Figure 7(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. (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. (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 ***. (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 *.