Androgen regulates the expression and distribution of TFPI and ADTRP, and TFPI activity. (A) mRNA expression of TFPI assayed by qRT-PCR in control ECs (open bars) and cells incubated with 30nM DHT for 24 hours (gray bars). The effect of DHT was studied in Native cells, and in ADTRP-shRNA–, ADTRP-FLAG–, and Cav-1-shRNA–expressing EAhy926 cells. Values are presented as fold-change from native control ECs after correction for 18S rRNA as internal control. Experiments were repeated twice and run in triplicate. (B) Immunostaining for ADTRP (FITC, green) and TFPI (Cy₃, red) on the cell surface, and Cav-1 (Cy₅, blue) after permeabilization on HUVECs treated with DHT. Overlap (white) designates the colocalization channel resulting from the image analysis. (C) Scatter-plot and statistics (1-way ANOVA) of MFI of cell surface TFPI and ADTRP measured on cells processed as in panel (B). Control cells were treated similarly to the Androgen-group but without DHT. Data are mean ± SEM. ***P < .001 for both sets of data in the Androgen-group compared with the Control-group. (D) Effect of DHT on the inhibitory capability of cell surface TFPI against TF-FVIIa–dependent FXa generation measured in TF-EC in the presence/absence of inhibitory anti-TFPI IgG. Values are normalized to 10⁶ cells. Data are mean ± SD of triplicates. The groups of cells and DHT treatment were as described in panel A. (E) Immunofluorescence and confocal microscopy show the effect of DHT on the distribution and colocalization of cell surface TFPI (red) with YFP-TF (green) and Cav-1 (blue; after permeabilization) on TF-EC. Triple overlap (white) channel resulted from the image analysis. (F) Immunofluorescence for cell surface TFPI (Cy₃, red) and ADTRP (FITC, green), and Cav-1 (Cy₅, blue; after permeabilization) in EC stable expressing Cav-1–shRNA, either native (Control) or incubated with DHT (Androgen). Overlap (white) designates the triple colocalization channel. White arrows: residual Cav-1 expression. (A, D) Statistical analysis was performed by 1-way ANOVA. *,#,♦: P < .05, **,##,♦♦: P < .01, ***,###,♦♦♦: P < .001, N.S. and ns, P ≥ .05. Asterisks (*) and ns: difference versus Native within the Control-group. Diamonds (♦): difference versus Native within the Androgen-group. Pound signs (#) and N.S: difference between Androgen-treated versus Control cells within each experimental group. Bars: 10 μm.