Fig. 4

Rictor/mTORC2 activity downregulates Rho guanine nucleotide dissociation inhibitor (RhoGDI2) and stimulates cellular invasion. a Whole cell lysates were assessed by western analysis. b-d Cells were treated with dimethyl sulfoxide (DMSO) or with PP242 (1 μM) for 4 h. Whole cell lysates or Pak-Pak binding domain pulldowns from whole cell lysates were assessed by western analysis (b) or assessed for migration through Matrigel-coated transwell filters 24 h after seeding, then stained with crystal violet for digital imaging (c); representative images shown) and quantitated (d). Midlines are the average number of cells on the lower side of the filter; points are average of three experimental replicates, experiments were repeated three times. e Cells stably expressing scrambled short hairpin shRNA sequences (shScr) or short hairpin RNA sequences against Raptor (shRaptor) were assessed by western analysis. f-g Cells seeded in the upper chambers of Matrigel-coated transwell filters were assessed for invasion and migration at 24 h after seeding using crystal-violet-staining and enumeration (g) as described in (d); N = 4. HER2 human epidermal growth factor receptor 2, GTP guanine triphosphate, shRictor short hairpin RNA sequences against Rictor