Fractions of mRNA reads RPFs mapping to each synthetic expression construct ( LacZ PK - LacZ ) E . coli transcripts , which are divided into three major categories : ribosomal , metabolic , other functions . Data shown for cultured in absence presence of IPTG ( 1 mM ) .

O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O

Fractions of mRNA reads and RPFs mapping to each synthetic expression construct (LacZ and PK-LacZ) and E. coli transcripts, which are divided into three major categories: ribosomal, metabolic, and other functions. Data shown for cells cultured in the absence and presence of IPTG (1 mM).


B HeLa were infected with x - Shigella mCherry for 4 h 40 min for quantitative confocal microscopy . IPTG was added 30 min prior to fixation , then samples were labelled with antibody for SEPT7 . scale bar represents 5 μm . Graph represents mean % ± of Shigella responding to IPTG outside ( - ) or inside ( + ) SEPT7 cages from 4 independent experiments . Student ' s t - , * * * = p < 0 . 001 .

O B-CELL_LINE O O O O O B-ORGANISM B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

B HeLa cells were infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for SEPT7. The scale bar represents 5 μm. Graph represents mean % ± SEM of Shigella responding to IPTG outside (-) or inside (+) SEPT7 cages from 4 independent experiments. Student's t-test, *** = p<0.001.


C HeLa were infected with x - Shigella mCherry for 4 h 40 min for quantitative confocal microscopy . IPTG was added 30 min prior to fixation , then samples were labelled with antibody for p62 . scale bar represents 5 μm . Graph represents mean % ± of Shigella responding to IPTG without ( - ) or with ( + ) p62 from at least 4 independent experiments . Student ' s t - , * * * = p < 0 . 001 .

O B-CELL_LINE O O O O O B-ORGANISM B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

C HeLa cells were infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for p62. The scale bar represents 5 μm. Graph represents mean % ± SEM of Shigella responding to IPTG without (-) or with (+) p62 from at least 4 independent experiments. Student's t-test, *** = p<0.001.


D Control ( CTRL ) or isopropanol - treated x - Shigella GFP were induced with IPTG for 30 min , then labelled with SYTOX Orange for 10 min . scale bar represents 5 μm .

O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

D Control (CTRL) or isopropanol-treated x-light Shigella GFP were induced with IPTG for 30 min, then labelled with SYTOX Orange for 10 min. The scale bar represents 5 μm.


E HeLa were infected for 1 h 30 min , treated with ethanol ( CTRL ) or erythromycin ( EM ) for 2 h prior to fixation , then labelled with antibody for SEPT7 . Graph represents mean % ± of Shigella in SEPT7 cages from 3 independent experiments . Student ' s t - , * * * = p < 0 . 001 .

O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

E HeLa cells were infected for 1 h 30 min, treated with ethanol (CTRL) or erythromycin (EM) for 2 h prior to fixation, and then labelled with antibody for SEPT7. Graph represents mean % ± SEM of Shigella in SEPT7 cages from 3 independent experiments. Student's t-test, *** = p<0.001.


B HeLa stably expressing SEPT6 - GFP were infected with S . flexneri AfaI for 4 h , harvested , then tested with Co - IP . Empty vector / or uninfected HeLa were used in parallel as control . GFP - trap magnetic agarose beads were used to isolate SEPT6 - GFP from , extracts were immunoblotted for SEPT6 , GFP , SEPT7 , or p62 . lysates were immunoblotted for GAPDH as control for .

O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O

B HeLa cells stably expressing SEPT6-GFP were infected with S. flexneri AfaI for 4 h, harvested, and then tested with Co-IP. Empty vector and/or uninfected HeLa cells were used in parallel as control. GFP-trap magnetic agarose beads were used to isolate SEPT6-GFP from cells, and extracts were immunoblotted for SEPT6, GFP, SEPT7, or p62. The lysates were immunoblotted for GAPDH as control for cellular protein levels.


A HeLa were labelled with MitoTracker Red CMXRos , treated with cytochalasin D for 30 min , then fixed labelled for endogenous SEPT7 for confocal microscopy . scale bar represents 0 . 5 μm . See also Fig EV3A .

O B-CELL_LINE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O

A HeLa cells were labelled with MitoTracker Red CMXRos, treated with cytochalasin D for 30 min, then fixed and labelled for endogenous SEPT7 for confocal microscopy. The scale bar represents 0.5 μm. See also Fig EV3A.


B HeLa were infected with S . flexneri for 4 h 40 min , labelled with MitoTracker Red CMXRos , fixed labelled with antibody for endogenous SEPT7 for quantitative confocal microscopy . scale bar represents 1 μm .

O B-CELL_LINE O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

B HeLa cells were infected with S. flexneri for 4 h 40 min, labelled with MitoTracker Red CMXRos, fixed and labelled with antibody for endogenous SEPT7 for quantitative confocal microscopy. The scale bar represents 1 μm.


C HeLa stably expressing SEPT6 - GFP were transfected with Mito - BFP for 24 h , infected with Shigella - mCherry for 4 h 40 min , processed for CLEM . SEPT6 is shown in green , mitochondria in red , Shigella - mCherry in blue . Septin cages identified by microscopy ( FLM ) were processed for . right - most image is enlarged from boxed region in image , shows bacterium ( Sf ) surrounded by a double of autophagy ( Au ) mitochondrial ( Mt ) . scale bar represents 1 μm . See also Fig EV3B .

O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-ORGANISM O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O

C HeLa cells stably expressing SEPT6-GFP were transfected with Mito-BFP for 24 h, infected with Shigella-mCherry for 4 h 40 min, and processed for CLEM. SEPT6 is shown in green, mitochondria in red, and Shigella-mCherry in blue. Septin cages identified by fluorescent light microscopy (FLM) were processed for TEM. The right-most image is enlarged from the boxed region in the TEM image, and shows the bacterium (Sf) surrounded by a double membrane of autophagy (Au) and the mitochondrial membrane (Mt). The scale bar represents 1 μm. See also Fig EV3B.


D HeLa stably expressing SEPT6 - GFP were transfected with Mito - BFP for 24 h , then infected with Shigella - mCherry for 2 h for live confocal microscopy . Time frame shows a septin cage assembling around Shigella . Mitochondria ( circle ) support sites of septin ring assembly around bacterium . Each frame was acquired every 2 min . scale bar represents 1 μm . See also Movie EV1 .

O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-ORGANISM O B-SUBCELLULAR O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O

D HeLa cells stably expressing SEPT6-GFP were transfected with Mito-BFP for 24 h, then infected with Shigella-mCherry for 2 h for live confocal microscopy. Time frame sequence shows a septin cage assembling around Shigella. Mitochondria (circle) support sites of septin ring assembly around the bacterium. Each frame was acquired every 2 min. The scale bar represents 1 μm. See also Movie EV1.


E HeLa were treated with control ( CTRL ) , Drp1 , or Mfn1 siRNA for 72 h . Whole lysates were immunoblotted for Drp1 or Mfn1 to show efficiency of depletion . GAPDH was used as loading control . siRNA - treated were infected with S . flexneri for 4 h 40 min , then fixed for microscopy stained for endogenous SEPT7 for quantitative confocal microscopy . Graph represents mean % ± of Shigella inside SEPT7 cages from at least 4 independent experiments per treatment . Student ' s t - , * * = p < 0 . 01 ; * * * = p < 0 . 001 .

O B-CELL_LINE O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O 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 HeLa cells were treated with control (CTRL), Drp1, or Mfn1 siRNA for 72 h. Whole cell lysates were immunoblotted for Drp1 or Mfn1 to show the efficiency of depletion. GAPDH was used as loading control. siRNA-treated cells were infected with S. flexneri for 4 h 40 min, then fixed for microscopy and stained for endogenous SEPT7 for quantitative confocal microscopy. Graph represents the mean % ± SEM of Shigella inside SEPT7 cages from at least 4 independent experiments per treatment. Student's t-test, ** = p <0.01; *** = p<0.001.


A HeLa expressing SEPT6 - GFP were stained with MitoTracker Red CMXRos , fixed for confocal microscopy . Inset image highlights location of septins at sites of mitochondrial fission . scale bar represents 5 μm .

O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O

A HeLa cells expressing SEPT6-GFP were stained with MitoTracker Red CMXRos, and fixed for confocal microscopy. Inset image highlights location of septins at sites of mitochondrial fission. The scale bar represents 5 μm.


B HeLa were fixed labelled with antibody against SEPT7 secondary antibody coupled to HRP , processed for . right image is enlarged from boxed region in image , shows a SEPT7 ( S7 ) filament ( labelled in black ) intersecting with mitochondrial ( Mt ) fission site . scale bar represents 100 nm . See also Fig EV4 .

O B-CELL_LINE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

B HeLa cells were fixed and labelled with antibody against SEPT7 and secondary antibody coupled to HRP, and processed for TEM. The right image is enlarged from the boxed region in the TEM image, and shows a SEPT7 (S7) filament (labelled in black) intersecting with the mitochondrial (Mt) fission site. The scale bar represents 100 nm. See also Fig EV4.


C HeLa expressing SEPT6 - GFP were stained with MitoTracker Red CMXRos for live SOFI - super resolution microscopy . Each image was reconstructed from 100 raw TIRF frames using a custom made SOFI - algorithm . Arrows highlight SEPT6 association with location of mitochondrial fission . scale bar represents 1 µm . See also Movie EV3 .

O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O

C HeLa cells expressing SEPT6-GFP were stained with MitoTracker Red CMXRos for live SOFI-based super resolution microscopy. Each image was reconstructed from 100 raw TIRF frames using a custom made SOFI-based algorithm. Arrows highlight SEPT6 association with location of mitochondrial fission. The scale bar represents 1 µm. See also Movie EV3.


D HeLa expressing SEPT6 - GFP were labelled with MitoTracker Red CMXRos , fixed for widefield immunofluorescence microscopy , labelled with antibodies to Drp1 . Arrows highlight one example of Drp1 SEPT6 association with location of mitochondrial fission . scale bar represents 1 μm .

O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O

D HeLa cells expressing SEPT6-GFP were labelled with MitoTracker Red CMXRos, fixed for widefield immunofluorescence microscopy, and labelled with antibodies to Drp1. Arrows highlight one example of Drp1 and SEPT6 association with location of mitochondrial fission. The scale bar represents 1 μm.


E HeLa were transfected with Mito - BFP ActA - SEPT7 - mCherry , fixed labelled with antibody to Drp1 for widefield immunofluorescence microscopy . scale bar represents 5 μm . Inset highlight Drp1 SEPT7 associated with sites of mitochondrial fission . Pearson ' s correlation coefficient from 3 independent experiments ( mean ± ) shows that mitochondria in ActA - SEPT7 transfected recruit significantly more Drp1 than control ( Mito - BFP transfected alone ) . Student ' s t - , * = p < 0 . 05 .

O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O

E HeLa cells were transfected with Mito-BFP and ActA-SEPT7-mCherry, fixed and labelled with antibody to Drp1 for widefield immunofluorescence microscopy. The scale bar represents 5 μm. Inset images highlight Drp1 and SEPT7 associated with sites of mitochondrial fission. Pearson's correlation coefficient from 3 independent experiments (mean ± SEM) shows that mitochondria in ActA-SEPT7 transfected cells recruit significantly more Drp1 than control cells (Mito-BFP transfected alone). Student's t-test, * = p<0.05.


A HeLa were treated with control ( CTRL ) , SEPT7 , or Drp1 siRNA for 72 h . Whole lysate of siRNA - treated were immunoblotted for GAPDH , SEPT7 , or Drp1 to show efficiency of siRNA depletion . GAPDH was used as loading control . siRNA - treated were labelled with MitoTracker Red CMXRos , fixed for confocal microscopy . scale bar represents 5 μm .

O B-CELL_LINE O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

A HeLa cells were treated with control (CTRL), SEPT7, or Drp1 siRNA for 72 h. Whole cell lysate of siRNA-treated cells were immunoblotted for GAPDH, SEPT7, or Drp1 to show the efficiency of siRNA depletion. GAPDH was used as loading control. siRNA-treated cells were labelled with MitoTracker Red CMXRos, and fixed for confocal microscopy. The scale bar represents 5 μm.


B HeLa were treated with control ( CTRL ) , SEPT2 , SEPT7 , SEPT9 , Drp1 , or SEPT7 + Drp1 siRNA for 72 h , labelled with MitoTracker Red CMXRos , fixed for quantitative confocal microscopy . Graph represents distribution in ( μm ; whiskers from min to max ) of mitochondria in siRNA - treated from 3 independent experiments ( of at least 100 measurements per biological replicate ) . Student ' s t - , * * * = p < 0 . 001 .

O B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O 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 HeLa cells were treated with control (CTRL), SEPT2, SEPT7, SEPT9, Drp1, or SEPT7+ Drp1 siRNA for 72 h, labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Graph represents the distribution in length (μm; whiskers from min to max) of mitochondria in siRNA-treated cells from 3 independent experiments (analysis of at least 100 measurements per biological replicate). Student's t-test, *** = p<0.001.


C HeLa stably expressing SEPT6 - GFP were harvested , then tested with Co - IP . HeLa expressing GFP were used in parallel as control . GFP - trap magnetic agarose beads were used to isolate SEPT6 - GFP from , extracts were immunoblotted for Drp1 , SEPT2 , SEPT7 , or GFP . lysates were immunoblotted for GAPDH as control for .

O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O

C HeLa cells stably expressing SEPT6-GFP were harvested, and then tested with Co-IP. HeLa cells expressing GFP were used in parallel as control. GFP-trap magnetic agarose beads were used to isolate SEPT6-GFP from cells, and extracts were immunoblotted for Drp1, SEPT2, SEPT7, or GFP. The lysates were immunoblotted for GAPDH as control for cellular protein levels.


D HeLa were treated with control ( CTRL ) or SEPT7 siRNA for 72 h , whole lysates were immunoblotted for SEPT7 , Drp1 , phosphorylated Drp1 ( P - Drp1 ) , or Mfn1 . GAPDH was used as loading control . Graph represents mean % ± of relative amount of Drp1 , P - Drp1 , Mfn1 , or SEPT7 quantified by densitometry from 4 independent experiments . Student ' s t - , ns = non - significant ; * * = p < 0 . 01 , * * * = p < 0 . 001 .

O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O 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 HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, and whole cell lysates were immunoblotted for SEPT7, Drp1, phosphorylated Drp1 (P-Drp1), or Mfn1. GAPDH was used as loading control. Graph represents the mean % ± SEM of the relative amount of Drp1, P-Drp1, Mfn1, or SEPT7 proteins quantified by densitometry from 4 independent experiments. Student's t-test, ns = non-significant; ** = p<0.01, *** = p<0.001.


E HeLa were treated with control ( CTRL ) or SEPT7 siRNA for 72 h , labelled with MitoTracker Red CMXRos , then fixed labelled for endogenous P - Drp1 for quantitative confocal microscopy . Representative shown here . scale bar represents 1 μm . recruitment of P - Drp1 to mitochondria was significantly decreased as compared to control as measured by Pearson ' s correlation coefficient from at least 5 independent experiments ( mean ± , of at least 250 per biological replicate ) . Student ' s t - , * = p < 0 . 05 .

O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, labelled with MitoTracker Red CMXRos, then fixed and labelled for endogenous P-Drp1 for quantitative confocal microscopy. Representative images shown here. The scale bar represents 1 μm. The recruitment of P-Drp1 to mitochondria was significantly decreased as compared to control cells as measured by Pearson's correlation coefficient from at least 5 independent experiments (mean ± SEM, analysis of at least 250 cells per biological replicate). Student's t-test, * = p<0.05.


F HeLa were treated with control ( CTRL ) or SEPT7 siRNA for 72 h , labelled with MitoTracker Red CMXRos , then fixed labelled for endogenous Mfn1 for quantitative confocal microscopy . Representative shown here . scale bar represents 1 μm . recruitment of Mfn1 to mitochondria was significantly increased as compared to control as measured by Pearson ' s correlation coefficient from at least 5 independent experiments ( mean ± , of at least 250 per biological replicate ) . Student ' s t - , * = p < 0 . 05 .

O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, labelled with MitoTracker Red CMXRos, then fixed and labelled for endogenous Mfn1 for quantitative confocal microscopy. Representative images shown here. The scale bar represents 1 μm. The recruitment of Mfn1 to mitochondria was significantly increased as compared to control cells as measured by Pearson's correlation coefficient from at least 5 independent experiments (mean ± SEM, analysis of at least 250 cells per biological replicate). Student's t-test, * = p<0.05.


A HeLa stably expressing SEPT6 - GFP were treated with Drp1 siRNA for 72 h , transfected with mito - BFP 24 h , then infected with S . flexneri - mCherry for 2 h for live confocal microscopy . Representative image shows interplay between two Shigella - septin cages fused mitochondria . scale bar represents 1 μm . See also Movie EV3 .

O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-ORGANISM O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O

A HeLa cells stably expressing SEPT6-GFP were treated with Drp1 siRNA for 72 h, transfected with mito-BFP 24 h, and then infected with S. flexneri-mCherry for 2 h for live confocal microscopy. Representative image shows interplay between two Shigella-septin cages and fused mitochondria. The scale bar represents 1 μm. See also Movie EV3.


B HeLa were treated with Drp1 siRNA for 72 h , then infected with x - Shigella mCherry for 4 h 40 min for quantitative confocal microscopy . IPTG was added 30 min prior to fixation , then samples were labelled with antibody for SEPT7 . Graph represents mean % ± of Shigella responding to IPTG outside ( - ) or inside ( + ) SEPT7 cages from 3 independent experiments . Student ' s t - , * * * = p < 0 . 001 .

O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

B HeLa cells were treated with Drp1 siRNA for 72 h, and then infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for SEPT7. Graph represents mean % ± SEM of Shigella responding to IPTG outside (-) or inside (+) SEPT7 cages from 3 independent experiments. Student's t-test, *** = p<0.001.


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

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


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

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


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

B-TISSUE B-GENEPROD O O O O O O O O O O O O O O O O O

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


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

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


CSF PGRN is increased in males compare to females.


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

B-TISSUE B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O

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


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

B-TISSUE B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-TISSUE B-GENEPROD O O O O O O O O O O O O O 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-TISSUE B-GENEPROD O O O O O O O O O O O O O O O O O O B-TISSUE B-DISEASE I-DISEASE I-DISEASE O O O O B-TISSUE O O B-GENEPROD O O O O O O B-TISSUE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O

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


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

O O O O B-TISSUE B-GENEPROD O O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O B-TISSUE B-GENEPROD O O O O O O O O O O O B-DISEASE O O O O O O O O O B-DISEASE O O O O O O O O B-DISEASE O O O O O O O O O O O O B-TISSUE B-GENEPROD O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-DISEASE O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O B-GENEPROD O O O O

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


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

O O O O O B-TISSUE B-GENEPROD O O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O O O B-TISSUE B-GENEPROD O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-TISSUE B-GENEPROD O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY

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


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

O O O O O B-TISSUE B-GENEPROD O O B-TISSUE O O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O

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


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

O O O O O B-TISSUE B-GENEPROD O B-TISSUE O O O B-DISEASE B-TISSUE O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O 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-TISSUE B-GENEPROD O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O B-TISSUE O O B-DISEASE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE O O

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


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

O O O O O B-TISSUE B-GENEPROD O B-TISSUE O O O B-DISEASE B-TISSUE O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O B-TISSUE B-GENEPROD O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O B-DISEASE I-DISEASE I-DISEASE O O

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


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

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

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

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

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O 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-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD 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 of GST - STIM1 ( domain ) pulled down by Cap - Gly - CC1 - p150Glued - V5 - coated beads , in presence of FLAG - EB1 C - terminal WT or ΔY . Negative control was performed using equal amount of empty GST , together with Cap - Gly - CC1 - p150Glued .

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

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

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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O

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


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

O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O B-SUBCELLULAR O O

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


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

O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O

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


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

O O O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O 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-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O 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-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O

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


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

O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O 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-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O

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


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

O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O 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-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O

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


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

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O

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


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

O O O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O 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-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O 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-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O

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


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

O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O 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-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O

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


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

O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O 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-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O

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


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

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O 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-GENEPROD B-EXP_ASSAY O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O

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


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

O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O B-CELL_TYPE O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O 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-CELL_TYPE O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O 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-CELL_TYPE O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-CELL_TYPE B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O

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


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

O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O 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 ***.