Fig. 6
From: Two distinct mTORC2-dependent pathways converge on Rac1 to drive breast cancer metastasis
Akt restoration partially rescues Rictor-mediated defects in cell migration and invasion. a-b Cells expressing scrambled short hairpin shRNA sequences (shScr) or short hairpin RNA sequences against Rictor (shRic) were infected with Ad.RFP or Ad.AktDD. Whole cell lysates, Pak binding domain (PBD) pulldowns from whole cell lysates, or RacG15A pulldowns from whole cell lysates were assessed by western analysis (a). Cells were assessed for their ability to invade through Matrigel-coated transwell filters at 24 hours after seeding (b). Cells on the lower side of the transwell filter were stained with crystal violet (representative images shown (b (upper panel)) and counted in digital images (b (lower panel)). Each data point represents the average value of three experimental replicates; midline represents the average of N = 4 repeats per condition, error bars are the standard error. Student’s t test used to assess significance. c-d Cells were treated with the Akt inhibitor AZD5365 (1 μM). Whole cell lysates, PBD pulldowns from whole cell lysates, or RacG15A pulldowns from whole cell lysates were assessed by western analysis (c). Cells were assessed for their ability to invade through Matrigel-coated transwell filters at 24 h after seeding (d). Cells on the lower side of the transwell filter were stained with crystal violet (representative images shown in d (upper panel) and counted in digital images (d (lower panel)) as described in (b). e-f Cells expressing shScr or shRictor were infected with Ad.RFP or Ad.PKCα. Whole cell lysates or PBD pulldowns from whole cell lysates were assessed by western analysis (e). Cells were assessed for their ability to invade through Matrigel-coated transwell filters at 24 h after seeding (f). Cells on the lower side of the transwell filter were stained with crystal violet (representative images shown in f (upper panel) and counted in digital images (f (lower panel)) as described in (b). (g). Proposed model of how mTORC2 controls cell motility and metastasis of HER2-overexpressing breast cancers. RhoGDI2 Rho guanine nucleotide dissociation inhibitor, PKC protein kinase C