Fig. 2: Rab35 mutant localization and rescue in endothelial sprouts.
From: Rab35 governs apicobasal polarity through regulation of actin dynamics during sprouting angiogenesis
a Quantification of GFP-Rab35 wild-type (WT), constitutively-active (CA), and dominant-negative (DN) localization in endothelial sprouts. Apical plasma membrane (PM, uniformly localized to apical membrane), basal PM (Rab35 uniformly located at the basal membrane), cytosolic (localized in the cytoplasm), equal PM (Rab35 equally distributed between the apical and basal membranes). n = number of cells. b GFP-Rab35 WT (top panels), CA (middle panels), and DN (bottom panels) localization in endothelial sprouts. Co-staining with podocalyxin (Podxl) and actin. Arrowheads in top panels denote Rab35 apical localization and puncta. Arrowheads in bottom panels denote abnormal accumulations of actin. c Rescue experiment using scrambled (Scram) or Rab35 siRNA (si)-mediated knockdown (KD) with overexpression of indicated constructs. Percentages represent quantification of lumen formation in described conditions. n = number of sprouts. d Representative images of Scram and Rab35 KD sprouts expressing either GFP (control), or GFP-Rab35-WT/CA/DN. Arrowheads denote actin accumulations. White dotted lines mark sprout exterior. L denotes lumen in all images. Insets are areas of higher magnification. All experiments were done using human umbilical vein endothelial cells in triplicate.
