BLOC-2–depleted immature WPBs lack endosomal input. (A) IF analyses of BLOC-2–depleted and control HUVECs dual-labeled with anti-VWF and CD63 antibodies, counterstained with DAPI. The insets highlight colabeling of mature WPBs with CD63 in control cells in contrast to perinuclear localization of the 2 in BLOC-2–depleted cells. Scale bars represent 10 μm. The bar graphs show Mander’s overlap coefficient. Fifteen cells each from 2 experiments were analyzed. The right panel shows super-resolution analysis using radial fluctuation (SRRF) of anti-VWF and anti-CD63 dual-labeled HUVECs confirming separation of VWF and CD63 signals in the perinuclear immature WPBs in BLOC-2–depleted cells compared with colabeled peripheral mature WPBs in control cells. Scale bars represent 2.5 μm. (B) IF analysis of HUVECs transduced with GFP-CD63 expressing lentiviral particles 48 hours after transfection with either control or HPS6 siRNA. Arrows show GFP-labeled mature rod-shaped WPBs in control cells missing in BLOC-2–depleted cells and the inset highlights a mature WPB. GFP labels endosomes in both cell types. Scale bars represent 10 μm. Bar graph compares total number of GFP-labeled mature WPBs per cell in control compared with BLOC-2–depleted cells. Fifteen cells each from 2 experiments were analyzed (***P < .001). (C) IF analyses of BLOC-2–depleted and control HUVECs dual-labeled with anti-P-selectin and CD63 antibodies, counterstained with DAPI. The insets highlight colabeling of VWF and P-selectin in both control and BLOC-2–depleted cells. Scale bars represent 10 μm. The bar graphs show Mander’s overlap coefficient. Fifteen cells each from 2 experiments were analyzed.