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Fig. 8. A. The development of 
-actinin/actin microspikes by IGF-I is blocked
by the inhibitors of PI-3 kinase signaling. MCF-7/IGF-IR/WT cells were
serum-starved (a), serum-starved and then stimulated with 50 ng/ml IGF-I for
15 minutes (b), pretreated for 30 minutes with 10 µM LY 294002 (c), for 3
hours with 40 µM D3-PI analog (d), 30 minutes with 50 µM PD 98059 (e) or
30 minutes with 50 µM UO 126 (f) and then stimulated with 50 ng/ml IGF-I
for 15 minutes. (a-f) Overlays of confocal images of cells stained with
TRITC-phalloidin (red) and antibody against -actinin (green). Examples
of actin/-actinin microspikes are indicated by arrows. Images are
representative of three experiments. Bar, 10 µm. (B) Differential effects
of the PI analog and UO 126 on IGF-I-stimulated loss of adherens junctions and
formation of microspikes. Serum-starved MCF-7/IGF-IR/WT cells were pretreated
with 40 µM D3-PI analog for 3 hours, stimulated with 50 ng/ml IGF-I for 15
minutes in the presence of the inhibitor, fixed and stained with antibody to
-catenin (a) or fascin (c) (Materials and Methods). In parallel, the
same serum-starved cells were pretreated with 50 µM UO 126 for 30 minutes,
stimulated with 50 ng/ml IGF-I for 15 minutes, then fixed and stained with an
antibody to -catenin (b) or fascin (d). The relocation of
-catenin from adherens junctions normally induced by IGF-I (compare
with Fig. 3Aa-d) is blocked by
the PI analog but not UO 126. Normal stimulation of fascin microspikes by
IGF-I (inset in c, 50 ng/ml IGF-I, 15 minutes) is also blocked only by the PI
analog. Confocal images are representative of four independent experiments.
Bar, 10 µm. (C) The MEK1/2 inhibitor affects function of stress fibers in
IGF-I-stimulated cells. Serum-starved cells not exposed to inhibitor (a) or
pretreated with 50 µM UO 126 for 30 minutes (b) were stimulated with 50
ng/ml IGF-I for 15 minutes, then fixed and stained with TRITC-phalloidin.
IGF-I normally promotes disassembly of stress fibers within 5 minutes followed
by their re-assembly typically after 15 minutes. In cells pretreated with the
MEK1/2 inhibitor, re-assembly of the prominent stress fibers is markedly
inhibited. The representative images of optical sections collected at the
basal level of the cells are shown. Bar, 10 µm.
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