We used U87MG cells, an established p110β^high GBM cell line. We starved cells overnight, then treated cells with growth factors, promoters or inhibitors of several pathways, including EGF, FGF-2 and GPCR. We collected cell lysates and measured PI3K/AKT pathway activity using immunoblotting. The ratio of pAKT/AKT/ACTB quantified PI3K/AKT pathway activity.

Serum starvation of U87MG cells depleted pAKT, and incubation with 1% FBS restored pAKT signaling in a time dependent manner.

Neither EGF nor FGF-2 activated PI3K/AKT signaling in U87MG cells after overnight starvation with serum free media. Furthermore, gefitinib, an EGFR inhibitor, failed to inhibit reactivation when co-treated with 1% FBS.

GPCR stimulation by Lysophosphatidic acid (LPA) increased pAKT/AKT signaling in a dose dependent manner. Additionally, Gallein, a GPCR inhibitor, inhibited PI3K/AKT signaling when co-treated with 1% FBS.

Both serum and GPCR stimulation are sufficient to activate the PI3K/AKT pathway in p110β^high cells. EGF and FGF-2 have no effect on PI3K/AKT pathway reactivation. These findings may provide novel therapeutic targets in GBM treatment.