( c , d ) . TEM analysis of mitochondria in mouse substantial nigra . Sections of substantial nigra from 18 - month - old PINK1KO and PINK1WT control mice were analyzed under TEM ( c , left panels , scale bar = 2 µm ) . Amplified images of mitochondria from dot line frames gated region are shown ( c , right panels , scale bar = 1 µm ) . Asterisks indicate abnormal elongated mitochondria . A quantitative analysis of mitochondria with perimeter > 2µm is shown ( d ) . Over 300 identifiable mitochondria ( cristae and / or double membrane ) per mice were randomly and blindly selected for perimeter measurement . Student ' s test . * p < 0 . 05 . Data was presented as mean ± SEM of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, d). TEM analysis of mitochondria in mouse substantial nigra. Sections of substantial nigra from 18-month-old PINK1KO and PINK1WT control mice were analyzed under TEM (c, left panels, scale bar=2 µm). Amplified images of mitochondria from dot line frames gated region are shown (c, right panels, scale bar=1 µm). Asterisks indicate abnormal elongated mitochondria. A quantitative analysis of mitochondria with perimeter >2µm is shown (d). Over 300 identifiable mitochondria (cristae and/or double membrane) per mice were randomly and blindly selected for perimeter measurement. Student's test. *p<0.05. Data was presented as mean ± SEM of three independent experiments.


( e , f ) . Overexpression of Drp1WT , but not phosphorylation mutant Drp1S616A , suppresses mitochondrial fusion in dendrites of PINK1KO primary neurons . ( e ) . DIV4 neurons generated from PINK1 WT control or PINK1KO mice were co - transfected mito - GFP with either pcDNA3 . 1 ( 3 . 1 ) or Drp1WT or Drp1S616A . Neurons were immuno - detected with an antibody against myc - tag ( Myc ) ( red , to detect exogenous Drp1 ) . Mito - GFP : green , to detect mitochondria . Representative images were shown , scale bar = 25 μm . Magnified dendritic shafts gated by a dot line frame are shown ( Zoom , right panels , scale bar = 5 μm ) . ( f ) . Quantification of Mito - index in dendrites of neurons from experiments of ( e ) . 10 neurons were randomly selected and analyzed in each experiment . One - way ANOVA followed with Tukey ' s test . * p < 0 . 05 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments .

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

(e, f). Overexpression of Drp1WT, but not phosphorylation mutant Drp1S616A, suppresses mitochondrial fusion in dendrites of PINK1KO primary neurons. (e). DIV4 neurons generated from PINK1 WT control or PINK1KO mice were co-transfected mito-GFP with either pcDNA3.1 (3.1) or Drp1WT or Drp1S616A. Neurons were immuno-detected with an antibody against myc-tag (Myc) (red, to detect exogenous Drp1). Mito-GFP: green, to detect mitochondria. Representative images were shown, scale bar=25 μm. Magnified dendritic shafts gated by a dot line frame are shown (Zoom, right panels, scale bar=5 μm).(f). Quantification of Mito-index in dendrites of neurons from experiments of (e). 10 neurons were randomly selected and analyzed in each experiment. One-way ANOVA followed with Tukey's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( a - c ) . Drp1 is required for mitochondrial fission induced by PINK1 . Drp1 - null HeLa cells ( Drp1KO ) and their wildtype controls ( Drp1WT ) were co - transfected Δ110 - PINK1 - GFP - FKBP with FRB - MTS . Cells were induced with 250 nM rapalog for 2 hours to activate PINK1 kinase , followed by immunodetecting mitochondria ( TOM20 , red ) and Δ110 - PINK1 - GFP - FKBP / FRB - MTS ( Δ110 - PINK1 , green ) . Nuclei were labeled with DAPI ( blue ) . Cells treated with solvent ( DMSO ) were used as a treatment control . Representative images were shown ( a , upper panel , scale bar = 25 μm ) . Higher magnification images are also included ( a , lower panel , scale bar = 10 μm ) . Mitochondrial morphology in different transfections was quantified . ( b , c ) . Student ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments . For each condition , > 100 cells were analyzed .

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

(a-c). Drp1 is required for mitochondrial fission induced by PINK1. Drp1-null HeLa cells (Drp1KO) and their wildtype controls (Drp1WT) were co-transfected Δ110-PINK1-GFP-FKBP with FRB-MTS. Cells were induced with 250 nM rapalog for 2 hours to activate PINK1 kinase, followed by immunodetecting mitochondria (TOM20, red) and Δ110-PINK1-GFP-FKBP /FRB-MTS (Δ110-PINK1, green). Nuclei were labeled with DAPI (blue). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown (a, upper panel, scale bar=25 μm). Higher magnification images are also included (a, lower panel, scale bar=10 μm). Mitochondrial morphology in different transfections was quantified. (b, c). Student's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( d - f ) . Drp1S616 phosphorylation is required for mitochondrial fission induced by PINK1 . Drp1KO HeLa cells were co - transfected Δ110 - PINK1 - GFP - FKBP / FRB - MTS with either a plasmid encoding Drp1WT or a Drp1 dephosphorylation mimic mutant Drp1S616A . Cells were treated with 250 nM rapalog for 2 hours to activate PINK1 kinase , followed by immunodetecting mitochondria ( TOM20 , blue ) , exogenous Drp1 ( myc , red ) , Δ110 - PINK1 - GFP - FKBP / FRB - MTS ( PINK1 - FKBP , green ) . Cells treated with solvent ( DMSO ) were used as a treatment control . Representative images were shown , scale bar = 25 μm ( d ) . Higher magnification images are also included ( d , Zoom , scale bar = 5 μm ) . Mitochondrial morphology in different transfections was quantified ( e , f ) . Student ' s test . * * p < 0 . 01 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments . For each condition , > 100 cells were analyzed .

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(d-f). Drp1S616 phosphorylation is required for mitochondrial fission induced by PINK1. Drp1KO HeLa cells were co-transfected Δ110-PINK1-GFP-FKBP /FRB-MTS with either a plasmid encoding Drp1WT or a Drp1 dephosphorylation mimic mutant Drp1S616A. Cells were treated with 250 nM rapalog for 2 hours to activate PINK1 kinase, followed by immunodetecting mitochondria (TOM20, blue), exogenous Drp1 (myc, red), Δ110-PINK1-GFP-FKBP /FRB-MTS (PINK1-FKBP, green). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown, scale bar=25 μm (d). Higher magnification images are also included (d, Zoom, scale bar=5 μm). Mitochondrial morphology in different transfections was quantified (e, f). Student's test. **p<0.01, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( g , h ) . Targeting PINK1 kinase domain to OMM increases mitochondrial localization of phosphorylated Drp1S616 . HEK293 cells co - transfected FRB - MTS with Δ110 - PINK1 - GFP - FKBP were treated with 250 nM rapalog ( Rapalog ) . After 2hours treatment , cytosol protion ( cyto ) and mitochondria ( Mito ) were fractionated and immunobloted with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) , Drp1 ( Drp1 ) , or GFP ( PINK1 ) . Tom20 and β - actin were detected as mitochondrial loading control and cytosol loading control , respectively ( g ) . Quantitation of pS616 / Drp1 in various conditions was shown ( h ) . Student ' s test . * * p < 0 . 01 , ns : no significance . Data was presented as mean ± SEM of three independent experiments .

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

(g, h). Targeting PINK1 kinase domain to OMM increases mitochondrial localization of phosphorylated Drp1S616. HEK293 cells co-transfected FRB-MTS with Δ110-PINK1-GFP-FKBP were treated with 250 nM rapalog (Rapalog). After 2hours treatment, cytosol protion (cyto) and mitochondria (Mito) were fractionated and immunobloted with antibodies against either phospho (Ser616)-Drp1 (pS616), Drp1 (Drp1), or GFP (PINK1). Tom20 and β-actin were detected as mitochondrial loading control and cytosol loading control, respectively (g). Quantitation of pS616/Drp1 in various conditions was shown (h). Student's test. **p<0.01, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( i , j ) . Targeting PINK1 kinase domain to the OMM increases mitochondrial localization of Drp1 . HeLa cells co - transfected FRB - MTS with Δ110 - PINK1 - GFP - FKBP ( FKBP - PINK1 ) were treated with 250 nM rapalog ( Rapalog ) for 2 hours to activate PINK1 kinase . Cells treated with solvent ( DMSO ) were included as a control . Cells were immunostained with antibodies against either Tom20 , Drp1 and GFP ( PINK1 - FKBP ) . Representative images were shown , scale bar = 2 μm ( i ) . Higher magnification images from gated box are shown ( bottom panels ) . Arrows indicate Drp1 puncta . Mitochondria - associated ( yellow ) and non - associated Drp1 ( Red ) are shown , scale bar = 1 μm . Mitochondria - associated Drp1 puncta were analyzed by using an ImageJ plugin - Coloc 2 and expressed as the Manders ' Colocalization Coefficients ( MCC ) ( j ) . Three different regions from each cell and > 15 cells for each experimental group were analyzed . Student ' s test . * * p < 0 . 01 . Data was presented as mean ± SEM of three independent experiments .

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

(i, j). Targeting PINK1 kinase domain to the OMM increases mitochondrial localization of Drp1. HeLa cells co-transfected FRB-MTS with Δ110-PINK1-GFP-FKBP (FKBP-PINK1) were treated with 250 nM rapalog (Rapalog) for 2 hours to activate PINK1 kinase. Cells treated with solvent (DMSO) were included as a control. Cells were immunostained with antibodies against either Tom20, Drp1 and GFP (PINK1-FKBP). Representative images were shown, scale bar=2 μm (i). Higher magnification images from gated box are shown (bottom panels). Arrows indicate Drp1 puncta. Mitochondria-associated (yellow) and non-associated Drp1(Red) are shown, scale bar=1 μm. Mitochondria-associated Drp1 puncta were analyzed by using an ImageJ plugin-Coloc 2 and expressed as the Manders' Colocalization Coefficients (MCC) (j). Three different regions from each cell and >15 cells for each experimental group were analyzed. Student's test. **p<0.01. Data was presented as mean ± SEM of three independent experiments.


( k , l ) . PINK1 regulates S616 and S637 phosphorylation of Drp1 via independent mechanisms . DRP1KO HEK293 cells co - transfected FRB - MTS / Δ110 - PINK1 - GFP - FKBP ( PINK1WT ) with either myc - Drp1WT or myc - Drp1S616A were treated with 250 nM rapalog ( Rapalog ) for 2 hours to activate PINK1 kinase . Cells treated with solvent ( DMSO ) were included as a control . Cell lysates were immunoblotting with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) , phospho - ( Ser637 ) - Drp1 ( pS637 ) , Drp1 and GFP ( to detect PINK1 - FKBP ) ( k ) . β - actin was detected as a loading control . Quantitation of pS637 / Drp1 in various conditions was shown ( l ) . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments .

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

(k, l). PINK1 regulates S616 and S637 phosphorylation of Drp1 via independent mechanisms. DRP1KO HEK293 cells co-transfected FRB-MTS/Δ110-PINK1-GFP-FKBP (PINK1WT) with either myc-Drp1WT or myc-Drp1S616A were treated with 250 nM rapalog (Rapalog) for 2 hours to activate PINK1 kinase. Cells treated with solvent (DMSO) were included as a control. Cell lysates were immunoblotting with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho-(Ser637)-Drp1 (pS637), Drp1 and GFP (to detect PINK1-FKBP) (k). β-actin was detected as a loading control. Quantitation of pS637/Drp1 in various conditions was shown (l). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( a - e ) . ATG5 is dispensable for PINK1 - mediated mitochondrial fission . MEF cells derived from ATG5 - null ( ATG5KO ) and their wildtype control mice ( ATG5WT ) were co - transfected Δ110 - PINK1 - GFP - FKBP / FRB - MTS with either PINK1WT or a kinase - dead mutant PINK1D384N . Cells were induced with 250 nM rapalog ( Rapalog ) for 2 hours to activate PINK1 kinase , followed by immunodetecting mitochondria ( TOM20 , red ) and Δ110 - PINK1 - GFP - FKBP / FRB - MTS ( PINK1 - FKBP , green ) . Cells treated with solvent ( DMSO ) were used as a treatment control . Representative images are shown , scale bar = 25 μm ( a ) . Higher magnification images are also included ( panels beneath the large cell images , scale bar = 10 μm ) . Mitochondrial morphology in different transfections was quantified ( b - e ) . Student ' s test . * p < 0 . 05 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments . For each condition , > 100 cells were analyzed .

O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-e). ATG5 is dispensable for PINK1-mediated mitochondrial fission. MEF cells derived from ATG5-null (ATG5KO) and their wildtype control mice (ATG5WT) were co-transfected Δ110-PINK1-GFP-FKBP /FRB-MTS with either PINK1WT or a kinase-dead mutant PINK1D384N. Cells were induced with 250 nM rapalog (Rapalog) for 2 hours to activate PINK1 kinase, followed by immunodetecting mitochondria (TOM20, red) and Δ110-PINK1-GFP-FKBP /FRB-MTS (PINK1-FKBP, green). Cells treated with solvent (DMSO) were used as a treatment control. Representative images are shown, scale bar=25 μm (a). Higher magnification images are also included (panels beneath the large cell images, scale bar=10 μm). Mitochondrial morphology in different transfections was quantified (b-e). Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( f - i ) . Drp1 is dispensable for recruitment of parkin to mitochondria . Drp1WT and Drp1KO HeLa cells were transfected with GFP - parkin . Cells were treated with 20 μM CCCP for 2 hours ( f ) followed by immunodetecting mitochondria ( TOM20 , red ) and parkin ( GFP - parkin , green ) . Nuclei were labeled with DAPI ( blue ) . Cells treated with solvent ( DMSO ) were used as a treatment control . Representative images were shown , scale bar = 25 μm . Cell with mitochondrial parkin ( % ) in different experimental group was quantified ( g , i ) . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments . For each condition , > 100 cells were analyzed .

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

(f-i). Drp1 is dispensable for recruitment of parkin to mitochondria. Drp1WT and Drp1KO HeLa cells were transfected with GFP-parkin. Cells were treated with 20 μM CCCP for 2 hours (f) followed by immunodetecting mitochondria (TOM20, red) and parkin (GFP-parkin, green). Nuclei were labeled with DAPI (blue). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown, scale bar=25 μm. Cell with mitochondrial parkin (%) in different experimental group was quantified (g, i). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( f - i ) . Drp1 is dispensable for recruitment of parkin to mitochondria . Drp1WT and Drp1KO HeLa cells were transfected with GFP - parkin . Cells were treated with 150 μM actinonin for 6 hours ( h ) , followed by immunodetecting mitochondria ( TOM20 , red ) and parkin ( GFP - parkin , green ) . Nuclei were labeled with DAPI ( blue ) . Cells treated with solvent ( DMSO ) were used as a treatment control . Representative images were shown , scale bar = 25 μm . Cell with mitochondrial parkin ( % ) in different experimental group was quantified ( g , i ) . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments . For each condition , > 100 cells were analyzed .

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

(f-i). Drp1 is dispensable for recruitment of parkin to mitochondria. Drp1WT and Drp1KO HeLa cells were transfected with GFP-parkin. Cells were treated with 150 μM actinonin for 6 hours (h), followed by immunodetecting mitochondria (TOM20, red) and parkin (GFP-parkin, green). Nuclei were labeled with DAPI (blue). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown, scale bar=25 μm. Cell with mitochondrial parkin (%) in different experimental group was quantified (g, i). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( a , b ) . Drp1WT and Drp1S616D , but not Drp1S616A , rescue PINK1 - null induced crushed thorax in Drosophila . Representative thorax images of PINK1WT and PINK1 - deficiency ( PINK1KO ) Drosophila expressing either mhc - gal4 ( Mhc / + ) , mhc - gal4 driven human Drp1wt ( Mhc > hDrp1WT ) or mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) are shown . Images were taken either under light microscopy ( a , upper panels , yellow arrowhead indicates the collapsed throax ) or SEM ( b , lower panels ) . Quantitation of crushed thorax is shown ( b ) . > 200 flies for each experiment are analyzed . Scale bar = 200µm . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 . Data was presented as mean ± SEM of three independent experiments . For each condition , > 200 flies were quantified .

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

(a, b). Drp1WT and Drp1S616D, but not Drp1S616A, rescue PINK1-null induced crushed thorax in Drosophila. Representative thorax images of PINK1WT and PINK1-deficiency (PINK1KO) Drosophila expressing either mhc-gal4 (Mhc/+), mhc-gal4 driven human Drp1wt (Mhc>hDrp1WT) or mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D) are shown. Images were taken either under light microscopy (a, upper panels, yellow arrowhead indicates the collapsed throax) or SEM (b, lower panels). Quantitation of crushed thorax is shown (b). > 200 flies for each experiment are analyzed. Scale bar=200µm. One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments. For each condition, >200 flies were quantified.


( c ) Drp1WT and Drp1S616D , but not Drp1S616A , restore abnormal ATP production in PINK1 - null flies . ATP production was measured using muscle lysates generated from PINK1WT flies expressing mhc - gal4 ( Mhc / + ) ( as a control ) and PINK1KO flies expressing either mhc - gal4 ( Mhc / + ) , mhc - gal4 driven human Drp1wt ( Mhc > hDrp1wt ) or mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments .

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

(c) Drp1WT and Drp1S616D, but not Drp1S616A, restore abnormal ATP production in PINK1-null flies. ATP production was measured using muscle lysates generated from PINK1WT flies expressing mhc-gal4 (Mhc/+) (as a control) and PINK1KO flies expressing either mhc-gal4 (Mhc/+), mhc-gal4 driven human Drp1wt (Mhc>hDrp1wt) or mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


Drp1WT and Drp1S616D , but not Drp1S616A , rescue mitochondrial structures in PINK1KO flies . Representative images of Mito - GFP ( green ) labeled mitochondria ( d ) from PINK1WT flies expressing mhc - gal4 ( Mhc / + ) ( as a control ) and PINK1KO flies expressing either mhc - gal4 ( Mhc / + ) , Mhc - gal4 driven human Drp1wt ( Mhc > hDrp1wt ) or Mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) are shown . Scale bar = 10 μm ( d ) Average mitochondrial size was quantified , > 3 flies / group and 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . One - way ANOVA followed with Tukey ' s test . * p < 0 . 05 , * * * p < 0 . 001 . Data was presented as mean ± SEM of three independent experiments ( e ) .

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

Drp1WT and Drp1S616D, but not Drp1S616A, rescue mitochondrial structures in PINK1KO flies. Representative images of Mito-GFP (green) labeled mitochondria (d) from PINK1WT flies expressing mhc-gal4 (Mhc/+) (as a control) and PINK1KO flies expressing either mhc-gal4 (Mhc/+), Mhc-gal4 driven human Drp1wt (Mhc>hDrp1wt) or Mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D) are shown. Scale bar=10 μm (d) Average mitochondrial size was quantified, >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. One-way ANOVA followed with Tukey's test. *p<0.05, ***p<0.001. Data was presented as mean ± SEM of three independent experiments (e).


Drp1WT and Drp1S616D , but not Drp1S616A , rescue mitochondrial structures in PINK1KO flies . Representative images of TEM analysis ( f ) of IFMs from PINK1WT flies expressing mhc - gal4 ( Mhc / + ) ( as a control ) and PINK1KO flies expressing either mhc - gal4 ( Mhc / + ) , Mhc - gal4 driven human Drp1wt ( Mhc > hDrp1wt ) or Mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) are shown . Scale bar = 1 μm ( f ) . Average mitochondrial size was quantified , > 3 flies / group and 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . Normal and abnormal mitochondrial number per mm2 were quantified . > 3 flies / group and 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . Bars represent the mean ± SEM of three independent experiments ( g ) .

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

Drp1WT and Drp1S616D, but not Drp1S616A, rescue mitochondrial structures in PINK1KO flies. Representative images of TEM analysis (f) of IFMs from PINK1WT flies expressing mhc-gal4 (Mhc/+) (as a control) and PINK1KO flies expressing either mhc-gal4 (Mhc/+), Mhc-gal4 driven human Drp1wt (Mhc>hDrp1wt) or Mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D) are shown. Scale bar= 1 μm (f). Average mitochondrial size was quantified, >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. Normal and abnormal mitochondrial number per mm2 were quantified. >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. Bars represent the mean ± SEM of three independent experiments (g).


( h , i ) . Drp1WT and Drp1S616D , but not Drp1S616A , suppress cell death in PINK1KO flies . Representative TUNEL staining ( Red ) images of IFM sections from PINK1WT and PINK1KO flies expressing either Mhc - gal4 ( Mhc / + ) , Mhc - gal4 driven human Drp1wt ( Mhc > hDrp1WT ) or Mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) are shown . Nuclei were counter - stained with DAPI ( blue ) , scale bar = 10 μm . Apoptotic cells ( % ) in different genotype flies were quantified ( i ) . > 5 flies / group and 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 . Data was presented as mean ± SEM of three independent experiments .

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

(h, i). Drp1WT and Drp1S616D, but not Drp1S616A, suppress cell death in PINK1KO flies. Representative TUNEL staining (Red) images of IFM sections from PINK1WT and PINK1KO flies expressing either Mhc-gal4 (Mhc/+), Mhc-gal4 driven human Drp1wt (Mhc>hDrp1WT) or Mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D) are shown. Nuclei were counter-stained with DAPI (blue), scale bar=10 μm. Apoptotic cells (%) in different genotype flies were quantified (i). >5 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat.One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments.


( j ) . Expression and phosphorylation of hDrp1 in Drosophila . Fly muscle lysates generated from PINK1WT or PINK1KO flies expressing mhc - gal4 driven hDrp1WT ( WT ) and hDrp1S616A ( S616A ) were immunoblotted with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) ( to detect phosphorylated hDrp1S616 ) , Drp1 ( to detect both endogenous and exogenous Drp1 ) , myc - tag ( to detect exogenous Drp1 ) . α - tubulin was detected as a loading control . Flies expressing mhc - gal4 ( - ) were included as an expression control .

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

(j). Expression and phosphorylation of hDrp1 in Drosophila. Fly muscle lysates generated from PINK1WT or PINK1KO flies expressing mhc-gal4 driven hDrp1WT (WT) and hDrp1S616A (S616A) were immunoblotted with antibodies against either phospho(Ser616)-Drp1(pS616) (to detect phosphorylated hDrp1S616), Drp1 (to detect both endogenous and exogenous Drp1), myc-tag (to detect exogenous Drp1). α-tubulin was detected as a loading control. Flies expressing mhc-gal4 (-) were included as an expression control.


( a , b ) . Drp1 rescues PINK1KO induced collapsed thorax phenotype in dATG7 - deficient Drosophila . Representative SEM images of thorax from PINK1WT , ( upper panels ) and PINK1KO ( lower panels ) flies combined with dATP7 deficiency ( dATG7KO ) or their controls ( dATG7WT ) , followed by expressing either mhc - gal4 ( Mhc / + ) alone , or Mhc - gal4 driven expression of human Drp1 ( Mhc > hDrp1WT ) , mhc - gal4 driven expression of human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven expression of human Drp1S616D ( Mhc > hDrp1S616D ) are shown ( a ) . Quantitation of crushed thorax in flies with indicated genotypes is presented ( b ) . > 100 flies for each experiment are analyzed . Scale bar = 200µm . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 . Data was presented as mean ± SEM of three independent experiments .

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

(a, b). Drp1 rescues PINK1KO induced collapsed thorax phenotype in dATG7-deficient Drosophila. Representative SEM images of thorax from PINK1WT, (upper panels) and PINK1KO (lower panels) flies combined with dATP7 deficiency (dATG7KO) or their controls (dATG7WT), followed by expressing either mhc-gal4 (Mhc/+) alone, or Mhc-gal4 driven expression of human Drp1 (Mhc>hDrp1WT), mhc-gal4 driven expression of human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven expression of human Drp1S616D (Mhc>hDrp1S616D) are shown (a). Quantitation of crushed thorax in flies with indicated genotypes is presented (b). > 100 flies for each experiment are analyzed. Scale bar=200µm. One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments.


( c , d ) Drp1 rescues PINK1KO induced mitochondrial defects in dATG7KO Drosophila . Representative TEM images of indirect flight muscle from PINK1WT ( upper panels ) and PINK1KO ( lower panels ) flies combined with dATG7KO or their controls ( dATG7WT ) , followed by expressing either mhc - gal4 ( Mhc / + ) alone , or Mhc - gal4 driven expression of human Drp1 ( Mhc > hDrp1WT ) , mhc - gal4 driven expression of human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven expression of human Drp1S616D ( Mhc > hDrp1S616D ) are shown ( c ) . Scale bar = 1 μm . Normal and abnormal mitochondrial number per mm2 from each experimental group were and quantified . > 3 flies / group and 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . Bars represent the mean ± SEM of three independent experiments ( d ) .

O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, d) Drp1 rescues PINK1KO induced mitochondrial defects in dATG7KO Drosophila. Representative TEM images of indirect flight muscle from PINK1WT (upper panels) and PINK1KO (lower panels) flies combined with dATG7KO or their controls (dATG7WT), followed by expressing either mhc-gal4 (Mhc/+) alone, or Mhc-gal4 driven expression of human Drp1(Mhc>hDrp1WT), mhc-gal4 driven expression of human Drp1S616A(Mhc>hDrp1S616A) or mhc-gal4 driven expression of human Drp1S616D(Mhc>hDrp1S616D) are shown (c). Scale bar=1 μm. Normal and abnormal mitochondrial number per mm2 from each experimental group were and quantified. >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. Bars represent the mean ± SEM of three independent experiments (d).


( e ) . Drp1 rescues PINK1KO induced ATP reduction in Drosophila . ATP contents of thorax muscle tissues from the indicated genotypes were measured and normalized against the protein levels One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments .

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). Drp1 rescues PINK1KO induced ATP reduction in Drosophila. ATP contents of thorax muscle tissues from the indicated genotypes were measured and normalized against the protein levels One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( g , h ) . Reduction of Drp1S616 phosphorylation in dermal fibroblasts derived from PD patients with PINK1 mutations . Lysates of cells derived from three unrelated normal control individuals ( Control , C ) and four familial PD patients with PINK1 mutations ( PINK1mutant ) were immunoblotted with antibodies recognizing either phosphor - Drp1S616 ( pS616 ) or Drp1 protein . β - actin was detected as a loading control ( g ) . A quantitative analysis of phospho - Drp1S616 ( pS616 / Drp1 ) is shown ( h ) . Student ' s test . * * p < 0 . 01 , ns : no significance . Data was presented as mean ± SEM of three independent experiments .

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

(g, h). Reduction of Drp1S616 phosphorylation in dermal fibroblasts derived from PD patients with PINK1 mutations. Lysates of cells derived from three unrelated normal control individuals (Control, C) and four familial PD patients with PINK1 mutations (PINK1mutant) were immunoblotted with antibodies recognizing either phosphor-Drp1S616 (pS616) or Drp1 protein. β-actin was detected as a loading control (g). A quantitative analysis of phospho-Drp1S616 (pS616/Drp1) is shown (h). Student's test. **p<0.01, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


Drp1S616 phosphorylation in dermal fibroblasts derived from sporadic PD patients . Lysates of cells derived from five unrelated normal control individuals ( Control , C ) and seven unrelated sporadic PD patients ( Sporadic PD , SP ) were immunoblotted with antibodies recognizing either phospho - Drp1S616 ( pS616 ) or Drp1 protein . β - actin was detected as a loading control ( i ) .

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

Drp1S616 phosphorylation in dermal fibroblasts derived from sporadic PD patients. Lysates of cells derived from five unrelated normal control individuals (Control, C) and seven unrelated sporadic PD patients (Sporadic PD, SP) were immunoblotted with antibodies recognizing either phospho-Drp1S616 (pS616) or Drp1 protein. β-actin was detected as a loading control (i).


Drp1S616 phosphorylation in dermal fibroblasts derived from sporadic PD patients . A quantitative analysis of phospho - Drp1S616 ( pS616 / Drp1 ) in sporadic cases is shown ( j ) . Student ' s test . * p < 0 . 05 . Data was presented as mean ± SEM of three independent experiments .

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

Drp1S616 phosphorylation in dermal fibroblasts derived from sporadic PD patients. A quantitative analysis of phospho-Drp1S616 (pS616/Drp1) in sporadic cases is shown (j). Student's test. *p<0.05. Data was presented as mean ± SEM of three independent experiments.


( 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 * .

O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

(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 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 .

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

(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 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 * .

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

(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 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 .

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

(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 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 ) .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 * * * .

O O O O O O O O O O O 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

(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 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 * * * .

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

(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 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 .

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

(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 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 ) .

O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-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 B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O 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 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 ) .

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

(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 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 ) .

O O O O O O O O O O O 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 B-CONTROLLED_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 O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O 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 B-CONTROLLED_VAR O 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 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 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_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 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 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 * * * .

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

(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 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 * * .

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

(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 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 * * .

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

(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 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 .

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

(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 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 * * * .

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

(B) 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 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 * .

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

(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 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 * * * .

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

(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 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 .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 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 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 * * * .

O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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 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 * * * .

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

(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 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 * .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-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 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 * .

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

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 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 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) 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 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 * * * .

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

(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 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 ) .

O O O O O O O O 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 O 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 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 .

O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 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 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 * * * .

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

(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 ***.