Effects of Tiam1 down-regulation on PAF-induced signaling.
A, Tiam1 down-regulation. HUVECs treated with siRNA specific for
Tiam1 showed a gradual decrease in the total amount of protein starting at 48
h, with a >80% reduction seen at 96 h, as demonstrated by Western blotting
of EC lysate probed with anti-Tiam1 Ab. Note that the scrambled sequence of
the specific siRNA did not change the levels of Tiam1 at any time point. The
specific siRNA sequence did not affect the expression of β-Pix and Vav2
Rac1 GEFs, as shown in the bottom two panels (n = 6).
B, Tiam1 down-regulation effects on Rac1 redistribution. The top
panels shows that there is no modification in the amount of Rac1 in the
membrane fraction (upper panel) as well as in its total amount
(lower panel), whereas the associated graph quantitatively
substantiates the lack of Rac1 redistribution at 96 h after Tim1 knockdown;
compare with Fig. 7A
(n = 7). Statistical significance was as follows: ‡,
p < 0.001 (paired Student's t test). C, Rac1
activation is affected by Tiam1. The activation of Rac1 as detected by the
pull-down assay is drastically impaired when Tiam1 is down-regulated and the
monolayers are stimulated with PAF, as illustrated in the upper
panel. However, the total amount of Rac1 is not affected under the same
conditions as shown in the lower panel (n = 6). Statistical
significance was as follows: *, p < 0.01; ‡,
p < 0.001 (paired Student's t test). D, Tiam1
knockdown effect on interendothelial gaps. When the distribution of
endothelial actin assessed by staining with fluorescein
isothiocyanate-phalloidin was investigated after Tiam1 down-regulation, we did
not found it, under basal conditions, changed (compare with the control
panel from Fig. 2A,
a). b-d show the distribution of cellular actin in
monolayers of HUVEC after Tiam1 depletion and exposure to PAF for different
periods of time (n = 4). Under these conditions, there is a
redistribution of actin toward a polymerized status; however, note the
dramatic reduction in the number of inter endothelial gaps, filopodia, and
lamellipodia (compare with Fig. 2,
a-e)(n = 8). Bar, 22 μm. E,
Tiam1 affects the actin shift. The Western blot shows inhibition of actin
shift after Tiam1 down-regulation. Challenging the Tiam1-depeleted monolayers
with PAF prevented the G- to F-actin shift (compare with
Fig. 2C)(n =
4). Statistical significance was as follows: *, p <
0.01; **, p < 0.05, by comparison with controls
(Student's t test).