Fig 2: KIF13A knockdown impairs endocytic recycling of VE-cadherin in ECs. (A) Confocal images of VE-cadherin and Rab5 double immunostained HUVECs showing increased VE-cadherin/Rab5 double positive early endosomes in KIF13A knockdown HUVECs. Scale bar: 20 μm (upper panel) and 8 μm (lower panel). (B) Quantification showing increased size of Rab5-positive early endosomes in KIF13A knockdown HUVECs (266 endosomes from control HUVECs, 289 endosomes from siHgs-1-treated HUVECs, and 275 endosomes from siHgs-2-treated HUVECs were analysed). (C) Western blot analyses of VE-cadherin and KIF13A expression in control and KIF13A knockdown HUVECs. (D–G) VE-cadherin internalization assay (D and E) and the corresponding quantifications (F and G) showing increased colocalization of VE-cadherin with Rab5-positive early endosomes and decreased colocalization of VE-cadherin with Rab11-positive recycling endosomes in KIF13A knockdown HUVECs (n = 5). Scale bars: 15 µm. (H) Immunostaining for VE-cadherin, Rab11, and F-actin showing impaired recruitment of VE-cadherin-containing recycling endosomes to the AMIS of KIF13A knockdown HUVECs. (Right) Quantification of VE-cadherin/Rab11-positive endosomes enriched at the AMIS of control and KIF13A knockdown HUVECs (n = 5). Scale bar: 15 µm. Values are presented as means ± standard deviation. **P < 0.01 by Mann–Whitney test.

Fig 3: Restoration of endosomal recycling rescues VE-cadherin polarity in Hgs knockdown ECs. (A–D) HUVECs were treated with control or Hgs-siRNA for 24 h and then transfected with control or KIF13A plasmids for 24–48 h before fixation in 4% PFA. (A) Confocal images showing that KIF13A overexpression reduced the intracellular accumulation of VE-cadherin and VE-cadherin-containing Rab5-positive early endosomes (upper panel) and normalized the size of Rab5-positive early endosomes (lower panel) in Hgs knockdown HUVECs. Scale bar: 10 µm. (B) Quantification of the size of Rab5-positive endosomes (333 endosomes from control HUVECs, 291 endosomes from siHgs-1-treated HUVECs, and 263 endosomes from siHgs-2-treated HUVECs transfected with KIF13A plasmids were analysed). (C) Quantification of junctionally distributed VE-cadherin (n = 5). (D) KIF13A overexpression restored the recruitment of VE-cadherin to the AMIS in Hgs knockdown HUVECs (n = 5). Scale bar: 10 µm. Values are presented as means ± standard deviation. **P < 0.01 by Mann–Whitney test and one-way analysis of variance.