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Over expression of exogenous wide-type p85α did not increase spore internalization, possibly because the amount of endogenous adaptors was sufficient to mediate spore internalization (Fig. 2, E and Fig. S1, D).
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The fact that both inhibitors caused significant reduction on spore internalization strongly suggests that PI3K activity is required for spore internalization.
We investigated which of the three Rho GTPases was responsible for spore internalization by epithelial cells.
Together the above results indicated that spore internalization by epithelial cells required actin polymerization.
The involvement of the actin cytoskeleton in spore internalization is supported by three lines of evidence.
We first examined if spore internalization by epithelial cells was dependent on the actin cytoskeleton.
Spore internalization was inhibited by PP2 (∼70%) but not by PP3 (Fig. 3, A).
Therefore, these three kinases are likely candidates involved in spore internalization.
LY294002 had a similar effect on spore internalization as that of wortmannin, ∼60% reduction on spore internalization by A549 cells (Fig. 2, B) and ∼70% by HeLa cells at the concentration used (Fig. S1, A).
Analysis of spore internalization showed that expression of Δp85α significantly decreased the spore internalization frequency (by ∼45%) in both A549 (Fig. 2, E, **, p<0.01) and HeLa cells (Fig. S1, D, ***, p<0.001) when compared to cells transfected with the vector control.
Together, these results further confirm that a class IA PI3K is recruited and activated during spore internalization.
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