C HeLa were infected with S . flexneri M90T for 4 h 40 min , labelled with MitoTracker Red CMXRos , fixed for quantitative confocal microscopy . Boxplots show of mitochondrial ( μm ; whiskers from min to max ) surrounding Shigella outside ( - ) or inside ( + ) SEPT7 cages from 3 independent experiments ( of at least 130 measurements per biological replicate ) . Student ' s t - ; * * * = p < 0 . 001 .

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C HeLa cells were infected with S. flexneri M90T for 4 h 40 min, labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Boxplots show the length of mitochondrial (μm; whiskers from min to max) surrounding Shigella outside (-) or inside (+) SEPT7 cages from 3 independent experiments (analysis of at least 130 measurements per biological replicate). Student's t-test; *** = p<0.001.


D HeLa were either kept uninfected as a control or infected with S . flexneri M90T or S . flexneri ΔIcsA for 4 h 40 min . Samples were labelled with MitoTracker Red CMXRos , fixed for quantitative confocal microscopy . Boxplots show of mitochondria ( μm ; whiskers from min to max ) in uninfected ( CTRL ) , surrounding S . flexneri M90T ( + M90T ) or S . flexneri ΔIcsA ( + ΔIcsA ) from 3 independent experiments ( of at least 250 measurements per biological replicate ) . Student ' s t - ; * * * = p < 0 . 001 .

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D HeLa cells were either kept uninfected as a control or infected with S. flexneri M90T or S. flexneri ΔIcsA for 4 h 40 min. Samples were labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Boxplots show length of mitochondria (μm; whiskers from min to max) in uninfected cells (CTRL), surrounding S. flexneri M90T (+M90T) or S. flexneri ΔIcsA (+ΔIcsA) from 3 independent experiments (analysis of at least 250 measurements per biological replicate). Student's t-test; *** = p<0.001.


CSF PGRN is increased in mutation carriers ( MC ) compared to non - carriers ( NC ) .

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CSF PGRN is increased in mutation carriers (MC) compared to non-carriers (NC).


CSF PGRN is not associated with in either NC or MC .

CSF PGRN is not associated with age in either NC or MC.


CSF PGRN is increased in males compare to females.


CSF PGRN do not differ among MC participants carrying a PSEN1 , PSEN2 or APP mutation .

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CSF PGRN levels do not differ among MC participants carrying a PSEN1, PSEN2 or APP mutation.


CSF PGRN as a function of EYO in mutation carriers ( MC , red ) non - carriers ( NC , blue ) . solid lines indicate regression for each of groups 95 % confidence interval ( CI ) calculated by a linear model adjusting by gender . interaction term of mutation status EYO is significant ( P = 0 . 041 ) , also when including PGRN outliers participants with EYO > + 20 ( P = 0 . 030 ) . data points are not displayed to prevent disclosure of mutation status .

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CSF PGRN as a function of EYO in mutation carriers (MC, red) and non-carriers (NC, blue). The solid lines indicate the regression line for each of the groups and the 95% confidence interval (CI) calculated by a linear model adjusting by gender. The interaction term of mutation status and EYO is significant (P = 0.041), also when including PGRN outliers and participants with EYO > +20 (P = 0.030). Individual data points are not displayed to prevent disclosure of mutation status.


graph depicts standardised differences of CSF PGRN between MCs NCs as a function of EYO , in context of other biomarker cognitive changes . curves were generated by linear model that best fit each marker ( see statistics section Appendix Table S2 ) . CSF PGRN is significantly increased in MC compared to NC 10 years before expected symptom onset ( shadowed ) after brain amyloidosis brain injury ( as measured by CSF T - tau ) have started , shortly before CSF sTREM2 starts to increase . Abbreviations : Aβ1 - 42 : amyloid - β 42 ; CSF , cerebrospinal fluid ; MC , mutation carriers ; MMSE , Mini - Mental State Examination ; NC , non - carriers ; T - tau , total tau .

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The graph depicts the standardised differences of CSF PGRN between MCs and NCs as a function of EYO, in the context of other biomarker and cognitive changes. The curves were generated by the linear model that best fit each marker (see statistics section and Appendix Table S2). CSF PGRN is significantly increased in MC compared to NC 10 years before the expected symptom onset (shadowed area) after brain amyloidosis and brain injury (as measured by CSF T-tau) have started, and shortly before CSF sTREM2 starts to increase. Abbreviations: Aβ1-42: amyloid-β 42; CSF, cerebrospinal fluid; MC, mutation carriers; MMSE, Mini-Mental State Examination; NC, non-carriers; T-tau, total tau.


Scatter plot representing of CSF PGRN in healthy controls ( depicted in blue ) different stages of Alzheimer ' s continuum ( depicted in red ) . graphs were performed excluding CSF PGRN outliers ( 1 ' healthy control ' , 1 ' Preclinical AD A + / TN - ' , 4 ' AD CDR = 0 . 5 ' 1 ' AD CDR = 1 ' ) . Including them yielded a similar result CSF PGRN was still significantly higher in ' AD CDR = 1 ' group compared to ' healthy controls ' ( P = 0 . 001 ) ' Preclinical AD A + / TN - ' ( P = 0 . 0001 ) groups . Abbreviations : A : amyloid - β biomarker status ; AD : Alzheimer ' s disease ; CDR : clinical dementia rating ; CSF , cerebrospinal fluid ; N : neurodegeneration biomarker status ; T : tau pathology biomarker status .

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Scatter plot representing the levels of CSF PGRN in healthy controls (depicted in blue) and the different stages of the Alzheimer's continuum (depicted in red). The analysis and graphs were performed excluding CSF PGRN outliers (1 'healthy control', 1 'Preclinical AD A+/TN-', 4 'AD CDR = 0.5' and 1 'AD CDR = 1'). Including them yielded a similar result and CSF PGRN was still significantly higher in the 'AD CDR = 1' group compared to the 'healthy controls' (P = 0.001) and 'Preclinical AD A+/TN-' (P = 0.0001) groups. Abbreviations: A: amyloid-β biomarker status; AD: Alzheimer's disease; CDR: clinical dementia rating; CSF, cerebrospinal fluid; N: neurodegeneration biomarker status; T: tau pathology biomarker status.


Scatter plots representing association of CSF PGRN with different cognitive . Only subjects of Alzheimer ' s continuum group ( n = 474 ) were included . In studied , higher of CSF PGRN were associated with worse cognitive performance ( namely , lower scores in ADNI - Mem , ADNI - EF MMSE higher scores in ADAS - Cog11 , ADAS - Cog13 CDR - SB ) . graphs are excluding PGRN outliers ; including them rendered similar results ( Appendix Table S6 ) . Abbreviations : ADAS - Cog , Alzheimer ' s disease Assessment Scale - cognitive subscale ; ADNI - Mem : ADNI memory composite score ; ADNI - EF : ADNI executive function composite score ; CDR - SB : clinical dementia rating Sum of Boxes ; MMSE , Mini - Mental State Examination .

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Scatter plots representing the association of CSF PGRN with different cognitive tests. Only the subjects of the Alzheimer's continuum group (n = 474) were included. In all tests studied, higher levels of CSF PGRN were associated with worse cognitive performance (namely, lower scores in ADNI-Mem, ADNI-EF and MMSE and higher scores in ADAS-Cog11, ADAS-Cog13 and CDR-SB). The analysis and the graphs are excluding PGRN outliers; including them rendered similar results (Appendix Table S6). Abbreviations: ADAS-Cog, Alzheimer's disease Assessment Scale - cognitive subscale; ADNI-Mem: ADNI memory composite score; ADNI-EF: ADNI executive function composite score; CDR-SB: clinical dementia rating Sum of Boxes; MMSE, Mini-Mental State Examination.


Scatter plots representing association of CSF PGRN with temporo - parietal FDG - PET uptake within subjects of Alzheimer ' s continuum group ( n = 474 ) . Abbreviations : FDG - PET : Fludeoxyglucose positron emission

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Scatter plots representing the association of CSF PGRN with temporo-parietal FDG-PET uptake within the subjects of the Alzheimer's continuum group (n = 474). Abbreviations: FDG-PET: Fludeoxyglucose positron emission tomography


Scatter plots representing association of CSF PGRN with total hippocampal volume ( B ) within subjects of Alzheimer ' s continuum group ( n = 474 ) .

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Scatter plots representing the association of CSF PGRN with total hippocampal volume (B) within the subjects of the Alzheimer's continuum group (n = 474).


Scatter plots representing associations of CSF PGRN with CSF sTREM2 each of AD CSF biomarkers ( T - tau , P - tau181P Aβ1 − 42 ) in non - carriers ( NC , blue ; A , C , E G ) in mutation carriers ( MC , red ; B , D , F H ) . Each point depicts value of CSF PGRN corresponding biomarker of a subject solid lines indicate regression 95 % confidence interval ( CI ) for each of groups . standardized regression coefficients ( β ) P - values are shown were computed using a linear model adjusting for , gender APOE ε4 . sample contained some outliers ( defined as 3 SDs below or above group mean ) of CSF markers of AD . Abbreviations : Aβ1 - 42 : amyloid - β 42 ; T - tau : total tau ; P - tau : tau phosphorylated at Threonine 181 .

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

Scatter plots representing the associations of CSF PGRN with CSF sTREM2 and each of the AD CSF core biomarkers (T-tau, P-tau181P and Aβ1−42) in non-carriers (NC, blue; A, C, E and G) and in mutation carriers (MC, red; B, D, F and H). Each point depicts the value of CSF PGRN and the corresponding biomarker of a subject and the solid lines indicate the regression line and the 95% confidence interval (CI) for each of the groups. The standardized regression coefficients (β) and the P-values are shown and were computed using a linear model adjusting for age, gender and APOE ε4. The sample contained some outliers (defined as 3 SDs below or above the group mean) of the CSF core markers of AD. Abbreviations: Aβ1-42: amyloid-β 42; T-tau: total tau; P-tau: tau phosphorylated at Threonine 181.


Scatter plots representing associations of CSF PGRN with CSF sTREM2 each of AD CSF biomarkers ( T - tau , P - tau181P Aβ1 − 42 ) in healthy controls ( blue ; A , D , G J ) , Alzheimer ' s continuum ( red ; B , E , H K ) ' suspected non - Alzheimer ' s pathophysiology ( SNAP ) ' groups ( green ; C , F , I L ) . Each point depicts value of CSF PGRN corresponding biomarker of a subject solid lines indicate regression 95 % confidence interval ( CI ) for each of groups . standardized regression coefficients ( β ) P - values are shown were computed using a linear model adjusting for , gender APOE ε4 . sample contained some outliers ( defined as 3 SDs below or above group mean ) of CSF markers of AD , including these outliers yielded similar results ( Appendix Table S8 ) . Aβ1 - 42 values used for associations are those on an extrapolation curve since upper technical limit is 1700pg / ml . We also tested associations with Aβ1 - 42 values truncated at upper technical limit result was similar . Abbreviations : Aβ1 - 42 : amyloid - β 42 ; T - tau : total tau ; P - tau : tau phosphorylated at Threonine 181 ; SNAP : suspected non - Alzheimer ' s pathophysiology .

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Scatter plots representing the associations of CSF PGRN with CSF sTREM2 and each of the AD CSF core biomarkers (T-tau, P-tau181P and Aβ1−42) in healthy controls (blue; A, D, G and J), Alzheimer's continuum (red; B, E, H and K) and 'suspected non-Alzheimer's pathophysiology (SNAP)' groups (green; C, F, I and L). Each point depicts the value of CSF PGRN and the corresponding biomarker of a subject and the solid lines indicate the regression line and the 95% confidence interval (CI) for each of the groups. The standardized regression coefficients (β) and the P-values are shown and were computed using a linear model adjusting for age, gender and APOE ε4. The sample contained some outliers (defined as 3 SDs below or above the group mean) of the CSF core markers of AD, the analysis including these outliers yielded similar results (Appendix Table S8). The Aβ1-42 values used for the associations test are those based on an extrapolation curve since the upper technical limit is 1700pg/ml. We also tested the associations with Aβ1-42 values truncated at the upper technical limit and the result was similar. Abbreviations: Aβ1-42: amyloid-β 42; T-tau: total tau; P-tau: tau phosphorylated at Threonine 181; SNAP: suspected non-Alzheimer's pathophysiology.


( A ) Representative Western Blot of endogenous p150Glued co - immunoprecipitated with EB1 in shCTL or shSTIM1 ECs ( left ) its by normalized densitometry ( right ) . Negative control ( CTL ) was performed incubating lysate with A - or G - Sepharose empty mouse IgG . Results are average ± SD of three independent assays . shCTL value of each biological replicate was normalized on itself so shSTIM1 experimental value . Results were analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for shCTL shSTIM1 p ≤ 0 , 05 * .

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(A) Representative Western Blot analysis of the endogenous p150Glued co-immunoprecipitated with EB1 in shCTL or shSTIM1 ECs (left) and its quantification by normalized densitometry (right). Negative control (CTL) was performed incubating cell lysate with protein A- or G-Sepharose and empty mouse IgG. Results are the average ± SD of three independent assays. shCTL value of each biological replicate was normalized on itself and so shSTIM1 experimental value. Results were analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for shCTL and shSTIM1 p≤ 0,05 *.


( B ) Representative of three Western Blot of endogenous p150Glued , - intermediate chain ( LIC ) of dynein 1 EB1 immunoprecipitated with STIM1 in wild - type ECs . Negative control ( CTL ) was performed incubating lysate with A - or G - Sepharose empty rabbit IgG .

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(B) Representative of three Western Blot analysis of endogenous p150Glued, light-intermediate chain (LIC) of cytoplasmic dynein 1 and EB1 immunoprecipitated with STIM1 in wild-type ECs. Negative control (CTL) was performed incubating cell lysate with protein A- or G-Sepharose and empty rabbit IgG.


( D ) Representative Western Blot of increasing amount of GST - STIM1 ( domain ) pulled down by Cap - Gly - CC1 - p150Glued - V5 - coated beads , in absence or presence of FLAG - EB1 C - terminal . Colors label as in ( c ) . Negative control was performed using equal amount of empty GST , together with Cap - Gly - CC1 - p150Glued .

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(D) Representative Western Blot analysis of increasing amount of GST-STIM1 (cytoplasmic domain) pulled down by Cap-Gly-CC1-p150Glued-V5-coated beads, in absence or presence of FLAG-EB1 C-terminal. Colors label the protein as in (c). Negative control was performed using equal amount of empty GST protein, together with Cap-Gly-CC1-p150Glued.


( E ) Representative Western Blot of mCherry - p150Glued WT co - immunoprecipitated with GFP - STIM1 WT , NN , ΔCC1 - 3 / WT ( ΔCC / WT ) or ΔCC1 - 3 / NN ( ΔCC / NN ) in co - transfected HEK 293T . Negative control ( CTL ) was performed incubating lysate from HEK 293T co - transfected with an empty GFP vector together with mCherry - P150Glued WT with pre - cleared A or G - Sepharose rabbit GFP antibody . Below , its by normalized densitometry . Results are average ± SD of three independent assays . value of P150Glued co - immunoprecipitated with GFP - STIM1 WT from each biological replicate was normalized on itself so those immunoprecipitated with GFP - STIM1 NN , ΔCC / WT or ΔCC / NN . Results were analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for STIM1 WT NN p > 0 . 05 not significant ( ns ) , for STIM1 WT ΔCC / WT p > 0 . 05 not significant ( ns ) for STIM1 WT ΔCC / NN p ≤ 0 , 05 * .

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(E) Representative Western Blot analysis of mCherry-p150Glued WT co-immunoprecipitated with GFP-STIM1 WT, NN, ΔCC1-3/WT (ΔCC/WT) or ΔCC1-3/NN (ΔCC/NN) in co-transfected HEK 293T cells. Negative control (CTL) was performed incubating cell lysate from HEK 293T co-transfected with an empty GFP vector together with mCherry-P150Glued WT with pre-cleared protein A or G-Sepharose and the rabbit GFP antibody. Below, its quantification by normalized densitometry. Results are the average ± SD of three independent assays. The value of P150Glued co-immunoprecipitated with GFP-STIM1 WT from each biological replicate was normalized on itself and so those immunoprecipitated with GFP-STIM1 NN, ΔCC/WT or ΔCC/NN. Results were analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for STIM1 WT and NN p> 0.05 not significant (ns), for STIM1 WT and ΔCC/WT p> 0.05 not significant (ns) and for STIM1 WT and ΔCC/NN p≤ 0,05 *.


( F ) Representative Western Blot of GST - STIM1 ( domain ) pulled down by Cap - Gly - CC1 - p150Glued - V5 - coated beads , in presence of FLAG - EB1 C - terminal WT or ΔY . Negative control was performed using equal amount of empty GST , together with Cap - Gly - CC1 - p150Glued .

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(F) Representative Western Blot analysis of GST-STIM1 (cytoplasmic domain) pulled down by Cap-Gly-CC1-p150Glued-V5-coated beads, in presence of FLAG-EB1 C-terminal WT or ΔY. Negative control was performed using equal amount of empty GST protein, together with Cap-Gly-CC1-p150Glued.


( G ) Confocal microscopy of wild - type ECs , transiently transfected with GFP - STIM1 WT or , NN or ΔCC1 - 3 / WT ( ΔCC / WT ) together with mCherry - P150Glued . Scale bar = 10 μm . Right insets are shown to highlight MT - bound p150Glued + ( arrows ) , colocalized with STIM1 . Scale bar = 5 μm . ( H ) Average number of mCherry - P150Glued + in same as in ( g ) . are average ± of three independent experiments for a total of 150 ( 30 ) . Results were analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for STIM1 WT NN p ≤ 0 , 001 * * * for STIM1 WT ΔCC / WT p > 0 . 05 not significant ( ns ) . ( I ) Co - localization of mCherry - P150Glued with GFP - STIM1 WT , NN or ΔCC1 - 3 / WT ( ΔCC / WT ) , transiently co - transfected in ECs , as in ( g ) . Results are average ± of three independent experiments for a total of 150 ( 30 ) analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for STIM1 WT NN p ≤ 0 , 001 * * * for STIM1 WT ΔCC / WT p > 0 . 05 not significant ( ns ) .

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(G) Confocal microscopy analysis of wild-type ECs, transiently transfected with GFP-STIM1 WT or, NN or ΔCC1-3/WT (ΔCC/WT) together with mCherry-P150Glued. Scale bar = 10 μm. Right insets are shown to highlight MT-bound p150Glued+ punctae (arrows), colocalized with STIM1. Scale bar = 5 μm. (H) Average number of the mCherry-P150Glued+ punctae in the same cells as in (g). Counts are the average ± SEM of three independent experiments for a total of 150 punctae (30 cells). Results were analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for STIM1 WT and NN p≤ 0,001 *** and for STIM1 WT and ΔCC/WT p> 0.05 not significant (ns). (I) Co-localization analysis of mCherry-P150Glued with GFP-STIM1 WT, NN or ΔCC1-3/WT (ΔCC/WT), transiently co-transfected in ECs, as in (g). Results are the average ± SEM of three independent experiments for a total of 150 punctae (30 cells) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for STIM1 WT and NN p≤ 0,001 *** and for STIM1 WT and ΔCC/WT p> 0.05 not significant (ns).


( G ) Confocal microscopy of wild - type ECs , transiently transfected with GFP - STIM1 WT or , NN or ΔCC1 - 3 / WT ( ΔCC / WT ) together with mCherry - P150Glued . Scale bar = 10 μm . Right insets are shown to highlight MT - bound p150Glued + ( arrows ) , colocalized with STIM1 . Scale bar = 5 μm .

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(G) Confocal microscopy analysis of wild-type ECs, transiently transfected with GFP-STIM1 WT or, NN or ΔCC1-3/WT (ΔCC/WT) together with mCherry-P150Glued. Scale bar = 10 μm. Right insets are shown to highlight MT-bound p150Glued+ punctae (arrows), colocalized with STIM1. Scale bar = 5 μm.


( A ) Confocal microscopy of ECs silenced with a control siRNA ( siCTL ) or one targeting STIM1 ( siSTIM1 ) stained for endogenous LAMP - 1 ( in green ) to visualize LEs DAPI ( in blue ) to highlight nucleus . yellow is drawn to define periphery . Scale bar = 20 μm . On right , inset panels to highlight respective perinuclear peripheral of . Scale bar = 5 μm .

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

(A) Confocal microscopy images of ECs silenced with a control siRNA (siCTL) or one targeting STIM1 (siSTIM1) and stained for endogenous LAMP-1 (in green) to visualize LEs and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm.


( B ) Distribution of , normalized on , quantified by image ( as in a ) segmentation ( see Material Method , Confocal microscopy early / late ) of LAMP - 1 + endosomes . Results are from three independent experiments for a total of 249 endosomes ( 15 siCTL ) 441 endosomes ( 13 siSTIM1 ) . In right inset , cumulative distribution used for Kolmogorov - Smirnov , p ≤ 0 , 001 * * * , is shown . p - value refers to rejection of null hypothesis in favor of alternative hypothesis that siCTL LE distribution has longer tail than siSTIM1 LE one .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Distribution of size, normalized on cell size, quantified by image (as in a) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of LAMP-1+ endosomes. Results are from three independent experiments for a total of 249 endosomes (15 siCTL cells) and 441 endosomes (13 siSTIM1 cells). In the right inset, the cumulative distribution used for the Kolmogorov-Smirnov test, p≤ 0,001 ***, is shown. The p-value refers to the rejection of the null hypothesis in favor of the alternative hypothesis that siCTL LE distribution has longer tail than siSTIM1 LE one.


( C ) Average number , normalized on , quantified by image ( as in a ) segmentation ( see Material Method , Confocal microscopy early / late ) of LAMP - 1 + endosomes . Results are average ± of three independent experiments for a total of 249 late endosomes in 15 siCTL ( 16 ± 2 endosomes per ) 441 late endosomes in 13 siSTIM1 ( 34 ± 3 endosomes per ) analyzed by a two - heteroscedastic Student ' s t - , p ≤ 0 , 001 * * * .

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

(C) Average number, normalized on cell size, quantified by image (as in a) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of LAMP-1+ endosomes. Results are the average ± SEM of three independent experiments for a total of 249 late endosomes in 15 siCTL cells (16 ± 2 endosomes per cell) and 441 late endosomes in 13 siSTIM1 cells (34 ± 3 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.


( D ) Distribution of to nucleus , normalized on , quantified by image ( as in a ) segmentation ( see Material Method , Confocal microscopy early / late ) of LAMP - 1 + endosomes . Results are from three independent experiments for a total of 249 late endosomes in 15 siCTL ( 16 ± 2 endosomes per ) 441 late endosomes in 13 siSTIM1 ( 34 ± 3 endosomes per ) analyzed by a two - heteroscedastic Student ' s t - , p ≤ 0 , 001 * * * .

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

(D) Distribution of distance to nucleus, normalized on cell size, quantified by image (as in a) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of LAMP-1+ endosomes. Results are from three independent experiments for a total of 249 late endosomes in 15 siCTL cells (16 ± 2 endosomes per cell) and 441 late endosomes in 13 siSTIM1 cells (34 ± 3 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.


( E ) Confocal microscopy of ECs silenced with a control shRNA ( shCTL ) or one targeting STIM1 ( shSTIM1 ) , rescued for GFP expression alone ( shCTL / GFP shSTIM1 / GFP ) or for GFP - STIM1 WT ( shSTIM1 / GFP - STIM1 WT ) , GFP - STIM1 NN ( shSTIM1 / GFP - STIM1 NN ) or GFP - STIM1 AA ( shSTIM1 / GFP - STIM1 AA ) stained for endogenous LAMP - 1 ( in red ) to visualize LEs DAPI ( in blue ) to highlight nucleus . yellow is drawn to define periphery . Scale bar = 20 μm . On right , inset panels to highlight respective perinuclear peripheral of . Scale bar = 5 μm .

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

(E) Confocal microscopy images of ECs silenced with a control shRNA (shCTL) or one targeting STIM1 (shSTIM1), rescued for GFP expression alone (shCTL/GFP and shSTIM1/GFP) or for GFP- STIM1 WT (shSTIM1/GFP-STIM1 WT), GFP- STIM1 NN (shSTIM1/GFP-STIM1 NN) or GFP- STIM1 AA (shSTIM1/GFP-STIM1 AA) and stained for endogenous LAMP-1 (in red) to visualize LEs and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm.


( F ) Upper panel , Distribution of to nucleus , normalized on , quantified by image ( as in e ) segmentation ( see Material Method , Confocal microscopy early / late ) of LAMP - 1 + endosomes . Results are from of two independent experiments for a total of 436 late endosomes in 13 shCTL / GFP ( 34 ± 6 endosomes per ) , 1452 late endosomes in 12 shSTIM1 / GFP ( 121 ± 13 endosomes per ) 498 late endosomes in 10 shSTIM1 / GFP - STIM1 WT ( 50 ± 5 endosomes per ) analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 01 ; Bonferroni for shCTL / GFP shSTIM1 / GFP p ≤ 0 , 001 * * * ( orange ) for shSTIM1 / GFP shSTIM1 / GFP - STIM1 WT p ≤ 0 , 001 * * * ( green ) . Lower panel , Distribution of to nucleus , normalized on , quantified by image ( as in e ) segmentation ( see Material Method , Confocal microscopy early / late ) of LAMP - 1 + endosomes . Results are from of two independent experiments for a total of 498 late endosomes in 10 shSTIM1 / GFP - STIM1 WT ( 50 ± 5 endosomes per ) , 1057 late endosomes in 11 shSTIM1 / GFP - STIM1 NN ( 96 ± 12 endosomes per ) 346 late endosomes in 10 shSTIM1 / GFP - STIM1 AA ( 35 ± 2 endosomes per ) analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 01 ; Bonferroni for shSTIM1 / GFP - STIM1 WT shSTIM1 / GFP - STIM1 NN p ≤ 0 , 05 * ( orange ) for shSTIM1 / GFP - STIM1 NN shSTIM1 / GFP - STIM1 AA p ≤ 0 , 05 * ( green ) .

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

(F) Upper panel, Distribution of distance to nucleus, normalized on cell size, quantified by image (as in e) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of LAMP-1+ endosomes. Results are from of two independent experiments for a total of 436 late endosomes in 13 shCTL/GFP cells (34 ± 6 endosomes per cell), 1452 late endosomes in 12 shSTIM1/GFP cells (121 ± 13 endosomes per cell) and 498 late endosomes in 10 shSTIM1/GFP-STIM1 WT cells (50 ± 5 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,01; Bonferroni for shCTL/GFP and shSTIM1/GFP p≤ 0,001 *** (orange) and for shSTIM1/GFP and shSTIM1/GFP-STIM1 WT p≤ 0,001 *** (green). Lower panel, Distribution of distance to nucleus, normalized on cell size, quantified by image (as in e) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of LAMP-1+ endosomes. Results are from of two independent experiments for a total of 498 late endosomes in 10 shSTIM1/GFP-STIM1 WT cells (50 ± 5 endosomes per cell), 1057 late endosomes in 11 shSTIM1/GFP-STIM1 NN cells (96 ± 12 endosomes per cell) and 346 late endosomes in 10 shSTIM1/GFP-STIM1 AA cells (35 ± 2 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,01; Bonferroni for shSTIM1/GFP-STIM1 WT and shSTIM1/GFP-STIM1 NN p≤ 0,05 * (orange) and for shSTIM1/GFP-STIM1 NN and shSTIM1/GFP-STIM1 AA p≤ 0,05 * (green).


( G ) Average , normalized on , quantified by image ( as in e ) segmentation ( see Material Method , Confocal microscopy early / late ) of LAMP - 1 + endosomes . Results are average ± of two independent experiments for a total of 436 late endosomes in 13 shCTL / GFP ( 34 ± 6 endosomes per ) , 1452 late endosomes in 12 shSTIM1 / GFP ( 121 ± 13 endosomes per ) , 615 late endosomes in 12 shSTIM1 / GFP - STIM1 WT ( 51 ± 5 endosomes per ) , 1278 late endosomes in 12 shSTIM1 / GFP - STIM1 NN ( 106 ± 15 endosomes per ) 506 late endosomes in 12 shSTIM1 / GFP - STIM1 AA ( 42 ± 7 endosomes per ) analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for shCTL / GFP shSTIM1 / GFP p ≤ 0 , 001 * * * , for shCTL / GFP shSTIM1 / GFP - STIM1 WT p > 0 . 05 not significant ( ns ) , for shSTIM1 / GFP - STIM WT shSTIM1 / GFP - STIM1 NN p ≤ 0 , 001 * * * , for shSTIM1 / GFP shSTIM1 / GFP - STIM1 NN p ≤ 0 , 001 * * * for shSTIM1 / GFP - STIM WT shSTIM1 / GFP - STIM1 AA p > 0 . 05 not significant ( ns ) .

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

(G) Average size, normalized on cell size, quantified by image (as in e) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of LAMP-1+ endosomes. Results are the average ± SEM of two independent experiments for a total of 436 late endosomes in 13 shCTL/GFP cells (34 ± 6 endosomes per cell), 1452 late endosomes in 12 shSTIM1/GFP cells (121 ± 13 endosomes per cell), 615 late endosomes in 12 shSTIM1/GFP-STIM1 WT cells (51 ± 5 endosomes per cell), 1278 late endosomes in 12 shSTIM1/GFP-STIM1 NN cells (106 ± 15 endosomes per cell) and 506 late endosomes in 12 shSTIM1/GFP-STIM1 AA cells (42 ± 7 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for shCTL/GFP and shSTIM1/GFP p≤ 0,001 ***, for shCTL/GFP and shSTIM1/GFP-STIM1 WT p> 0.05 not significant (ns), for shSTIM1/GFP-STIM WT and shSTIM1/GFP-STIM1 NN p≤ 0,001 ***, for shSTIM1/GFP and shSTIM1/GFP-STIM1 NN p≤ 0,001 *** and for shSTIM1/GFP-STIM WT and shSTIM1/GFP-STIM1 AA p> 0.05 not significant (ns).


( H ) Average number , normalized on , quantified by image ( as in e ) segmentation ( see Material Method , Confocal microscopy early / late ) of LAMP - 1 + endosomes . Results are average ± of two independent experiments for a total of 436 late endosomes in 13 shCTL / GFP ( 34 ± 6 endosomes per ) , 1452 late endosomes in 12 shSTIM1 / GFP ( 121 ± 13 endosomes per ) , 615 late endosomes in 12 shSTIM1 / GFP - STIM1 WT ( 51 ± 5 endosomes per ) , 1278 late endosomes in 12 shSTIM1 / GFP - STIM1 NN ( 106 ± 15 endosomes per ) 506 late endosomes in 12 shSTIM1 / GFP - STIM1 AA ( 42 ± 7 endosomes per ) analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for shCTL / GFP shSTIM1 / GFP p ≤ 0 , 001 * * * , for shCTL / GFP shSTIM1 / GFP - STIM1 WT p ≤ 0 , 001 * * * , for shSTIM1 / GFP - STIM WT shSTIM1 / GFP - STIM1 NN p ≤ 0 , 05 * , for shSTIM1 / GFP shSTIM1 / GFP - STIM1 NN p ≤ 0 , 01 * * for shSTIM1 / GFP - STIM WT shSTIM1 / GFP - STIM1 AA p > 0 . 05 not significant ( ns ) .

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

(H) Average number, normalized on cell size, quantified by image (as in e) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of LAMP-1+ endosomes. Results are the average ± SEM of two independent experiments for a total of 436 late endosomes in 13 shCTL/GFP cells (34 ± 6 endosomes per cell), 1452 late endosomes in 12 shSTIM1/GFP cells (121 ± 13 endosomes per cell), 615 late endosomes in 12 shSTIM1/GFP-STIM1 WT cells (51 ± 5 endosomes per cell), 1278 late endosomes in 12 shSTIM1/GFP-STIM1 NN cells (106 ± 15 endosomes per cell) and 506 late endosomes in 12 shSTIM1/GFP-STIM1 AA cells (42 ± 7 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for shCTL/GFP and shSTIM1/GFP p≤ 0,001 ***, for shCTL/GFP and shSTIM1/GFP-STIM1 WT p≤ 0,001 ***, for shSTIM1/GFP-STIM WT and shSTIM1/GFP-STIM1 NN p≤ 0,05 *, for shSTIM1/GFP and shSTIM1/GFP-STIM1 NN p≤ 0,01 ** and for shSTIM1/GFP-STIM WT and shSTIM1/GFP-STIM1 AA p> 0.05 not significant (ns).


( A ) Confocal microscopy of ECs silenced with a control siRNA ( siCTL ) or one targeting STIM1 ( siSTIM1 ) stained for endogenous Rab5 ( in green ) EEA - 1 ( in red ) to visualize EEs DAPI ( in blue ) to highlight nucleus . yellow is drawn to define periphery . Scale bar = 20 μm . On right , inset panels to highlight respective perinuclear peripheral of . Scale bar = 5 μm .

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

(A) Confocal microscopy images of ECs silenced with a control siRNA (siCTL) or one targeting STIM1 (siSTIM1) and stained for endogenous Rab5 (in green) and EEA-1 (in red) to visualize EEs and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm.


( B ) Average , normalized on , of EEA - 1 + endosomes as in a . Results are average ± of three independent experiments for a total 4483 early endosomes in 18 siCTL ( 299 ± 33 endosomes per ) 2454 early endosomes in 13 siSTIM1 ( 189 ± 17 endosomes per ) analyzed by a two - heteroscedastic Student ' s t - , p ≤ 0 , 001 * * * .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Average size, normalized on cell size, of EEA-1+ endosomes as in a. Results are the average ± SEM of three independent experiments for a total 4483 early endosomes in 18 siCTL cells (299 ± 33 endosomes per cell) and 2454 early endosomes in 13 siSTIM1 cells (189 ± 17 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.


( C ) Average number , normalized on , of EEA - 1 + endosomes as in a . Results are average ± of three independent experiments for a total 4483 early endosomes in 18 siCTL ( 299 ± 33 endosomes per ) 2454 early endosomes in 13 siSTIM1 ( 189 ± 17 endosomes per ) analyzed by a two - heteroscedastic Student ' s t - , p ≤ 0 , 01 * * .

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

(C) Average number, normalized on cell size, of EEA-1+ endosomes as in a. Results are the average ± SEM of three independent experiments for a total 4483 early endosomes in 18 siCTL cells (299 ± 33 endosomes per cell) and 2454 early endosomes in 13 siSTIM1 cells (189 ± 17 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,01 **.


( D ) Distribution of to nucleus overtime , normalized on , quantified by image ( data correspond to movies EV1 EV2 ) segmentation ( see Material Method , Live imaging experiments ) of Rab5 + endosomes , appearing for 60 seconds . Results are from three independent experiments for a total of 903 early endosomes in 18 siCTL ( 50 ± 5 endosomes per ) 935 early endosomes in 20 siSTIM1 ( 47 ± 9 endosomes per ) . Data are analyzed by a two - heteroscedastic Student ' s t - , p ≤ 0 , 01 * * .

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

(D) Distribution of distance to nucleus overtime, normalized on cell size, quantified by image (data correspond to movies EV1 and EV2) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 60 seconds. Results are from three independent experiments for a total of 903 early endosomes in 18 siCTL cells (50 ± 5 endosomes per cell) and 935 early endosomes in 20 siSTIM1 cells (47 ± 9 endosomes per cell). Data are analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,01 **.


( A ) Confocal microscopy of ECs silenced with a control shRNA ( shCTL ) or one targeting STIM1 ( shSTIM1 ) , rescued for GFP expression alone ( shCTL / GFP shSTIM1 / GFP ) or for GFP - STIM1 WT ( shSTIM1 / GFP - STIM1 WT ) or GFP - STIM1 NN ( shSTIM1 / GFP - STIM1 NN ) stained for endogenous EEA - 1 ( in red ) to visualize EEs DAPI ( in blue ) to highlight nucleus . yellow is drawn to define periphery . Scale bar = 20 μm . On right , inset panels to highlight respective perinuclear peripheral of . Scale bar = 5 μm .

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

(A) Confocal microscopy images of ECs silenced with a control shRNA (shCTL) or one targeting STIM1 (shSTIM1), rescued for GFP expression alone (shCTL/GFP and shSTIM1/GFP) or for GFP-STIM1 WT (shSTIM1/GFP-STIM1 WT) or GFP- STIM1 NN (shSTIM1/GFP-STIM1 NN) and stained for endogenous EEA-1 (in red) to visualize EEs and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm.


( B ) Upper panel , Distribution of to nucleus , normalized on , quantified by image ( as in a ) segmentation ( see Material Method , Confocal microscopy early / late ) of EEA - 1 + endosomes . Results are from two independent experiments for a total of 830 early endosomes in 13 shCTL / GFP ( 64 ± 6 endosomes per ) , 719 early endosomes in 10 shSTIM1 / GFP ( 60 ± 6 endosomes per ) 820 early endosomes in 12 shSTIM1 / GFP - STIM1 WT ( 68 ± 6 endosomes per ) analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for shCTL / GFP shSTIM1 / GFP p ≤ 0 , 001 * * * ( orange ) for shSTIM1 / GFP shSTIM1 / GFP - STIM1 WT p ≤ 0 , 001 * * * ( green ) . Lower panel , Distribution of to nucleus , normalized on , quantified by image ( as in a ) segmentation ( see Material Method , Confocal microscopy early / late ) of EEA - 1 + endosomes . Results are from two independent experiments for a total of 820 early endosomes in 12 shSTIM1 / GFP - STIM1 WT ( 68 ± 6 endosomes per ) 400 early endosomes in 12 shSTIM1 / GFP - STIM1 NN ( 33 ± 7 endosomes per ) analyzed a two - heteroscedastic Student ' s t - , p ≤ 0 , 001 * * * .

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

(B) Upper panel, Distribution of distance to nucleus, normalized on cell size, quantified by image (as in a) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of EEA-1+ endosomes. Results are from two independent experiments for a total of 830 early endosomes in 13 shCTL/GFP cells (64 ± 6 endosomes per cell), 719 early endosomes in 10 shSTIM1/GFP cells (60 ± 6 endosomes per cell) and 820 early endosomes in 12 shSTIM1/GFP-STIM1 WT cells (68 ± 6 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for shCTL/GFP and shSTIM1/GFP p≤ 0,001 *** (orange) and for shSTIM1/GFP and shSTIM1/GFP-STIM1 WT p≤ 0,001 *** (green). Lower panel, Distribution of distance to nucleus, normalized on cell size, quantified by image (as in a) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of EEA-1+ endosomes. Results are from two independent experiments for a total of 820 early endosomes in 12 shSTIM1/GFP-STIM1 WT cells (68 ± 6 endosomes per cell) and 400 early endosomes in 12 shSTIM1/GFP-STIM1 NN cells (33 ± 7 endosomes per cell) and analyzed a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.


( C ) Average , normalized on , quantified by image ( as in a ) segmentation ( see Material Method , Confocal microscopy early / late ) of EEA - 1 + endosomes . Results are average ± of two independent experiments for a total 830 early endosomes in 13 shCTL / GFP ( 64 ± 6 endosomes per ) , 719 early endosomes in 10 shSTIM1 / GFP ( 60 ± 6 endosomes per ) , 820 early endosomes in 12 shSTIM1 / GFP - STIM1 WT ( 68 ± 6 endosomes per ) 400 early endosomes in 12 shSTIM1 / GFP - STIM1 NN ( 33 ± 7 endosomes per ) analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for shCTL / GFP shSTIM1 / GFP p ≤ 0 , 001 * * * , for shCTL / GFP shSTIM1 / GFP - STIM1 WT p > 0 . 05 not significant ( ns ) , for shSTIM1 / GFP - STIM WT shSTIM1 / GFP - STIM1 NN p ≤ 0 , 001 * * * for shSTIM1 / GFP shSTIM1 / GFP - STIM1 NN p ≤ 0 , 05 * .

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

(C) Average size, normalized on cell size, quantified by image (as in a) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of EEA-1+ endosomes. Results are the average ± SEM of two independent experiments for a total 830 early endosomes in 13 shCTL/GFP cells (64 ± 6 endosomes per cell), 719 early endosomes in 10 shSTIM1/GFP cells (60 ± 6 endosomes per cell), 820 early endosomes in 12 shSTIM1/GFP-STIM1 WT cells (68 ± 6 endosomes per cell) and 400 early endosomes in 12 shSTIM1/GFP-STIM1 NN cells (33 ± 7 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for shCTL/GFP and shSTIM1/GFP p≤ 0,001 ***, for shCTL/GFP and shSTIM1/GFP-STIM1 WT p> 0.05 not significant (ns), for shSTIM1/GFP-STIM WT and shSTIM1/GFP-STIM1 NN p≤ 0,001 *** and for shSTIM1/GFP and shSTIM1/GFP-STIM1 NN p≤ 0,05 *.


( D ) Average number , normalized on , quantified by image ( as in a ) segmentation ( see Material Method , Confocal microscopy early / late ) of EEA - 1 + endosomes . Results are average ± of two independent experiments for a total 830 early endosomes in 13 shCTL / GFP ( 64 ± 6 endosomes per ) , 719 early endosomes in 10 shSTIM1 / GFP ( 60 ± 6 endosomes per ) , 820 early endosomes in 12 shSTIM1 / GFP - STIM1 WT ( 68 ± 6 endosomes per ) 400 early endosomes in 12 shSTIM1 / GFP - STIM1 NN ( 33 ± 7 endosomes per ) analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for shCTL / GFP shSTIM1 / GFP p ≤ 0 , 01 * * , for shCTL / GFP shSTIM1 / GFP - STIM1 WT p > 0 . 05 not significant ( ns ) , for shSTIM1 / GFP - STIM WT shSTIM1 / GFP - STIM1 NN p ≤ 0 , 001 * * * for shSTIM1 / GFP shSTIM1 / GFP - STIM1 NN p ≤ 0 , 001 * * * .

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

(D) Average number, normalized on cell size, quantified by image (as in a) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of EEA-1+ endosomes. Results are the average ± SEM of two independent experiments for a total 830 early endosomes in 13 shCTL/GFP cells (64 ± 6 endosomes per cell), 719 early endosomes in 10 shSTIM1/GFP cells (60 ± 6 endosomes per cell), 820 early endosomes in 12 shSTIM1/GFP-STIM1 WT cells (68 ± 6 endosomes per cell) and 400 early endosomes in 12 shSTIM1/GFP-STIM1 NN cells (33 ± 7 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for shCTL/GFP and shSTIM1/GFP p≤ 0,01 **, for shCTL/GFP and shSTIM1/GFP-STIM1 WT p> 0.05 not significant (ns), for shSTIM1/GFP-STIM WT and shSTIM1/GFP-STIM1 NN p≤ 0,001 *** and for shSTIM1/GFP and shSTIM1/GFP-STIM1 NN p≤ 0,001 ***.


( E ) Confocal microscopy of untreated ECs ( UT ) or treated with Ciliobrevin D ( CilioD ) stained for endogenous EEA - 1 ( in green ) LAMP - 1 ( in red ) to visualize EEs LEs , respectively , DAPI ( in blue ) to highlight nucleus . yellow is drawn to define periphery . Scale bar = 20 μm . On right , inset panels to highlight respective perinuclear peripheral of . Scale bar = 5 μm .

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

(E) Confocal microscopy images of untreated ECs (UT) or treated with Ciliobrevin D (CilioD) and stained for endogenous EEA-1 (in green) and LAMP-1 (in red) to visualize EEs and LEs, respectively, and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm.


( F ) Distribution of to nucleus overtime , normalized on , quantified by image ( data correspond to movies EV3 EV4 ) segmentation ( see Material Method , Live imaging experiments ) of Rab5 + endosomes , appearing for 30 seconds , during Ciliobrevin D ( CilioD ) treatment . Results are from three independent experiments for a total of 853 early endosomes in 6 untreated ( UT ) ( 142 ± 22 endosomes per ) 657 early endosomes in 9 CilioD ( 73 ± 23 endosomes per ) analyzed by a two - heteroscedastic Student ' s t - , p ≤ 0 , 001 * * * .

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

(F) Distribution of distance to nucleus overtime, normalized on cell size, quantified by image (data correspond to movies EV3 and EV4) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 30 seconds, during Ciliobrevin D (CilioD) treatment. Results are from three independent experiments for a total of and 853 early endosomes in 6 untreated (UT) cells (142 ±22 endosomes per cell) and 657 early endosomes in 9 CilioD cells (73 ± 23 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.


( G ) Distribution of to nucleus , normalized on , quantified by image ( as in e ) segmentation ( see Material Method , Confocal microscopy early / late ) of LAMP - 1 + endosomes . Results are from three independent experiments for a total of 432 late endosomes in 12 untreated ( UT ) ( 36 ± 9 endosomes per ) 1184 late endosomes in 18 CilioD ( 66 ± 9 endosomes per ) analyzed by a two - heteroscedastic Student ' s t - , p ≤ 0 , 001 * * * .

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

(G) Distribution of distance to nucleus, normalized on cell size, quantified by image (as in e) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of LAMP-1+ endosomes. Results are from three independent experiments for a total of 432 late endosomes in 12 untreated (UT) cells (36 ± 9 endosomes per cell) and 1184 late endosomes in 18 CilioD cells (66 ± 9 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.


( A ) mRNA of KIFC1 , KIFC2 KIFC3 in ECs . Values are normalized on GAPDH mRNA . Results are average ± SD of three independent experiments analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 01 ; Bonferroni for KIFC1 KIFC2 p ≤ 0 , 01 * * for KIFC1 KIFC3 p ≤ 0 , 05 * .

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

(A) mRNA levels of KIFC1, KIFC2 and KIFC3 in ECs. Values are normalized on GAPDH mRNA levels. Results are the average ± SD of three independent experiments and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,01; Bonferroni for KIFC1 and KIFC2 p≤ 0,01 ** and for KIFC1 and KIFC3 p≤ 0,05 *.


( B ) Representative Western Blot of endogenous KIFC1 STIM1 ( both normalized on total tubulin ) in ECs silenced with a control siRNA ( siCTL ) or one targeting KIFC1 ( siKIFC1 ) or two control siRNAs ( siCTLsiCTL ) or two targeting KIFC1 STIM1 ( siKIFC1siSTIM1 ) . Results are average ± SD of three independent experiments analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 01 ; Bonferroni for siCTL siKIFC1 , KIFC1 expression p ≤ 0 , 001 * * * , STIM1 expression p > 0 . 05 not significant ( ns ) for siCTLsiCTL siKIFC1siSTIM1 , KIFC1 expression p ≤ 0 , 01 * * , STIM1 expression p ≤ 0 , 05 * .

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

(B) Representative Western Blot analysis of endogenous KIFC1 and STIM1 proteins (both normalized on total tubulin levels) in ECs silenced with a control siRNA (siCTL) or one targeting KIFC1 (siKIFC1) or two control siRNAs (siCTLsiCTL) or two targeting KIFC1 and STIM1 (siKIFC1siSTIM1). Results are the average ± SD of three independent experiments and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,01; Bonferroni for siCTL and siKIFC1, KIFC1 expression p≤ 0,001 ***, STIM1 expression p> 0.05 not significant (ns) and for siCTLsiCTL and siKIFC1siSTIM1, KIFC1 expression p≤ 0,01 **, STIM1 expression p≤ 0,05 *.


( C ) Confocal microscopy of ECs silenced with a control siRNA ( siCTL ) or one targeting KIFC1 ( siKIFC1 ) or one targeting STIM1 ( siSTIM1 ) or two targeting both ( siKIFC1siSTIM1 ) stained for endogenous EEA - 1 ( in green ) LAMP - 1 ( in red ) to visualize EEs LEs , respectively , DAPI ( in blue ) to highlight nucleus . yellow is drawn to define periphery . Scale bar = 20 μm . On right , inset panels to highlight respective perinuclear peripheral of . Scale bar = 5 μm .

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

(C) Confocal microscopy images of ECs silenced with a control siRNA (siCTL) or one targeting KIFC1 (siKIFC1) or one targeting STIM1 (siSTIM1) or two targeting both (siKIFC1siSTIM1) and stained for endogenous EEA-1 (in green) and LAMP-1 (in red) to visualize EEs and LEs, respectively, and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm.


( D ) Distribution of to nucleus , normalized on , quantified by image ( as in c ) segmentation ( see Material Method , Confocal microscopy early / late ) of EEA - 1 + endosomes in siCTL siKIFC1 ECs . Results are from three independent experiments for a total of 4091 early endosomes in 19 siCTL ( 215 ± 20 endosomes per ) 2275 early endosomes in 20 siKIFC1 ( 114 ± 10 endosomes per ) analyzed by a two - heteroscedastic Student ' s t - , p ≤ 0 , 001 * * * .

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

(D) Distribution of distance to nucleus, normalized on cell size, quantified by image (as in c) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of EEA-1+ endosomes in siCTL and siKIFC1 ECs. Results are from three independent experiments for a total of 4091 early endosomes in 19 siCTL cells (215 ± 20 endosomes per cell) and 2275 early endosomes in 20 siKIFC1 cells (114 ± 10 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.


( E ) Distribution of to nucleus quantified by image ( as in c ) segmentation ( see Material Method , Confocal microscopy early / late ) of EEA - 1 + endosomes . Results are from three independent experiments for a total of 4091 early endosomes in 19 siCTL ( 215 ± 20 endosomes per ) , 2275 early endosomes in 20 siKIFC1 ( 114 ± 10 endosomes per ) 3257 early endosomes in 18 siKIFC1 / siSTIM1 ( 181 ± 14 endosomes per ) . Data are analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for siCTL siKIFC1 ECs p ≤ 0 , 001 * * * ( orange ) for siKIFC1 siSTIM1 / siKIFC1 ECs p ≤ 0 , 001 * * * ( green ) . On right - side of plot , colored bands represent average diameter , significantly changing throughout conditions . Data are analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 01 ; Bonferroni for siCTL siKIFC1 ECs p > 0 . 05 not significant ( orange ns ) for siKIFC1 siSTIM1 / siKIFC1 ECs p ≤ 0 , 01 * * ( green ) .

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

(E) Distribution of distance to nucleus quantified by image (as in c) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of EEA-1+ endosomes. Results are from three independent experiments for a total of 4091 early endosomes in 19 siCTL cells (215 ± 20 endosomes per cell), 2275 early endosomes in 20 siKIFC1 cells (114 ± 10 endosomes per cell) and 3257 early endosomes in 18 siKIFC1/siSTIM1 cells (181 ± 14 endosomes per cell). Data are analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for siCTL and siKIFC1 ECs p≤ 0,001 *** (orange) and for siKIFC1 and siSTIM1/siKIFC1 ECs p≤ 0,001 *** (green). On the right-hand side of the plot, colored bands represent the average cell diameter, significantly changing throughout the conditions. Data are analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,01; Bonferroni for siCTL and siKIFC1 ECs p> 0.05 not significant (orange ns) and for siKIFC1 and siSTIM1/siKIFC1 ECs p≤ 0,01 ** (green).


( A ) Confocal microscopy of untreated ( UT ) or treated with Ciliobrevin D ( CilioD ) or AZ82 or both treatments simultaneously ( CilioD + AZ82 ) ECs stained for endogenous EEA - 1 ( in green ) LAMP - 1 ( in red ) to visualize EEs LEs , respectively , Draq5 ( in blue ) to highlight nucleus . yellow is drawn to define periphery . Scale bar = 20 μm . On right , inset panels to highlight respective perinuclear peripheral of . Scale bar = 5 μm .

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

(A) Confocal microscopy images of untreated (UT) or treated with Ciliobrevin D (CilioD) or AZ82 or both treatments simultaneously (CilioD + AZ82) ECs and stained for endogenous EEA-1 (in green) and LAMP-1 (in red) to visualize EEs and LEs, respectively, and Draq5 (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm.


( B ) Distribution of to nucleus overtime , normalized on , quantified by image ( data correspond to movies EV3 EV5 ) segmentation ( see Material Method , Live imaging experiments ) of Rab5 + endosomes , appearing for 30 seconds , during AZ82 treatment in ECs , compared to untreated ( UT ) . Results are from two independent experiments for a total of 853 early endosomes in 6 untreated ( 142 ± 22 endosomes per ) 355 early endosomes in 7 AZ82 ( 51 ± 12 endosomes per ) . Data are analyzed by a two - heteroscedastic Student ' s t - , p ≤ 0 , 001 * * * .

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

(B) Distribution of distance to nucleus overtime, normalized on cell size, quantified by image (data correspond to movies EV3 and EV5) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 30 seconds, during AZ82 treatment in ECs, compared to untreated cells (UT). Results are from two independent experiments for a total of 853 early endosomes in 6 untreated cells (142 ± 22 endosomes per cell) and 355 early endosomes in 7 AZ82 cells (51±12 endosomes per cell). Data are analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.


( C ) Distribution of retrograde velocity overtime , quantified by image ( data correspond to movies EV3 , EV4 EV5 ) segmentation ( see Material Method , Live imaging experiments ) of Rab5 + endosomes , appearing for 30 seconds , during Ciliobrevin D ( CilioD D ) or AZ82 treatment in ECs , compared to untreated ( UT ) . Results are from three independent experiments for a total of 580 EEs in 6 UT ( 97 ± 18 endosomes per ) , 432 EEs in 9 CilioD ( 48 ± 15 endosomes per ) from two independent experiments for a total of 223 EEs in 7 AZ82 ( 32 ± 6 endosomes per ) . Data are analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for UT CilioD treated ECs p ≤ 0 , 01 * * for UT AZ82 treated ECs p ≤ 0 , 001 * * * .

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

(C) Distribution of retrograde velocity overtime, quantified by image (data correspond to movies EV3, EV4 and EV5) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 30 seconds, during Ciliobrevin D (CilioD D) or AZ82 treatment in ECs, compared to untreated cells (UT). Results are from three independent experiments for a total of 580 EEs in 6 UT cells (97 ± 18 endosomes per cell), 432 EEs in 9 CilioD cells (48 ± 15 endosomes per cell) and from two independent experiments for a total of 223 EEs in 7 AZ82 cells (32 ± 6 endosomes per cell). Data are analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for UT and CilioD treated ECs p≤ 0,01 ** and for UT and AZ82 treated ECs p≤ 0,001 ***.


( D ) Distribution of to nucleus overtime , normalized on , quantified by image ( data correspond to movies EV3 , EV5 EV6 ) segmentation ( see Material Method , Live imaging experiments ) of Rab5 + endosomes , appearing for 30 seconds , during AZ82 treatment ( as in b ) , alone or in combination with CilioD treatment ( CilioD + AZ82 ) , compared to untreated EC ( UT ) . Results are from two independent experiments for a total of 853 early endosomes in 6 UT ( 142 ± 22 endosomes per ) , 355 EEs in 7 AZ82 ( 51 ± 12 endosomes per ) 1106 EEs in 8 CilioD + AZ82 ( 136 ± 42 endosomes per ) analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for UT AZ82 treated ECs p ≤ 0 , 001 * * * ( orange ) for CilioD CilioD + AZ82 treated ECs p ≤ 0 , 001 * * * ( green ) .

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

(D) Distribution of distance to nucleus overtime, normalized on cell size, quantified by image (data correspond to movies EV3, EV5 and EV6) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 30 seconds, during AZ82 treatment (as in b), alone or in combination with CilioD treatment (CilioD + AZ82), compared to untreated EC cells (UT). Results are from two independent experiments for a total of 853 early endosomes in 6 UT cells (142 ±22 endosomes per cell), 355 EEs in 7 AZ82 cells (51 ± 12 endosomes per cell) and 1106 EEs in 8 CilioD+AZ82 cells (136 ± 42 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for UT and AZ82 treated ECs p≤ 0,001 *** (orange) and for CilioD and CilioD + AZ82 treated ECs p≤ 0,001 *** (green).


( A ) Representative of three Western Blot of endogenous - intermediate chain ( LIC ) of dynein 1 , KIFC1 p150Glued immunoprecipitated with HOOK1 in wild - type ECs . Negative control ( CTL ) was performed incubating lysate with A - or G - Sepharose empty rabbit IgG .

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(A) Representative of three Western Blot analysis of endogenous light-intermediate chain (LIC) of cytoplasmic dynein 1, KIFC1 and p150Glued immunoprecipitated with HOOK1 in wild-type ECs. Negative control (CTL) was performed incubating cell lysate with protein A- or G-Sepharose and empty rabbit IgG.


( B ) Representative of three Western Blot of endogenous p150Glued , HOOK3 p50 ( or Dynamitin ) immunoprecipitated with KIFC1 in wild - type ECs . Negative control ( CTL ) was performed incubating lysate with A - or G - Sepharose empty mouse IgG .

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(B) Representative of three Western Blot analysis of endogenous p150Glued, HOOK3 and p50 (or Dynamitin) immunoprecipitated with KIFC1 in wild-type ECs. Negative control (CTL) was performed incubating cell lysate with protein A- or G-Sepharose and empty mouse IgG.


( C ) Representative Western Blot of endogenous HOOK1 or HOOK3 total tubulin in ECs , silenced with a control siRNA ( siCTL ) or one targeting HOOK1 ( siHOOK1 ; on left ) or one targeting HOOK3 ( siHOOK3 ; on right ) .

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(C) Representative Western Blot analysis of endogenous HOOK1 or HOOK3 and total tubulin proteins in ECs, silenced with a control siRNA (siCTL) or one targeting HOOK1 (siHOOK1; on the left) or one targeting HOOK3 (siHOOK3; on the right).


( D ) Confocal microscopy of ECs silenced with a control siRNA ( siCTL ) or one targeting HOOK1 ( siHOOK1 ) or one targeting HOOK3 ( siHOOK3 ) or two targeting both ( siHOOK1siHOOK3 ) stained for endogenous EEA - 1 ( in green ) LAMP - 1 ( in red ) to visualize EEs LEs , respectively , DAPI ( in blue ) to highlight nucleus . yellow is drawn to define periphery . Scale bar = 20 μm . On right , inset panels to highlight respective perinuclear peripheral of . Scale bar = 5 μm .

(D) Confocal microscopy images of ECs silenced with a control siRNA (siCTL) or one targeting HOOK1 (siHOOK1) or one targeting HOOK3 (siHOOK3) or two targeting both (siHOOK1siHOOK3) and stained for endogenous EEA-1 (in green) and LAMP-1 (in red) to visualize EEs and LEs, respectively, and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm.


( E ) Distribution of to nucleus , normalized on , quantified by image ( as in d ) segmentation ( see Material Method , Confocal microscopy early / late ) of EEA - 1 + endosomes . Results are from three independent experiments for a total of 1193 early endosomes in 12 siCTL ( 99 ± 9 endosomes per ) 1104 early endosomes in 21 siHOOK1 ( 53 ± 4 endosomes per ) analyzed by a two - heteroscedastic Student ' s t - , p ≤ 0 , 001 * * * .

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(E) Distribution of distance to nucleus, normalized on cell size, quantified by image (as in d) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of EEA-1+ endosomes. Results are from three independent experiments for a total of 1193 early endosomes in 12 siCTL cells (99 ± 9 endosomes per cell) and 1104 early endosomes in 21 siHOOK1 cells (53 ± 4 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.


( F ) Distribution of to nucleus , normalized on , quantified by image ( as in d ) segmentation ( see Material Method , Confocal microscopy early / late ) of EEA - 1 + endosomes . Results are from three independent experiments for a total of 1193 early endosomes in 12 siCTL ( 99 ± 7 endosomes per ) 1282 early endosomes in 22 siHOOK3 ( 58 ± 5 endosomes per ) analyzed by a two - heteroscedastic Student ' s t - , p ≤ 0 , 001 * * * .

(F) Distribution of distance to nucleus, normalized on cell size, quantified by image (as in d) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of EEA-1+ endosomes. Results are from three independent experiments for a total of 1193 early endosomes in 12 siCTL cells (99 ± 7 endosomes per cell) and 1282 early endosomes in 22 siHOOK3 cells (58 ± 5 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.


( G ) Distribution of to nucleus quantified by image ( as in d ) segmentation ( see Material Method , Confocal microscopy early / late ) of EEA - 1 + endosomes . Results are from three independent experiments for a total of 1193 early endosomes in 12 siCTL ( 99 ± 7 endosomes per ) , 1104 EEs in 21 siHOOK1 ( 53 ± 4 endosomes per ) 2340 EEs in 19 siHOOK1 / siHOOK3 ( 123 ± 11 endosomes per ) analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for siCTL siHOOK1 ECs p ≤ 0 , 001 * * * ( orange ) for siHOOK1 siHOOK1 / siHOOK3 ECs p ≤ 0 , 001 * * * ( green ) . On right side of plot , colored bands represent average diameter , significantly changing throughout conditions . Data are analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for siCTL siHOOK1 ECs p > 0 . 05 not significant ( orange ns ) for siHOOK1 siHOOK1 / siHOOK3 ECs p ≤ 0 , 001 * * * ( green ) .

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(G) Distribution of distance to nucleus quantified by image (as in d) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of EEA-1+ endosomes. Results are from three independent experiments for a total of 1193 early endosomes in 12 siCTL cells (99 ± 7 endosomes per cell), 1104 EEs in 21 siHOOK1 cells (53 ± 4 endosomes per cell) and 2340 EEs in 19 siHOOK1/siHOOK3 cells (123 ± 11 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for siCTL and siHOOK1 ECs p≤ 0,001 *** (orange) and for siHOOK1 and siHOOK1/siHOOK3 ECs p≤ 0,001 *** (green). On the right hand side of the plot, colored bands represent the average cell diameter, significantly changing throughout the conditions. Data are analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for siCTL and siHOOK1 ECs p> 0.05 not significant (orange ns) and for siHOOK1 and siHOOK1/siHOOK3 ECs p≤ 0,001 *** (green).


( H ) Representative Western Blot of endogenous P150Glued co - immunoprecipitated with HOOK1 in untreated ( UT ) AZ82 treated ECs ( left ) its by normalized densitometry ( right ) . Negative control ( CTL ) was performed incubating lysate with A - or G - Sepharose empty rabbit IgG . Results are average ± SD of three independent assays . UT value of each biological replicate was normalized on itself so AZ82 experimental value . Results were analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for UT AZ82 p ≤ 0 , 05 * .

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(H) Representative Western Blot analysis of the endogenous P150Glued co-immunoprecipitated with HOOK1 in untreated (UT) and AZ82 treated ECs (left) and its quantification by normalized densitometry (right). Negative control (CTL) was performed incubating cell lysate with protein A- or G-Sepharose and empty rabbit IgG. Results are the average ± SD of three independent assays. UT value of each biological replicate was normalized on itself and so AZ82 experimental value. Results were analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for UT and AZ82 p≤ 0,05 *.


( A ) Confocal microscopy of ECs silenced with a control shRNA ( shCTL ) or one targeting STIM1 ( shSTIM1 ) , to which pH - sensitive dye pHrodo Green was given then were stained for LAMP - 1 ( in red ) to visualize LEs DAPI ( in blue ) to highlight nucleus . Scale bar = 20 μm . ( B ) Relative amount of LAMP - 1 + LEs positive to pH - sensitive dye pHrodo ( as in a ) in shCTL shSTIM1 ECs . Results are average ± of three independent experiments for a total of 90 ( 30 for experiment ) analyzed by a two - heteroscedastic Student ' s t - , p ≤ 0 , 001 * * * .

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(A) Confocal microscopy images of ECs silenced with a control shRNA (shCTL) or one targeting STIM1 (shSTIM1), to which the pH-sensitive dye pHrodo Green was given and then cells were stained for LAMP-1 (in red) to visualize LEs and DAPI (in blue) to highlight the nucleus. Scale bar = 20 μm. (B) Relative amount of LAMP-1+ LEs positive to the pH-sensitive dye pHrodo (as in a) in shCTL and shSTIM1 ECs. Results are the average ± SEM of three independent experiments for a total of 90 cells (30 cell for experiment) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.


( A ) Confocal microscopy of ECs silenced with a control shRNA ( shCTL ) or one targeting STIM1 ( shSTIM1 ) , to which pH - sensitive dye pHrodo Green was given then were stained for LAMP - 1 ( in red ) to visualize LEs DAPI ( in blue ) to highlight nucleus . Scale bar = 20 μm .

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(A) Confocal microscopy images of ECs silenced with a control shRNA (shCTL) or one targeting STIM1 (shSTIM1), to which the pH-sensitive dye pHrodo Green was given and then cells were stained for LAMP-1 (in red) to visualize LEs and DAPI (in blue) to highlight the nucleus. Scale bar = 20 μm.


( C ) Representative Western Blot of endogenous Raptor , total phospho - Thr389 S6K1 ( normalized on total tubulin ) in ECs silenced with a control shRNA ( shCTL ) or one targeting STIM1 ( siSTIM1 ) . Numbers above each band represent respective N . O . D . average ( two biological replicates ) .

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(C) Representative Western Blot analysis of endogenous Raptor, total and phospho-Thr389 S6K1 proteins (normalized on total tubulin levels) in ECs silenced with a control shRNA (shCTL) or one targeting STIM1 (siSTIM1). Numbers above each band represent respective N.O.D. average (two biological replicates) quantification.


( D ) Representative Western Blot of endogenous LC3 - I - II ( both normalized on total tubulin represented as LC3 - II / LC3 - I ) in ECs silenced with a control shRNA ( shCTL ) or one targeting STIM1 ( siSTIM1 ) . Results are average ± SD of three independent experiments analyzed by a two - heteroscedastic Student ' s t - , p ≤ 0 , 01 * * .

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(D) Representative Western Blot analysis of endogenous LC3-I and -II proteins (both normalized on total tubulin levels and represented as ratio LC3-II/LC3-I) in ECs silenced with a control shRNA (shCTL) or one targeting STIM1 (siSTIM1). Results are the average ± SD of three independent experiments and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,01 **.


a , Autophagosome formation in heart indicated by GFP - LC3 . Hearts were isolated from GFP - LC3 transgenic mice at multiple stages , including 18 . 5 day 0 . 5 h , 3 h , 6 h , 24 h 2 . 5 day neonates , immediately fixed , cryosectioned , analysed by microscopy . Scale bar , 10 µm .

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a, Autophagosome formation in the heart indicated by GFP-LC3. Hearts were isolated from GFP-LC3 transgenic mice at multiple stages, including 18.5 day embryos and 0.5 h, 3 h, 6 h, 24 h and 2.5 day neonates, immediately fixed, cryosectioned, and analysed by fluorescence microscopy. Scale bar, 10 µm.


b , of GFP - LC3 dots in neonatal tissues . Cryosections were prepared from tissues isolated at indicated times . of total of GFP - LC3 dots to total is shown as a . Values represent mean ± s . d . of three mice . Diaph . , diaphragm ; Panc . , pancreas ; Muscle ; gastrocnemius muscle .

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b, Quantification of GFP-LC3 dots in neonatal tissues. Cryosections were prepared from tissues isolated at the indicated times. The ratio of the total area of GFP-LC3 dots to the total cellular area is shown as a percentage. Values represent mean ± s.d. of three mice. Diaph., diaphragm; Panc., pancreas; Muscle; gastrocnemius muscle.


c , Electron microscopic of heart from wild - type neonates . Typical autophagosomes ( arrows ) autolysosomes ( arrowhead ) were observed in heart at 10 h after birth . Scale bar , 1 µm .

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c, Electron microscopic analysis of the heart from wild-type neonates. Typical autophagosomes (arrows) and autolysosomes (arrowhead) were observed in the heart at 10 h after birth. Scale bar, 1 µm.


d , Conversion of LC3 - I to LC3 - II in neonatal hearts . Heart homogenates from five / neonates were pooled analysed by immunoblotting using an anti - LC3 antibody . bands representing LC3 - I LC3 - II are marked with arrows .

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d, Conversion of LC3-I to LC3-II in neonatal hearts. Heart homogenates from five embryos/neonates were pooled and analysed by immunoblotting using an anti-LC3 antibody. The bands representing LC3-I and LC3-II are marked with arrows.


a , Detection of wild - type ( WT ) targeted alleles . Genomic was prepared from mouse tails analysed by Southern blot . Restriction enzymes used were HpaI for 5 ′ probe SpeI for 3 ′ probe ( see Supplementary Fig . S2 for restriction map ) . KO , knockout ( targeted ) allele .

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a, Detection of wild-type (WT) and targeted alleles. Genomic DNA was prepared from mouse tails and analysed by Southern blot analysis. Restriction enzymes used were HpaI for the 5′ probe and SpeI for the 3′ probe (see Supplementary Fig.S2 for the restriction map). KO, knockout (targeted) allele.


b , Photograph of a representative Atg5 - / - neonate compared to a wild - type littermate .

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b, Photograph of a representative Atg5-/- neonate compared to a wild-type littermate.


c , Immunoblot of Atg5 LC3 . Heart homogenates were prepared from neonates 10 h after birth . MEFs were cultured in Dulbeccos modified Eagles medium supplemented with 10 % FBS ( unstarved ) or Hanks ' solution for 2 h ( starved ) , total lysates were prepared . Immunoblot was performed using anti - Atg5 anti - LC3 antibodies . position of Atg12 - Atg5 conjugate on blot is indicated .

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c, Immunoblot analysis of Atg5 and LC3. Heart homogenates were prepared from neonates 10 h after birth. MEFs were cultured in Dulbeccos modified Eagles medium supplemented with 10% FBS (unstarved) or Hanks' solution for 2 h (starved), and total cell lysates were prepared. Immunoblot analysis was performed using anti-Atg5 and anti-LC3 antibodies. The position of the Atg12-Atg5 conjugate on the blot is indicated.


d , Absence of GFP - LC3 dot formation in Atg5 - / - mouse heart . Various tissues from Atg5 - / - mice expressing GFP - LC3 were analysed for dot formation by microscopy . A representative result from a neonatal heart at 3 h after birth is shown .

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d, Absence of GFP-LC3 dot formation in Atg5-/- mouse heart. Various tissues from Atg5-/- mice expressing GFP-LC3 were analysed for dot formation by fluorescence microscopy. A representative result from a neonatal heart at 3 h after birth is shown.


Neonates were obtained by caesarean delivery . a , b , Successfully resuscitated pups were monitored in a humidified chamber without milk feeding ( a ) or with artificial milk feeding provided every 3 - 6 h through a tube inserted into stomach ( b ) .

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Neonates were obtained by caesarean delivery. a, b, Successfully resuscitated pups were monitored in a humidified chamber without milk feeding (a) or with artificial milk feeding provided every 3-6 h through a tube inserted into the stomach (b).


a , Plasma tissue acid concentrations . acid concentrations were measured at 0 10 h after caesarean delivery under fasting conditions . ' Total ' indicates sum of Asp , Thr , Ser , Asn , Glu , Gln , Pro , Gly , Ala , Val , Cys , Met , Ile , Leu , Tyr , Phe , Lys , His Arg concentrations ; ' Essential ' indicates sum of Thr , Val , Met , Ile , Leu , Phe , Lys , His Arg concentrations ; ' BCAA ' indicates sum of Val , Ile Leu concentrations . Tissue acid concentrations are expressed as mmol kg - 1 of wet . Bars represent mean ± s . d . of three mice . Single double asterisks indicate a significant difference between mutant control ( wild - type plus heterozygous ) mice at P 0 . 05 P 0 . 01 , respectively .

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a, Plasma and tissue amino acid concentrations. Amino acid concentrations were measured at 0 and 10 h after the caesarean delivery under fasting conditions. 'Total' indicates the sum of the Asp, Thr, Ser, Asn, Glu, Gln, Pro, Gly, Ala, Val, Cys, Met, Ile, Leu, Tyr, Phe, Lys, His and Arg concentrations; 'Essential' indicates the sum of Thr, Val, Met, Ile, Leu, Phe, Lys, His and Arg concentrations; 'BCAA' indicates the sum of the Val, Ile and Leu concentrations. Tissue amino acid concentrations are expressed as mmol kg-1 of wet weight. Bars represent mean ± s.d. of three mice. Single and double asterisks indicate a significant difference between mutant and control (wild-type plus heterozygous) mice at P 0.05 and P 0.01, respectively.


b , Activation of AMPK in Atg5 - / - mice . Hearts were isolated from unfed neonates at 0 h ( lanes 1 , 3 , 5 ) 10 h ( lanes 2 , 4 , 6 ) , force - fed mice at 10 h ( lane 7 ) after caesarean delivery . Homogenates were prepared in presence of phosphatase inhibitors analysed by immunoblotting using anti - AMPK anti - phospho - AMPK antibodies .

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b, Activation of AMPK in Atg5-/- mice. Hearts were isolated from unfed neonates at 0 h (lanes 1, 3, 5) and 10 h (lanes 2, 4, 6), and force-fed mice at 10 h (lane 7) after the caesarean delivery. Homogenates were prepared in the presence of phosphatase inhibitors and analysed by immunoblotting using anti-AMPK and anti-phospho-AMPK antibodies.


c , ECGs from neonatal mice at indicated time after caesarean delivery . ST elevation was observed in Atg5 - / - mice ( n = 4 ) at 8 - 9 h after delivery .

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c, ECGs from neonatal mice at the indicated time after caesarean delivery. ST elevation was observed in all Atg5-/- mice (n = 4) at 8-9 h after delivery.


B , Mouse embryonic fibroblasts genetically modified using CRISPR to introduce a GFP immediately after start codon of Syne2 display at envelope .

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B, Mouse embryonic fibroblasts genetically modified using CRISPR to introduce a GFP sequence immediately after the start codon of Syne2 display fluorescence at the nuclear envelope.


C , Brightfield of a migrating through a 2 µm constriction . Arrows indicate areas of accumulation of nesprin - 2 at front of nucleus as it is deformed through constriction .

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C, Brightfield and fluorescence images of a cell migrating through a 2 µm constriction. Arrows indicate areas of accumulation of nesprin-2 at the front of the nucleus as it is deformed through the constriction.


E , Brightfield of a migrating along a 15 µm control channel .

E, Brightfield and fluorescence images of a cell migrating along a 15 µm control channel.


F , of of signal around perimeter of nucleus in a 2 µm constriction a 15 µm control channel . average at front back is normalized to at sides , demonstrating an increase in signal at front of nucleus as it is deformed a reduction after deformation . Error bars correspond to standard deviation . Constriction : n = 17 over 4 biological replicates , 15 µm channels : n = 20 over 3 biological replicates . A Wilcoxon signed - rank is used to compare matched samples , stars ( * P < 0 . 05 , * * P < 0 . 01 ) denote comparisons to measurement at side , pounds ( # P < 0 . 05 , # # P < 0 . 01 ) denote a comparison to " before " time point .

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F, Quantification of the intensity of the fluorescent signal around the perimeter of the nucleus in a 2 µm constriction and a 15 µm control channel. The average intensity at the front and the back is normalized to the intensity at the sides, demonstrating an increase in the signal at the front of the nucleus as it is deformed and a reduction after deformation. Error bars correspond to the standard deviation. Constriction: n=17 cells over 4 biological replicates, 15 µm channels: n=20 cells over 3 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars (*P < 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.


G , GFP - nesprin - 2 deformed by pressure gradient in a micropipette aspiration device . Note that GFP signal does not increase at tip but increases at sides of nucleus .

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G, GFP-nesprin-2 cells deformed by the pressure gradient in a micropipette aspiration device. Note that the GFP signal does not increase at the tip but increases at sides of the nucleus.


A , CRISPR - modified ( Brightfield , left ) that express GFP - nesprin - 2 ( middle ) mCh - LAC ( right ) show nesprin ( orange arrow ) but not lamin accumulation at front of nucleus as it is deformed through a narrow constriction . B , of of GFP - nesprin - 2 mCh - LAC , normalized to at sides . Error bars correspond to standard deviation . Constriction : n = 19 over 5 biological replicates , 15 µm : n = 27 over 6 biological replicates . A Wilcoxon signed - rank is used to compare matched samples , stars ( * P < 0 . 05 , * * P < 0 . 01 ) denote comparisons to measurement at side , pounds ( # P < 0 . 05 , # # P < 0 . 01 ) denote a comparison to " before " time point .

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

A, CRISPR-modified cells (Brightfield, left) that express GFP-nesprin-2 (middle) and mCh-LAC (right) show nesprin (orange arrow) but not lamin accumulation at the front of the nucleus as it is deformed through a narrow constriction. B, Quantification of intensity of GFP-nesprin-2 and mCh-LAC, normalized to the intensity at the sides. Error bars correspond to the standard deviation. Constriction: n=19 cells over 5 biological replicates, 15 µm: n= 27 over 6 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars (*P < 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.