Fig. 3

Rictor supports Rac-mediated migration and invasion of human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells. a-b In situ detection of Rac-guanosine triphosphate (GTP) using indirect immunofluorescence via glutathione-S transferase-Pak binding domain (GST-PBD) in paraffin-embedded sections of Rictor ^+/+ NIC and Rictor ^FL/FL NIC mammary tumors. GST-PBD binding is shown in red, while 4',6-diamidino-2-phenylindole (DAPI) counterstaining of nuclei is shown in blue. Representative images are shown (a), original magnification, ×200. Quantitation (b) shows the average fluorescent pixel area/group (midlines) ± S.D. and the average fluorescence in five random fields/sample (points); N = 3–4. c Whole cell lysates and PAK-PBD pull-downs from whole cell lysates were assessed by western analysis; N = 3 replicates. d-f Cells were treated with dimethyl sulfoxide (DMSO) or with Rac inhibitor (0.5 μM) and assessed by western analysis of whole cell lysates or PAK-PBD pulldowns (d) or assessed for migration through Matrigel-coated transwell filters 24 h after seeding, then stained with crystal violet for digital imaging (e); representative images shown) and quantitated (f). 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. g-i Cells expressing Ad.RFP or Ad.Rac^G12V were assessed by western analysis of whole cell lysates or PAK-PBD pulldowns (g), or for migration through Matrigel-coated transwell filters (h-i); N = 3. Quantitation is shown i. Midlines are the average number of migrating cells; points are replicates each assessed in triplicate