Figure 4(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 ***. (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 ***. (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 ***. (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 ***.