Figure 7 is a western blot that demonstrates that inhibiting integrin α5β1 binding with blocking antibody or blocking peptide P1 had no effect on Akt phosphorylation. An inhibitor of PI3K, LY294002, was used as a positive control. These data suggest that PI3K activation by FGF-2 is mediated directly by FGF-2-mediated signaling, independent of signaling by integrin α5β1. Fig. 7 Akt activation by FGF-2 in dormant cells is independent of integrin α5β1 ligation. Western blots of lysates

from cells incubated on fibronectin with and without FGF-2 10 ng/ml or blocking antibodies to integrin α5β1 or integrin α2β1 2 μg/ml, blocking peptide P1 to fibronectin 100 nm, or PI3K inhibitor LY294002 25 μM on day 3, as described in Materials and Methods, were stained MK1775 with antibody to phospho-Akt or total Akt PI3K Activation is Necessary for Cortical Actin Redistribution Gemcitabine in Dormant Cells To determine if dual signaling by FGF-2 through PI3K as well as ligation

of the upregulated integrin α5β1 is required for the cortical actin rearrangement in the dormant cells, we incubated the cells with the PI3K inhibitor LY294002. Figure 8a demonstrates that dormant cells incubated with LY294002 lost their spread appearance and their cortical actin rearrangement and developed stress fibers. Figure 8b shows that the percentage of cells with cortical actin increased from 33.1 + 11.5% in growing cells to 74.2 + 7.7 in the dormant cells (p < 0.01), an effect reversed by the PI3K inhibitor to 30.88 + 15.5% (p < 0.01). These data suggest that dual signaling by FGF-2 DOK2 directly through PI3K and through integrin α5β1 is necessary for cortical rearrangement in dormant cells. Fig. 8 Cortical actin stabilization in dormant breast cancer cells is PI3K-dependent. a MCF-7 cells incubated with or without FGF-2 10 ng/ml on fibronectin-coated cover slips at clonogenic density, with and without addition of LY294002 25 μM on day 3 were stained on day 6 with BODIPY-Phallacidin (green actin staining) and DAPI (blue nuclear

staining) and photographed at 400 x magnification. The figure demonstrates cortical actin distribution that appears in dormancy and is reversed by PI3K inhibition. The appearance of stress fibers and loss of the characteristic cell spreading is evident in dormant cells inhibited by LY294002. b Quantitative representation of manually counted cells with cortical actin on triplicate slides from a duplicate experiment demonstrating an increase in cortical actin with dormancy and reversal with PI3K inhibition. Error bars are + standard deviations. *p < 0.01 (Student’s t test) Membrane Localization of GRAF and Inactivation of RhoA Require PI3K Activity Since guanine exchange factors and GTP activating proteins have both been linked to PI3K activity, we investigated whether the inactivation of RhoA in dormant cells was dependent on activation of PI3K.

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