Distinct retrograde microtubule motor sets drive early and late endosome transport (2020)
Figure 5

Figure 5. KIFC1 acts as an additional early endosome-dedicated MT retrograde motor.

(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 *.

(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 ***.

(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).