits low levels of activity likely due to reduced expression from the low copy plasmid . The HChim and HLoop mutants lead to a loss of activity silencing , displaying nearly the same activity as ScV1ΔH . E SDS - PAGE and Western Blot analysis of ScV1 preparations . Left panel , 15 % SDS PAGE of ScV1 purified from the subunit H mutant and WT strains . Note that H WTch is purified from a yeast strain deleted for the C subunit ( used for crystallization ) . The remaining strains contain subunit C , which is co - purified with ScV1 in varying levels . Note that while H WTch ScV1 , which is expressed from a strain deleted for subunit C , displays no detectable MgATPase activity , the H subunit mutants co - purified with the most C subunit display the least activity , suggesting that absence or presence of subunit C has no effect on MgATPase activity . Right panel , immunoblot probed with an antibody directed against the N - terminal Myc tagged mutant H subunits expressed from a plasmid . Note that the levels of mutant H expression does not appear to vary from strain to strain .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5C Growth phenotype of H subunit mutants. Deletion of V-ATPase subunits from yeast results in a conditional lethal phenotype (Vma-) characterized by an inability to grow at pH 7.5 in the presence of 60 mM CaCl2 (upper panel), but growth is observed at pH 5 (lower panel). Subunit H mutations were introduced on a plasmid into a yeast strain deleted for the H subunit and their phenotypes assessed. Both humanH subunit isoforms (HsH1, HsH2), a chimeric H subunit (HChim) containing ScHNT and HsHCT, mutations in yeast intended to mimic the length of the human H loop sequence and accessibility (Hloop), and a point mutation of the conserved aspartic acid residue in the loop sequence (HD410A) are shown compared to the H subunit deletion (ΔH) and wild type H on a plasmid (H WTpl). All grow on pH 5 medium, but the human isoforms failed to complement the deletion phenotype.D ATPase activity of H subunit mutants. The ScV1 sectors were purified from each strain that complemented the phenotype as well as from ScV1ΔH, for comparison of MgATPase activity. Average specific activities are shown plotted +/- s.e.m from two independent purifications. The ScV1 sector containing chromosomally encoded H subunit shows no detectable MgATPase activity (Parra et al, 2000; Zhang et al, 2003), whereas ScV1containing plasmid-borne wild type H (H WTpl) exhibits low levels of activity likely due to reduced expression from the low copy plasmid. The HChim and HLoop mutants lead to a loss of activity silencing, displaying nearly the same activity as ScV1ΔH.E SDS-PAGE and Western Blot analysis of ScV1 preparations. Left panel, 15% SDS PAGE of ScV1 purified from the subunit H mutant and WT strains. Note that H WTch is purified from a yeast strain deleted for the C subunit (used for crystallization). The remaining strains contain subunit C, which is co-purified with ScV1 in varying levels. Note that while H WTch ScV1, which is expressed from a strain deleted for subunit C, displays no detectable MgATPase activity, the H subunit mutants co-purified with the most C subunit display the least activity, suggesting that absence or presence of subunit C has no effect on MgATPase activity. Right panel, immunoblot probed with an antibody directed against the N-terminal Myc tagged mutant H subunits expressed from a plasmid. Note that the levels of mutant H expression does not appear to vary from strain to strain.


Figure 6A Overlay of ScV1 and the cryo - EM structure of the V1Vo holoenzyme in " state 2 " ( Zhao et al , 2015 ) . The structures of ScV1 and ScV1Vo were overlaid by aligning the A3B3 catalytic hexamers , thus highlighting structural differences in the central rotor and peripheral stator subunits . Coloring of ScV1Vo ( shown as semi - transparent ribbons ) is as used throughout for ScV1 for all but the A3B3F ( tan ) , subunit H ( dark green ) , subunit C and Vo domain ( gray ) . The EG1 - HNT interaction is boxed . Note the shift in position of HNT and EG1 between ScV1 and ScV1Vo . B Comparison of peripheral stalk EG1 and HNT in ScV1 versus ScV1Vo . The boxed view in A is shown enlarged . The N - terminal α helices of peripheral stalk EG1 and HNT change in conformation , likely induced by the movement of HCT when going from the holo enzyme to the autoinhibited conformation . C View as in A rotated 150º around the vertical . Comparisons of peripheral stalk EG3 and HCT are boxed . D Comparison of peripheral stalk EG3 in ScV1 versus ScV1Vo . In ScV1Vo , EG3 binds to the C subunit head domain ( Chead ; gray ) whereas in ScV1 , EG3 is bent towards the C - terminal domain of a catalytic A subunit ( enlarged view of box labeled EG3 in panel C ) . E Comparison of HCT ' s conformation in ScV1 versus ScV1Vo . Enlarged view of box labeled HCT in panel C highlighting the conformational changes and binding interactions of HCT ; for clarity only aNT , HCT and the central rotor ( DF ) are shown . In ScV1Vo , the most C - terminal α helix ( dark green ) of HCT ( green ) binds to aNT ( light gray ) , with the inhibitory loop ( green spheres ) rotated away and inaccessible for binding the B subunit and the central stalk ( transparent red ) . In ScV1 , the HCT ( yellow ) inhibitory loop ( yellow spheres ) binds to the central stalk subunit D ( red ) and the B subunit of the open ( AB ) 1 catalytic site . The difference in conformation between ScV1Vo and ScV1 involves a ~ 150 ° rotation of HCT . Note that the HCT loop ( spheres ) is rotated away from the central stalk in ScV1Vo whereas in ScV1 , the C - terminal α helix ( orange ) involved in binding aNT is rotated away and unavailable for rebinding to the membrane . F HCT conformational changes . Available subunit H structures as obtained for isolated H ( Sagermann et al , 2001 ) ( purple ) , in ScV1Vo ( Zhao et al , 2015 ) ( green ) and in ScV1 ( yellow ) are shown aligned by their N - terminal domains ( HNT ) illustrating the conformational flexibility of HCT . The loop involved in inhibition of ScV1 is shown as red spheres , highlighting the 180º rotation of HCT when going from the conformation of isolated H ( HCT 1ho8 ) to the inhibitory conformation in ScV1 ( HCT ScV1 ) .

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 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 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

Figure 6A Overlay of ScV1 and the cryo-EM structure of the V1Vo holoenzyme in "state 2" (Zhao et al, 2015). The structures of ScV1 and ScV1Vo were overlaid by aligning the A3B3 catalytic hexamers, thus highlighting structural differences in the central rotor and peripheral stator subunits. Coloring of ScV1Vo (shown as semi-transparent ribbons) is as used throughout for ScV1 for all but the A3B3F (tan), subunit H (dark green), subunit C and Vo domain (gray). The EG1-HNT interaction is boxed. Note the shift in position of HNT and EG1 between ScV1 and ScV1Vo.B Comparison of peripheral stalk EG1 and HNT in ScV1 versus ScV1Vo. The boxed view in A is shown enlarged. The N-terminal α helices of peripheral stalk EG1 and HNT change in conformation, likely induced by the movement of HCT when going from the holo enzyme to the autoinhibited conformation.C View as in A rotated 150º around the vertical. Comparisons of peripheral stalk EG3 and HCT are boxed.D Comparison of peripheral stalk EG3 in ScV1 versus ScV1Vo. In ScV1Vo, EG3 binds to the C subunit head domain (Chead; gray) whereas in ScV1, EG3 is bent towards the C-terminal domain of a catalytic A subunit (enlarged view of box labeled EG3 in panel C).E Comparison of HCT's conformation in ScV1 versus ScV1Vo. Enlarged view of box labeled HCT in panel C highlighting the conformational changes and binding interactions of HCT; for clarity only aNT, HCT and the central rotor (DF) are shown. In ScV1Vo, the most C-terminal α helix (dark green) of HCT (green) binds to aNT (light gray), with the inhibitory loop (green spheres) rotated away and inaccessible for binding the B subunit and the central stalk (transparent red). In ScV1, the HCT (yellow) inhibitory loop (yellow spheres) binds to the central stalk subunit D (red) and the B subunit of the open (AB)1 catalytic site. The difference in conformation between ScV1Vo and ScV1 involves a ~150° rotation of HCT. Note that the HCT loop (spheres) is rotated away from the central stalk in ScV1Vo whereas in ScV1, the C-terminal α helix (orange) involved in binding aNT is rotated away and unavailable for rebinding to the membrane.F HCT conformational changes. Available subunit H structures as obtained for isolated H (Sagermann et al, 2001) (purple), in ScV1Vo (Zhao et al, 2015) (green) and in ScV1 (yellow) are shown aligned by their N-terminal domains (HNT) illustrating the conformational flexibility of HCT. The loop involved in inhibition of ScV1 is shown as red spheres, highlighting the 180º rotation of HCT when going from the conformation of isolated H (HCT 1ho8) to the inhibitory conformation in ScV1(HCT ScV1).


Figure 1IFNα dose - dependency of STAT1 and STAT2 phosphorylation in Huh7 . 5 cells . Cells were seeded 24 hours prior to the start of the experiment . Three hours before stimulation , cells were growth factor - depleted and were subsequently stimulated with the indicated concentrations of IFNα . Cytoplasmic protein lysates were collected one hour after the stimulation and phosphorylation of STAT1 and STAT2 was detected by immunoblot utilizing antibodies recognizing STAT1 phosphorylated on tyrosine residue 701 , or STAT2 phosphorylated on tyrosine residue 690 . Data points are displayed as dots with 1σ confidence interval estimated from biological replicates ( N = 1 to N = 38 ) using a combined scaling and error model . Data is approximated with a sigmoidal function and signals corresponding to a low dose ( 2 . 8 pM IFNα ) , a medium dose ( 28 pM IFNα ) and a high dose ( 1400 pM IFNα ) are displayed with dashed lines . Representative immunoblot of IFNα - induced phosphorylation of STAT1 and STAT2 upon stimulation of Huh7 . 5 cells prestimulated for 24 hours with 2 . 8 pM IFNα , 1400 pM IFNα or without prestimulation . Time points after presimulation are indicated . 20 µg of cytoplasmic lysates were analyzed using antibodies for the indicated targets . Phosphorylation of STAT1 and STAT2 was detected by immunoblot utilizing antibodies recognizing STAT1 phosphorylated on tyrosine residue 701 , or STAT2 phosphorylated on tyrosine residue 690 . An asterisk indicates pSTAT1β . Calnexin served as loading control . Molecular weights are indicated on the left . Immunoblot detection was performed with chemiluminescence employing a CCD camera device ( ImageQuant ) . Quantification of immunoblots of IFNα - induced phosphorylation of cytoplasmic and nuclear STAT1 and STAT2 in Huh7 . 5 cells prestimulated with 2 . 8 pM IFNα , 1400 pM IFNα or without pretreatment . Time points after presimulation are displayed . Data is displayed as fold change relative to untreated cells . Errors were estimated with a combined scaling and error model , comprising 1σ confidence interval estimated from biological replicates ( N = 3 ) . Dashed lines indicate smoothing splines . Induction of interferon - stimulated genes upon prestimulation with 1400 pM IFNα ( yellow background ) and stimulation with 1400 pM IFNα ( white background ) in Huh7 . 5 cells , assessed by qRT - PCR . RNA levels were normalized to the geometric mean of reference genes GAPDH , HPRT and TBP and were displayed as fold change . Peak of gene expression is indicated . Data points displayed as dots with 1σ confidence - interval estimated from biological replicates ( N = 4 to N = 14 ) using a combined scaling and error model . Dashed lines indicate smoothing splines . Induction of interferon - stimulated genes upon prestimulation with 2 . 8 pM IFNα ( yellow background ) and stimulation with 1400 pM IFNα ( white background ) in Huh7 . 5 cells , assessed by qRT - PCR . RNA levels were normalized to the geometric mean of reference genes GAPDH , HPRT and TBP and were displayed as fold change . Peak of gene expression is indicated . Data points are displayed as dots with 1σ confidence interval estimated from biological replicates ( N = 4 to N = 6 ) using a combined scaling and error model . Dashed lines indicate smoothing splines .

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 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 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

Figure 1IFNα dose-dependency of STAT1 and STAT2 phosphorylation in Huh7.5 cells. Cells were seeded 24 hours prior to the start of the experiment. Three hours before stimulation, cells were growth factor-depleted and were subsequently stimulated with the indicated concentrations of IFNα. Cytoplasmic protein lysates were collected one hour after the stimulation and phosphorylation of STAT1 and STAT2 was detected by immunoblot utilizing antibodies recognizing STAT1 phosphorylated on tyrosine residue 701, or STAT2 phosphorylated on tyrosine residue 690. Data points are displayed as dots with 1σ confidence interval estimated from biological replicates (N=1 to N=38) using a combined scaling and error model. Data is approximated with a sigmoidal function and signals corresponding to a low dose (2.8 pM IFNα), a medium dose (28 pM IFNα) and a high dose (1400 pM IFNα) are displayed with dashed lines.Representative immunoblot of IFNα-induced phosphorylation of STAT1 and STAT2 upon stimulation of Huh7.5 cells prestimulated for 24 hours with 2.8 pM IFNα, 1400 pM IFNα or without prestimulation. Time points after presimulation are indicated. 20 µg of cytoplasmic lysates were analyzed using antibodies for the indicated targets. Phosphorylation of STAT1 and STAT2 was detected by immunoblot utilizing antibodies recognizing STAT1 phosphorylated on tyrosine residue 701, or STAT2 phosphorylated on tyrosine residue 690. An asterisk indicates pSTAT1β. Calnexin served as loading control. Molecular weights are indicated on the left. Immunoblot detection was performed with chemiluminescence employing a CCD camera device (ImageQuant).   Quantification of immunoblots of IFNα-induced phosphorylation of cytoplasmic and nuclear STAT1 and STAT2 in Huh7.5 cells prestimulated with 2.8 pM IFNα, 1400 pM IFNα or without pretreatment. Time points after presimulation are displayed. Data is displayed as fold change relative to untreated cells. Errors were estimated with a combined scaling and error model, comprising 1σ confidence interval estimated from biological replicates (N=3). Dashed lines indicate smoothing splines.   Induction of interferon-stimulated genes upon prestimulation with 1400 pM IFNα (yellow background) and stimulation with 1400 pM IFNα (white background) in Huh7.5 cells, assessed by qRT-PCR. RNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT and TBP and were displayed as fold change. Peak of gene expression is indicated. Data points displayed as dots with 1σ confidence-interval estimated from biological replicates (N=4 to N=14) using a combined scaling and error model. Dashed lines indicate smoothing splines.   Induction of interferon-stimulated genes upon prestimulation with 2.8 pM IFNα (yellow background) and stimulation with 1400 pM IFNα (white background) in Huh7.5 cells, assessed by qRT-PCR. RNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT and TBP and were displayed as fold change. Peak of gene expression is indicated. Data points are displayed as dots with 1σ confidence interval estimated from biological replicates (N=4 to N=6) using a combined scaling and error model. Dashed lines indicate smoothing splines.


Figure 3Growth factor - depleted Huh7 . 5 cells were prestimulated with 2 . 8 pM , 28 pM , 1400 pM IFNα or left untreated and were stimulated with 1400 pM IFNα 24 hours later . IFNα - induced signaling was measured by time - resolved quantitative immunoblotting and detected with chemiluminescence using a CCD - based camera . Data was normalized to reference proteins Calnexin or HDAC1 , scaled and subjected to model calibration . Model calibration with time - resolved IFNα - induced phosphorylation of STAT1 and STAT2 and induced feedback proteins upon prestimulation with 0 , 2 . 8 pM , 28 pM or 1400 pM IFNα . Cytoplasmic lysates were subjected to quantitative immunoblotting . Experimental data is represented by filled circles with errors representing 1σ confidence intervals estimated from biological replicates ( N = 3 to N = 23 ) using a combined scaling and error model . Model trajectories are represented by lines . pSTAT1 , pSTAT2 represent phosphorylated STAT1 and STAT2 on residue Tyr701 and Tyr690 , respectively . tSTAT1 and tSTAT2 represent total form of STAT1 and STAT2 comprising both phosphorylated and unphosphorylated STAT1 , STAT2 , respectively . Model calibration with time - resolved IFNα - induced feedback transcripts upon prestimulation with 0 , 2 . 8 pM , 28 pM or 1400 pM IFNα , assessed by qRT - PCR . mRNA levels were normalized to the geometric mean of reference genes GAPDH , HPRT and TBP . Experimental data is represented by filled circles with errors representing 1σ confidence - interval estimated from biological replicates ( N = 3 to N = 14 ) using a combined scaling and error model . Model trajectories are represented by lines . Model calibration with the amount of molecules per cell of STAT1 , STAT2 , IRF9 and USP18 determined 24 hours after prestimulation with 0 , 2 . 8 pM , 28 pM or 1400 pM IFNα . Calibrator proteins were spiked into 10 µg of total protein lysate and subjected to quantitative immunoblotting . Immunoblot detection was performed by chemiluminescence using a CCD - camera - based device . Averaged values ( N = 4 ) are displayed with standard deviations . Green squares indicate amounts estimated by the mathematical model . Model calibration of IFNα - induced phosphorylation of STAT1 and STAT2 upon stimulation of Huh7 . 5 prestimulated with 0 , 2 . 8 pM , 28 pM or 1400 pM IFNα . Nuclear lysates were subjected to quantitative immunoblotting . Experimental data is represented by filled circles with errors representing 1σ confidence - interval estimated from biological replicates ( N = 4 to N = 22 ) using a combined scaling and error model . Model trajectories are represented by lines .

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 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 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 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

Figure 3Growth factor-depleted Huh7.5 cells were prestimulated with 2.8 pM, 28 pM, 1400 pM IFNα or left untreated and were stimulated with 1400 pM IFNα 24 hours later. IFNα-induced signaling was measured by time-resolved quantitative immunoblotting and detected with chemiluminescence using a CCD-based camera. Data was normalized to reference proteins Calnexin or HDAC1, scaled and subjected to model calibration.   Model calibration with time-resolved IFNα-induced phosphorylation of STAT1 and STAT2 and induced feedback proteins upon prestimulation with 0, 2.8 pM, 28 pM or 1400 pM IFNα. Cytoplasmic lysates were subjected to quantitative immunoblotting. Experimental data is represented by filled circles with errors representing 1σ confidence intervals estimated from biological replicates (N=3 to N=23) using a combined scaling and error model. Model trajectories are represented by lines. pSTAT1, pSTAT2 represent phosphorylated STAT1 and STAT2 on residue Tyr701 and Tyr690, respectively. tSTAT1 and tSTAT2 represent total form of STAT1 and STAT2 comprising both phosphorylated and unphosphorylated STAT1, STAT2, respectively.   Model calibration with time-resolved IFNα-induced feedback transcripts upon prestimulation with 0, 2.8 pM, 28 pM or 1400 pM IFNα, assessed by qRT-PCR. mRNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT and TBP. Experimental data is represented by filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=3 to N=14) using a combined scaling and error model. Model trajectories are represented by lines.Model calibration with the amount of molecules per cell of STAT1, STAT2, IRF9 and USP18 determined 24 hours after prestimulation with 0, 2.8 pM, 28 pM or 1400 pM IFNα. Calibrator proteins were spiked into 10 µg of total protein lysate and subjected to quantitative immunoblotting. Immunoblot detection was performed by chemiluminescence using a CCD-camera-based device. Averaged values (N=4) are displayed with standard deviations. Green squares indicate amounts estimated by the mathematical model.Model calibration of IFNα-induced phosphorylation of STAT1 and STAT2 upon stimulation of Huh7.5 prestimulated with 0, 2.8 pM, 28 pM or 1400 pM IFNα. Nuclear lysates were subjected to quantitative immunoblotting. Experimental data is represented by filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=4 to N=22) using a combined scaling and error model. Model trajectories are represented by lines.