PDI silencing had no effect on TF-FVIIa coagulant and cell-signaling functions. (A) Immunoblot analysis of MDA-MB-231 cell extracts that were stably transfected with PDI shRNA or a nonspecific shRNA. Blots were probed with anti-PDI or anti-β-actin antibodies. (B) FACS analysis of MDA-MB-231 cells transfected with nonspecific shRNA and PDI-specific shRNA for TF (left) and PDI (right) expression, respectively. For PDI staining, cells were permeabilized with 0.1% Triton X-100, whereas nonpermeabilized cells were used to stain cell surface TF expression. (C) MDA-MB-231 cells stably transfected with PDI-specific shRNA or nonspecific shRNA control vector were stimulated with FVIIa (10 nM) for 60 minutes and TF-FVIIa-induced IL-8 expression was determined by Northern blot analysis. NS shRNA indicates nonspecific shRNA; and GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) Wild-type (WT) MDA 231 cells or MDA 231 cells stably transfected with nonspecific or PDI-specific shRNA were treated with control vehicle or HgCl2, and the cell surface TF-FVIIa coagulant activity was determined as described in Figure 1. Data are expressed as mean plus or minus SEM (n = 3).
