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Fig. 3 Rab10 and Rab25 work in combination to traffic LH3 to CIVC vesicles. (a) Representative images of ECs expressing RFP-Rab10 wild-type (WT) and stained for collagen IV (Col IV) (green). Green arrowheads indicate collagen IV-containing (CIVC) vesicles only and yellow arrowheads indicate Rab10 puncta only. (b) Representative images of scramble, Rab10, Rab25, or LH3 siRNA-treated ECs and stained for Col IV (red), actin (green), and DNA (blue). Arrowheads denote extracellular Col IV secretion. (c) Graph of extracellular Col IV ratio of scramble, Rab10, Rab25, or LH3 siRNA-treated ECs cultured in serum-starve (SS) media. (d) Representative images of fibrin-bead sprouts between indicated siRNA treatment groups. Sprouts were stained for actin (grey) to delineate sprout morphology. (e–g) Graphs of sprouting parameters for scramble, Rab10, Rab25, or LH3 siRNA-treated sprouts. (h) Representative images of ECs cultured in VEGF-containing or SS media and stained for Col IV (green), LH3 (red), and DNA (blue). Green arrowheads indicate CIVC vesicles only and yellow arrowheads indicate co-localized puncta. (i) Graph of percent CIVC vesicles co-localized with LH3 in ECs cultured in VEGF-containing or SS media. (j) Representative images of scramble, Rab10, Rab25, or both Rab10/25 siRNA-treated ECs and stained for Col IV (green), LH3 (red), actin (grey), and DNA (blue). Yellow arrowheads indicate co-localized puncta only, green arrowheads indicate Col IV only puncta, and red arrowheads indicate LH3 puncta only. (k) Graph of percent CIVC vesicles co-localized with LH3 in scramble, Rab10, Rab25, or LH3 siRNA-treated conditions cultured in SS media or SS media with CLQ (10 µM). (l) Representative images of ECs expressing RFP-Rab10 WT, RFP-Rab10 constitutively active (CA), or RFP-Rab10 dominant negative (DN), stained for Col IV (green) and LH3 (blue). Yellow arrowheads indicate co-localized puncta only, green arrowheads indicate Col IV puncta, and red arrowheads indicate LH3 puncta. (m) Graph of percent CIVC vesicles co-localized with LH3 in ECs expressing RFP-Rab10 WT/CA/DN. (n) Graph of percent Rab10 wild-type (WT) puncta co-localized with Rab25 in either VEGF-containing or SS media. (o) Graph of percent Rab10 puncta co-localized with Rab25 in ECs transfected with indicated constructs. (p) Schematic diagram showing how Rab10 and Rab25 function to coordinate delivery of LH3 to CIVC vesicles for proper secretion of Col IV. For all graphs n number of cells unless otherwise indicated. For all experiments, data represented as mean ± 95% confidence intervals. Black bars indicate comparison groups with indicated p-values. All p-values are from two-tailed Student’s t-test from at least three experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; ns not significant