Influence of PDI inhibitors on VWF processing. HUVECs (A) were incubated with 80 nM PMA for 25 min at 37°C/5% CO₂ to induce VWF release from WPBs (B). Cells were washed 5-fold with phosphate-buffered saline, and either medium (C,F) or medium containing 2 µM 16F16 (D,G) or 100 nM PAO (E,H) was added. After 4 h (C-E) and 8 h (F-H), VWF was detected by immunofluorescence. Images that combine all planes of the cells were imaged, using the quick-full-focus function of the BZ9000 fluorescence microscope (Keyence) equipped with a CFI Plan Apo λ 60× H oil objective (Nikon). Representative images of 1 of 5 independent experiments are shown. Scale bar represents 10 µm. The western blot shows VWF dimers in HUVEC lysates before PMA treatment (U) and after 4 and 8 hours of recovery without PDI inhibitors (C) or in the presence of 16F16 (F) or PAO (P).