C1GALT1 influences VWF OLG determinants and WPB morphology. (A) Inhibitory effect of ITZ on O-glycan biosynthetic processing. (B) C1GALT1 immunoblot in HUVEC cell lysates after ITZ treatment (α-tubulin loading control; representative blot, n = 3). VWF immunoblot in HUVEC cell lysates and conditioned media after ITZ treatment (GAPDH loading control; representative blot, n = 3). (C) Unstimulated VWF:Ag secretion levels from HUVECs incubated with or without ITZ (2 μM for 48-72 hours; n = 12, from 4 independent experiments; Mann-Whitney test; P < .0001). (D) Immunofluorescent images of HUVECs treated with ITZ (2 μM for 48-72 hours) compared with untreated CTRLs (VWF in gray; DAPI in blue; representative images of 3 independent experiments). Scale bars are set at 10 μm for overview images and at 5 μm for the zoomed regions. (E) Automated assessment of WPB length (data are shown as normalized to CTRL) in CTRL and ITZ-treated cells (n = 10 images, from 3 independent experiments; t test; P = .0026).