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Blood, Vol. 110, Issue 1, 161-170, July 1, 2007
Tissue factor trafficking in fibroblasts: involvement of protease-activated receptor–mediated cell signaling
Blood Mandal et al.
110: 161
Supplemental materials for: Mandal et al, Vol 110, Issue 1, 161-170
Files in this Data Supplement:
- Figure S1. Effect of brefeldin A on TF localization in the Golgi, and factor VIIa–induced increased tissue factor activity at the cell surface (PDF, 1.64 MB) -
(A) Fibroblasts (WI-38) were treated with control vehicle or brefeldin A (1 µM) for 1 hour. Cells were fixed, permeabilized, and immunostained with rabbit polyclonal anti–human TF and monoclonal anti–human golgin-97 antibodies, followed by Rhodamine Red–labeled antirabbit and Oregon Green–labeled antimouse antibodies as secondary reporter antibodies. Left panel images represent TF staining; middle panel images, golgin-97 staining; and right panel images, the overlay of TF and golgin-97 staining (colocalization). (B) Fibroblasts were first treated with a control vehicle or brefeldin A as in panel A, followed by FVIIa (10 nM) or PAR2 AP (50 µM) for 1 hour, and cell-surface TF activity was measured as described in “Materials and methods.”
- Figure S2. Factor VIIa or PAR2-mediated cell signaling does not affect the localization of PITPß in the Golgi (PDF, 2.41 MB) -
WI-38 fibroblasts treated with control buffer, FVIIa (10 nM) or PAR2 AP (50 µM) for 2 hours at 37°C. The cells were fixed, permeabilized, and immunostained with rabbit polyclonal anti–human PITPß and monoclonal anti–human golgin-97 antibodies, followed by Rhodamine Red–labeled antirabbit and Oregon Green–labeled antimouse antibodies as secondary reporter antibodies. Left panel images represent PITPß staining; middle panel images, Golgi marker staining; and right panel images, the overlay of PITPß and Golgi marker staining. Unlike TF, PITPß remains in the Golgi in cells stimulated with factor VIIa or PAR2 agonist peptide.
- Figure S3. Effect of cycloheximide, serum starvation, or TF gene silencing on TF pool in the Golgi (PDF, 2.27 MB) -
De novo TF protein synthesis was inhibited by treating fibroblasts (WI-38) with cycloheximide (10 µg/mL for 1 hour) or inhibiting TF mRNA transcription by serum deprivation or TF siRNA transfection (24 hours). Cells were fixed, permeabilized, and immunostained with rabbit polyclonal anti–human TF and monoclonal anti–human golgin-97 antibodies, followed by Rhodamine Red–labeled antirabbit and Oregon Green–labeled antimouse antibodies as secondary reporter antibodies. Left panel images represent TF staining; middle panel images, Golgi marker staining; and right panel images, the overlay of TF and Golgi marker staining. Perinuclear TF staining was seen in serum-deprived and TF siRNA–transfected cells but not in cycloheximide-treated cells.
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