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

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

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


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

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

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

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


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

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

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

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


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

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

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

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


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

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

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


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

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

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


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

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

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

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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD 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 B-GENEPROD O O O B-GENEPROD O O O O O O 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 O O O O B-GENEPROD O B-GENEPROD O 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 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 cell size , quantified by image ( as in a ) segmentation ( see Material and Method , Confocal microscopy and early / late quantification ) of EEA - 1 + endosomes . Results are the average ± SEM of two independent experiments for a total 830 early endosomes in 13 shCTL / GFP cells ( 64 ± 6 endosomes per cell ) , 719 early endosomes in 10 shSTIM1 / GFP cells ( 60 ± 6 endosomes per cell ) , 820 early endosomes in 12 shSTIM1 / GFP - STIM1 WT cells ( 68 ± 6 endosomes per cell ) and 400 early endosomes in 12 shSTIM1 / GFP - STIM1 NN cells ( 33 ± 7 endosomes per cell ) and analyzed by a parametric two - tailed analysis of variance ( ANOVA ) with Bonferroni post hoc analysis . ANOVA p ≤ 0 , 001 ; Bonferroni for shCTL / GFP and shSTIM1 / GFP p ≤ 0 , 01 * * , for shCTL / GFP and shSTIM1 / GFP - STIM1 WT p > 0 . 05 not significant ( ns ) , for shSTIM1 / GFP - STIM WT and shSTIM1 / GFP - STIM1 NN p ≤ 0 , 001 * * * and for shSTIM1 / GFP and shSTIM1 / GFP - STIM1 NN p ≤ 0 , 001 * * * .

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

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

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


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

O O O O O O O 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 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 O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE 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

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


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

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

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


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

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

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

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

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


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

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

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


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

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

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

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


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

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


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

O 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 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 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 O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE 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 B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O O O

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


( a ) Brains of the ' rescued ' mice ( Becn1 − / − ; Becn1EGFP / + ) and of control Becn1 + / + littermates were lysed and subjected to immune isolation using an anti - GFP antibody . Proteins bound to anti - GFP antibody were isolated , detected and identified by SDS - PAGE , Coomassie brilliant blue staining and mass spectrometry . Seven bands pulled down by anti - GFP antibody specifically from ' rescued ' mice lysate are numbered ( this is a copy of image from our previous report with modification ) .

O O O B-TISSUE O O O O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD 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 B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

(a) Brains of the 'rescued' mice (Becn1−/−; Becn1EGFP/+) and of control Becn1+/+ littermates were lysed and subjected to immune isolation using an anti-GFP antibody. Proteins bound to anti-GFP antibody were isolated, detected and identified by SDS-PAGE, Coomassie brilliant blue staining and mass spectrometry. Seven bands pulled down by anti-GFP antibody specifically from 'rescued' mice lysate are numbered (this is a copy of image from our previous report with modification).


( b ) Mouse brain lysates were subjected to co - immunoprecipitation ( co - IP ) followed by WB analysis using the indicated antibodies .

O O O B-ORGANISM B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

(b) Mouse brain lysates were subjected to co-immunoprecipitation (co-IP) followed by WB analysis using the indicated antibodies.


( c ) Mouse brain lysates were subjected to co - IP by NRBF2 antibody and followed by WB analysis using the indicated antibodies .

O O O B-ORGANISM B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O

(c) Mouse brain lysates were subjected to co-IP by NRBF2 antibody and followed by WB analysis using the indicated antibodies.


( d ) Gel filtration analysis . The supernatant fraction of mouse brain lysates was subjected to a Superdex 200 HR 10 / 30 column and each fraction was immunoblotted with the indicated antibodies . Relative amounts of each fraction determined by Odyssey software are plotted .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM B-TISSUE 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

(d) Gel filtration analysis. The supernatant fraction of mouse brain lysates was subjected to a Superdex 200 HR 10/30 column and each fraction was immunoblotted with the indicated antibodies. Relative amounts of each fraction determined by Odyssey software are plotted.


( e ) The deficiency of Atg14L impairs the protein interaction between Beclin 1 and NRBF2 . NIH3T3 cells were transfected with Atg14L or NRBF2 RNAi for 72 h , respectively , and then were lysed and subjected to IP using Beclin 1 antibody . The immunoprecipitants were immunoblotted using the indicated antibodies .

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

(e) The deficiency of Atg14L impairs the protein interaction between Beclin 1 and NRBF2. NIH3T3 cells were transfected with Atg14L or NRBF2 RNAi for 72 h, respectively, and then were lysed and subjected to IP using Beclin 1 antibody. The immunoprecipitants were immunoblotted using the indicated antibodies.


( f ) Direct binding between Atg14L and NRBF2 . Flag - tagged Atg14L purified from HEK293T cells overexpressing Flag - Atg14L was incubated with GST or NRBF2 - GST purified from Escherichia coli . The GST pull - down assay was performed to examine the direct binding between Flag - Atg14L and NRBF2 - GST .

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

(f) Direct binding between Atg14L and NRBF2. Flag-tagged Atg14L purified from HEK293T cells overexpressing Flag-Atg14L was incubated with GST or NRBF2-GST purified from Escherichia coli. The GST pull-down assay was performed to examine the direct binding between Flag-Atg14L and NRBF2-GST.


( b ) NRBF2 KO MEFs were transfected with CFP , NRBF2 - CFP , dMIT - CFP or dCCD - CFP for 48 h . Atg14L - linked Vps34 was pulled down by an anti - Atg14L antibody and subjected to KA . IP products and inputs were immunoblotted using the antibodies indicated . ( c ) Quantification of IPed Vps34 kinase activity ( PI ( 3 ) P ) , Vps34 and Vps15 protein versus IPed Atg14L protein . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , multiple t - test are followed by Bonferroni correction , n = 4 ( otherwise indicated , n means number of independent experiments ; NS , not significant ) ) .

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

(b) NRBF2 KO MEFs were transfected with CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP for 48 h. Atg14L-linked Vps34 was pulled down by an anti-Atg14L antibody and subjected to KA. IP products and inputs were immunoblotted using the antibodies indicated. (c) Quantification of IPed Vps34 kinase activity (PI(3)P), Vps34 and Vps15 protein versus IPed Atg14L protein. (Data are shown as mean±s.e.m., P values are indicated on the figures, multiple t-test are followed by Bonferroni correction, n=4 (otherwise indicated, n means number of independent experiments; NS, not significant)).


( d ) Overexpression of NRBF2 enhances Atg14L - linked Vps34kinase activity . HEK293T cells were co - transfected with Myc - Vps34 - Vps15 - His and Flag - Atg14L in the presence of CFP or NRBF2 - CFP . Atg14L - linked Vps34 is pulled down by an anti - Flag antibody and subjected to KA ( upper panel ) . IP products and inputs were immunoblotted using the antibodies indicated ( upper panel ) . ( e ) Quantification of Vps34kinase activity ( lower panel ) shows the significant difference between two groups . ( Data are shown as mean ± s . e . m . , P value is indicated on the figures , one sample t - test versus hypothetical mean 1 , n = 5 ) .

O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD 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 O O O 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 O O O O O O O O O O O O O O O O O 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) Overexpression of NRBF2 enhances Atg14L-linked Vps34kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and Flag-Atg14L in the presence of CFP or NRBF2-CFP. Atg14L-linked Vps34 is pulled down by an anti-Flag antibody and subjected to KA (upper panel). IP products and inputs were immunoblotted using the antibodies indicated (upper panel). (e) Quantification of Vps34kinase activity (lower panel) shows the significant difference between two groups. (Data are shown as mean±s.e.m., P value is indicated on the figures, one sample t-test versus hypothetical mean 1, n=5).


( a ) Starvation - induced long - lived protein degradation is impaired after NRBF2 knockdown . Compared with control ( con ) siRNA , NRBF2 siRNA decreases long - lived protein degradation ( proteolysis rate ) in NIH3T3 cells under starvation ( ST ) condition . This difference is diminished when the starved cells are co - treated with wortmannin ( WM , 100 nM ) . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , one - way analysis of variance with Dunnett as post hoc test , n = 4 ; NS , not significant ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Starvation-induced long-lived protein degradation is impaired after NRBF2 knockdown. Compared with control (con) siRNA, NRBF2 siRNA decreases long-lived protein degradation (proteolysis rate) in NIH3T3 cells under starvation (ST) condition. This difference is diminished when the starved cells are co-treated with wortmannin (WM, 100 nM). (Data are shown as mean±s.e.m., P values are indicated on the figures, one-way analysis of variance with Dunnett as post hoc test, n=4; NS, not significant).


( b ) Rapamycin ( Rap ) - induced autophagy is impaired in the NRBF2 KO cells . The MEFs were treated with ( dimethyl sulfoxide ) DMSO or 50 μg ml − 1 of Rap for 4 h then the cell lysates were immunoprobed with anti - LC3 and anti - p62 antibodies . ( c ) LC3 - II and p62 levels were quantified and normalized with β - actin . Quantification results show that Rap - induced LC3 lipidation ( upper ) and p62 degradation ( lower ) were markedly impaired in the NRBF2 KO MEFs . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , one sample t - test ( WT con versus KO con ) or unpaired t - test ( WT Rap versus KO Rap ) ) . p62 blots quantification , n = 6 ; LC3 blots quantification , n = 3 ) .

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

(b) Rapamycin (Rap)-induced autophagy is impaired in the NRBF2 KO cells. The MEFs were treated with (dimethyl sulfoxide) DMSO or 50 μg ml−1 of Rap for 4 h then the cell lysates were immunoprobed with anti-LC3 and anti-p62 antibodies. (c) LC3-II and p62 levels were quantified and normalized with β-actin. Quantification results show that Rap-induced LC3 lipidation (upper) and p62 degradation (lower) were markedly impaired in the NRBF2 KO MEFs. (Data are shown as mean±s.e.m., P values are indicated on the figures, one sample t-test (WT con versus KO con) or unpaired t-test (WT Rap versus KO Rap)). p62 blots quantification, n=6; LC3 blots quantification, n=3).


( d , e ) The MEFs were subjected to Rap ( 50 μg ml − 1 ) or starvation ( HBSS ) treatment then stained with anti - LC3 ( d ) or anti - WIPI2 ( e ) antibody . ( f ) The LC3 and WIPI2 puncta per cell were counted and quantified . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , independent samples Kruskal - Wallis test followed by pairwise comparisons , n = 30 randomly selected cells , a representative result from three independent repeats ) .

O O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE 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 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 O O O O O O O O O O O O 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,e) The MEFs were subjected to Rap (50 μg ml−1) or starvation (HBSS) treatment then stained with anti-LC3 (d) or anti-WIPI2 (e) antibody. (f) The LC3 and WIPI2 puncta per cell were counted and quantified. (Data are shown as mean±s.e.m., P values are indicated on the figures, independent samples Kruskal-Wallis test followed by pairwise comparisons, n=30 randomly selected cells, a representative result from three independent repeats).


( g ) NRBF2 - CFP stable cells were starved in HBSS for 4 h and then subjected to WIPI2 staining . Under starved condition , NRBF2 - CFP forms puncta structures that co - localized with WIPI2 . This is a representative image from three independent experiments .

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

(g) NRBF2-CFP stable cells were starved in HBSS for 4 h and then subjected to WIPI2 staining. Under starved condition, NRBF2-CFP forms puncta structures that co-localized with WIPI2. This is a representative image from three independent experiments.


( h ) The NRBF2 KO MEFs were transfected with CFP , NRBF2 - CFP , dMIT - CFP or dCCD - CFP for 24 h and then followed by co - immunostaining with GFP and WIPI2 antibodies . ( i ) The WIPI2 puncta number per cell in GFP - positive cells were counted and quantified . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , independent samples Kruskal - Wallis test followed by pairwise comparisons , n = 30 randomly selected cells , a representative result from two independent repeats ) . Scale bars , 20 μm , or length indicated on the figure . CM , culture medium .

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

(h) The NRBF2 KO MEFs were transfected with CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP for 24 h and then followed by co-immunostaining with GFP and WIPI2 antibodies. (i) The WIPI2 puncta number per cell in GFP-positive cells were counted and quantified. (Data are shown as mean±s.e.m., P values are indicated on the figures, independent samples Kruskal-Wallis test followed by pairwise comparisons, n=30 randomly selected cells, a representative result from two independent repeats). Scale bars, 20 μm, or length indicated on the figure. CM, culture medium.


( a ) NRBF2 KO MEFs display impaired autophagosome acidification . WT and NRBF2 KO MEFs are transiently transfected with mCherry - GFP - LC3B . WT MEFs expressing mCherry - GFP - LC3B contain many red - only puncta along with yellow ( presence of both red and green ) puncta . The number of both red - only puncta and yellow puncta markedly increases after rapamycin ( Rap ; 50 μg ml − 1 , 4 h ) or starvation ( HBSS , 4 h ) treatment , suggesting the increased autophagolysosomes and nascent autophagosomes . In contrast , NRBF2 KO MEFs expressing mCherry - GFP - LC3B contain quite fewer red - only puncta and yellow puncta , even after Rap or starvation treatment . ( b ) The quantification results show that both the numbers of red - only puncta and percentage of red - only puncta ( verses total puncta ) in KO MEFs were markedly reduced . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , Mann - Whitney U - test , n = 20 randomly selected cells , a representative result from three independent repeats ) , indicating both the autophagolysome number and autophagosome acidification rate are reduced in KO cells . Scale bars , 20 μm or 10 μm ( amplified regions ) .

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

(a) NRBF2 KO MEFs display impaired autophagosome acidification. WT and NRBF2 KO MEFs are transiently transfected with mCherry-GFP-LC3B. WT MEFs expressing mCherry-GFP-LC3B contain many red-only puncta along with yellow (presence of both red and green) puncta. The number of both red-only puncta and yellow puncta markedly increases after rapamycin (Rap; 50 μg ml−1, 4 h) or starvation (HBSS, 4 h) treatment, suggesting the increased autophagolysosomes and nascent autophagosomes. In contrast, NRBF2 KO MEFs expressing mCherry-GFP-LC3B contain quite fewer red-only puncta and yellow puncta, even after Rap or starvation treatment. (b) The quantification results show that both the numbers of red-only puncta and percentage of red-only puncta (verses total puncta) in KO MEFs were markedly reduced. (Data are shown as mean±s.e.m., P values are indicated on the figures, Mann-Whitney U-test, n=20 randomly selected cells, a representative result from three independent repeats), indicating both the autophagolysome number and autophagosome acidification rate are reduced in KO cells. Scale bars, 20 μm or 10 μm (amplified regions).


( c ) Autophagolysome formation is impaired in NRBF2 KO MEFs . WT and NRBF2 KO MEFs were transiently transfected with RFP - LC3 then stained with LAMP2 antibody . The co - localization between RFP - LC3 and LAMP2 are illustrated by line profile . RFP - LC3 puncta recruit much stronger LAMP2 signal in WT MEFs than in NRBF2 KO MEFs ( as indicated by white arrows ) , suggesting the impairment of autophagolysosome formation in KO cells . Scale bars , 20 μm or 10 μm ( amplified regions ) . This is a representative image from three independent experiments .

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

(c) Autophagolysome formation is impaired in NRBF2 KO MEFs. WT and NRBF2 KO MEFs were transiently transfected with RFP-LC3 then stained with LAMP2 antibody. The co-localization between RFP-LC3 and LAMP2 are illustrated by line profile. RFP-LC3 puncta recruit much stronger LAMP2 signal in WT MEFs than in NRBF2 KO MEFs (as indicated by white arrows), suggesting the impairment of autophagolysosome formation in KO cells. Scale bars, 20 μm or 10 μm (amplified regions). This is a representative image from three independent experiments.


( d ) Overexpression of NRBF2 enhances UVRAG - linked Vps34 kinase activity . HEK293T cells were co - transfected with Myc - Vps34 - Vps15 - His and FLAG - UVRAG , in the presence of CFP or NRBF2 - CFP . UVRAG - linked Vps34 was pulled down by an anti - FLAG antibody and subjected to KA . IP products and inputs are immunoblotted using the antibodies indicated . ( e ) Quantification of Vps34 kinase activity normalized with immunoprecipitated Vps34 shows the significant difference between two groups . ( Data are shown as mean ± s . e . m . , P value is indicated on the figures , one sample t - test versus hypothetical mean 1 , n = 5 ) .

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

(d) Overexpression of NRBF2 enhances UVRAG-linked Vps34 kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and FLAG-UVRAG, in the presence of CFP or NRBF2-CFP. UVRAG-linked Vps34 was pulled down by an anti-FLAG antibody and subjected to KA. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of Vps34 kinase activity normalized with immunoprecipitated Vps34 shows the significant difference between two groups. (Data are shown as mean±s.e.m., P value is indicated on the figures, one sample t-test versus hypothetical mean 1, n=5).


( a ) The gross anatomical views of representative 4 % paraformaldehyde - fixed livers and quantification of liver index . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , unpaired Student ' s t - test , n = 3 mice each group ) . Scale bar , 1 cm .

O O O O O O O O O O O O O O B-TISSUE O O O B-TISSUE O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O

(a) The gross anatomical views of representative 4% paraformaldehyde-fixed livers and quantification of liver index. (Data are shown as mean±s.e.m., P values are indicated on the figures, unpaired Student's t-test, n=3 mice each group). Scale bar, 1 cm.