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Figure legends
Fig. 1. Chemical structure of viscolin and its putative functional groups ( ).
Fig. 2. Viscolin inhibits the TNF-α-induced increase in VCAM-1 mRNA and protein levels in HUVECs. (A) HUVECs were treated with TNF-α (10 ng/mL) for the indicated
time, then protein levels in cell lysates were measured on Western blots. (B) HUVECs
were incubated with the indicated concentrations of viscolin for 24 h, then with 10 ng/mL of TNF-α for 6 h in the continued presence of the same concentration of viscolin, and
VCAM-1 protein in cell lysates was measured by Western blot. GAPDH was used as the
loading control. (C) Analysis of VCAM-1 mRNA levels in untreated HUVECs or
HUVECs preincubated with or without 10 µM viscolin for 24 h, then incubated with 10 ng/mL of TNF-α for 6 h. Total RNA was analyzed by RT-PCR after normalization to 18S
levels. (D) NIH 3T3 cells were transfected with a luciferase plasmid containing the
VCAM-1 promoter for 24 h, then were incubated with or without 10 µM viscolin for 24 h
prior to the addition of TNF-α (10 ng/mL) for a further 6 h. In A-D, the data are
expressed as a fold value compared to the control value and are the mean±SEM for three
separate experiments. *P<0.05 compared to the untreated cells. †P<0.05 compared to the TNF-α-treated cells.
Fig. 3. The viscolin-mediated reduction in TNF-α-induced VCAM-1 expression is partly
dependent on inhibition of JNK phosphorylation. (A-C) HUVECs were treated with 10
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ng/mL TNF-α for the indicated time, then the cell lysate was analyzed for MAPK
phosphorylation by Western blot using antibodies against p-ERK1/2 (A), p-JNK (B), or
p-p38(C). (D-F) The cells were preincubated for 1h with the indicated concentration of
PD98059 (ERK1/2 inhibitor) (D), SP600125 (JNK inhibitor) (E), or SB203580 (p38 inhibitor) (F), then were treated with TNF-α for 6 h and the cell lysate analyzed for
VCAM-1 expression by Western blot. (G-I) Western blot analysis showing the effect of
viscolin treatment on the phosphorylation of p-ERK1/2 (G), p-JNK (H), or p-p38 (I) in
TNF-α-treated HUVECs. HUVECs were incubated for 24 h with or without 30 µM viscolin, then the cells were incubated with 10 ng/mL of TNF-α for 15 min and aliquots
of cell lysate containing equal amounts of protein subjected to immunoblotting with the
indicated antibodies. The data are expressed as a fold of the control value and are the
mean±SEM for 3 separate experiments. GAPDH was used as the loading control.
*P<0.05 compared to the untreated cells. †P<0.05 compared to the TNF-α-treated cells.
Fig. 4. The viscolin-induced downregulation of VCAM-1 expression in
TNF-α-stimulated HUVECs is mediated by inhibition of NF-κB activation and NF-κB
p65 nuclear translocation. (A-B) Nuclear extracts prepared from untreated cells or from cells with or without 24 h pretreatment with 30 µM viscolin, then incubated with 10
ng/mL of TNFα for 30 min were tested for AP-1 (A) or NF-κB (B) DNA binding activity
by EMSA. (C, D) Western blot and immunofluorescent staining for NF-κB p65.
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HUVECs were preincubated for 24 h with 30 µM viscolin or for 2 h with 10 mM NAC,
10 µM DPI, or 100 µM APO, then were treated with 10 ng/mL of TNF-α for 30 min. A
representative result from three separate experiments is shown. Bar=100 µM. (E) Cells
were coincubated for 24 h with 0-10 µM parthenolide (Par, NF-κB inhibitor) and 10 ng/mL of TNF-α, then cell lysates were prepared and assayed for VCAM-1 by Western
blot. The data are expressed as a fold of the control value and are the mean±SEM for 3
separate experiments. GAPDH was used as the loading control. *P<0.05 compared to the
untreated cells. †P<0.05 compared to the TNF-α-treated cells.
Fig. 5. Viscolin reduces TNF-α-induced ROS production, NADPH oxidase activity
and p47phox translocation in HUVECs. (A, B) HUVECs were incubated with DHE and
amplex red/HRP, and then 10 ng/mL of TNF-α was added to the well for the indicated
time, and ethidium and resorufin fluorescence were measured for the generation of
O2•-
and H2O2, respectively. (C, D) The cells were pretreated with 30 µM viscolin for 24 h or with 10 µM DPI for 2 h prior to addition of 10 ng/mL TNF-α for 15 min. The
methods for the measurement of O2•-
and H2O2 was described in the Materials and
Methods. (E) The cytosolic and membrane fractions from unstimulated HUVECs
were incubated with the reaction mixture for NADPH oxidase assembly as described
in the Materials and Methods, then were left untreated or were incubated for 2 min with 30 µM viscolin or100 µM APO, then ferricytochrome c reduction was measured
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at 550 nm. (F) Control cells or cells pretreated with 30 µM viscolin for 24 h or 10 µM
DPI for 2 h were incubated with 10 ng/mL of TNF-α for 20 min, then the plasma
membrane protein was assayed with a superoxide-dependent lucigenin
chemiluminescent assay. (G) Western blot of p47phox levels in the membrane fractions of HUVECs pretreated with viscolin for 24 h or with 10 µM DPI for 2 h, then
stimulated with of 10 ng/mL of TNF-α for 20 min. (H-J) The cells were incubated for
2 h with the indicated concentration of 0-10 mM NAC (H), 0-10 µM DPI (I), or 0-200 µM APO (J), then were treated with TNF-α for 6 h and the cell lysate analyzed for
VCAM-1 expression by Western blot. (K) The cells were preincubated for 2 h with 10 mM NAC, 10 µM DPI, or 100 µM APO, then were treated with TNF-α for 15 min
and the cell lysate analyzed for JNK phosphorylation by Western blotting. Values
were presented as the mean±SEM. *P<0.05 compared to the untreated cells. †P<0.05 compared to the TNF-α-treated cells.
Fig. 6. Viscolin reduces the adhesion of U937 cells to TNF-α-stimulated HUVECs. Cells
were left untreated or were pretreated for 24 h with 30 µM viscolin, or for 1h with 1
µg/mL or with 2µg/mL of anti-VCAM-1 antibodies, or for 1h with 10 µM PD98059, SP600125, SB203580 or parthenolide, or for 2 h with 10 mM NAC, 10 µM DPI, or100
µM APO, then were incubated with 10 ng/mL of TNF-α for 6 h in the continued presence of the inhibitor. (A) Representative fluorescent photomicrographs showing the effect on
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TNF-α-induced adhesion of fluorescein-labeled U937 cells to HUVECs. C is untreated
cells. Bar=100 µm. (B) The number of U937 cells bound per high power field was
counted. The data are expressed as the mean±SEM for three separate experiments.
*P<0.05 compared to the untreated cells. †P<0.05 compared to the TNF-α-treated cells.
Fig. 7. Immunohistochemical staining for vWF or VCAM-1 expression in serial
sections of thoracic aortas from mice. Mice were treated with DMSO (C), TNF-α,
TNF-α+viscolin, or viscolin alone as described in the Materials and Methods, then
serial sections were stained for vWF (endothelial cell marker) or VCAM-1. The
lumen is uppermost in all sections. The reaction product and the internal elastic membrane are indicated by an arrowhead and an arrow, respectively. Bar=50 µM.