Fig. 6

a Maximum intensity confocal z-projections showing the distribution of Tfn-488 (black dots) upon 30 min of incubation in COS-1 cells expressing mCherry-tagged ARF3^WT and all identified mutants. Red circle indicates Tfn signal at the perinuclear region (PN). Outlines (black in a and yellow in c) depict the boundaries of representative transfected cells. The black and white images are rendered by inverting the original LUT in Fiji and nuclei are pseudo-colored (purple) in the images. The images are representative of two independent experiments. b–b’ Incidence of cells showing “clustered”, “semi-clustered” or “dispersed” Tfn staining (b) and the ratio of the cells (%) showing “clustered” Tfn phenotype normalized by not-transfected cells (NT) with the same phenotype (b’, internal control). No. of cells = 42 (WT); 22 (K127E, ****p < 0.0001); 33 (L12V/D67V, ***p = 0.0002); 31 (P47S); 26 (D93N); and 25 (T32N). Data are expressed as mean ± SEM (b, b’) of three (WT) and two (all the other mutants) independent experiments. c Maximum intensity confocal z-projections showing COS-1 cells expressing ARF3^WT and all identified mutants, incubated with Tfn-488 for 30 min followed by immunostaining against Rab5 (marker of early endosomes). For all the panels single channels (ARF3mCherry: gray, Tfn-488: magenta, Rab5: green), the merge showing the co-localization between Tfn and Rab5 are shown. The insets in the white square show a zoom on the PN co-localization signal. Nuclei are stained with DAPI. The images are representative of cells from a single experiment. d Colocalization analysis showing the spatial co-occurrence of Tfn and Rab5+ signals at the PN region in the z-stacks analyzed, no. of cells = 13 (WT; K127E ****p < 0.0001; T32N), 16 (L12V/D67V, **p = 0.0016; D93N), 21 (P47S). The fraction (%) of Tfn⁺ signal co-localized with Rab5⁺ vesicles at the PN (thresholded Mander’s coefficient M1) is reported as box-and-whisker with median (middle line), 25th–75th percentiles (box), and min–max values (whiskers) of a single experiment. All the data points and the mean (“+”) are also shown. Two-sided Chi-square’s test in a 2 × 2 contingency table (semi-clustered and dispersed vs. clustered, b), One-way ANOVA followed by Dunnett’s multiple comparison post hoc test (b‘, d) are used to assess the statistical significance. Source data are provided as a Source Data file.