Figure 3.

Ras and PI3K inhibition cooperatively normalize gain-of-function Shp2-induced GM-CSF hypersensitivity. (A) Proliferation of WT Shp2- and Shp2 E76K-transduced Pik3r1+/+ or Pik3r1−/− fetal liver cells in response to GM-CSF 1 ng/mL in the presence of increasing concentrations of the farnesyltransferase inhibitor, tipifarnib; representative of 2 independent experiments, n=8, *P<0.02 comparing Pik3r1+/+, Shp2 E76K versus Pik3r1−/−, Shp2 E76K in response to 1 ng/mL GM-CSF in the absence of tipifarnib, ^P<0.001 comparing Pik3r1+/+, Shp2 E76K-expressing cells in the absence to the presence of tipifarnib at 1 μM and 5 μM; ^^P<0.005 comparing Pik3r1−/−, Shp2 E76K-expressing cells in the absence to the presence of 1 μM tipifarnib; ^^^P<0.001 comparing Pik3r1−/−, Shp2 E76K-expressing cells in the absence to the presence of 5 μM tipifarnib; statistics performed using unpaired, two-tailed students’ t-test. (B) Immunoblots demonstrating p110α, p110δ, phospho-Akt, and phospho-Erk levels in Shp2 E76K-expressing Pik3r1+/+ cells compared to Pik3r1−/−cells in the absence and presence of tipifarnib, experiment repeated on 3 independent occasions. (C) Proliferation of Shp2 E76K-transduced cells in response to GM-CSF 1 ng/mL in the presence of increasing concentrations of tipifarnib and GDC-0941; representative of 2 independent experiments, n=7, *P<0.01 and **P<0.001 comparing GDC-0941 0.5 μM and 1 μM to no GDC-0941 in the absence of tipifarnib; ^P<0.005 and ^^P<0.001 comparing GDC-0941 0.5 μM and 1 μM to no GDC-0941 in the presence of 1 μM tipifarnib; #P<0.01, ##P<0.001, and ###P<0.001 comparing GDC-0941 0.1 μM, 0.5 μM, and 1 μM to no GDC-0941 in the presence of 5 μM tipifarnib; statistics performed using unpaired, two-tailed students’ t-test. (D) Immunoblots demonstrating reduced phospho-Akt and phospho-Erk in Shp2 E76K-expressing cells treated with increasing concentrations of GDC-0941 in the absence and presence of increasing tipifarnib concentrations, experiment repeated on 2 independent occasions.