PF-271 FAK inhibition prevents anchorage-independent ID8-IP growth without effects on c-Src Y416 phosphorylation. (A) Adherent proliferation of ID8 or ID8-IP cells over 6 days in the presence of vehicle (dimethylsulfoxide, DMSO) or increasing concentrations of PF-271 (0.1 to 1.0 μM). Values are average fold-change above 50,000 starting cells from triplicate points (+/- SD), (***p<0.001). (B) Anchorage-independent growth of 250,000 ID8-IP cells on poly-HEMA coated plates over 5 days in the presence of DMSO or increasing concentrations of PF-271 (0.1 to 1.0 μM). Values are means (+/- SD) of triplicate points, (*p<0.05, *** p<0.001). (C) ID8-IP soft agar colony number after 7 days in the presence of DMSO or increasing concentrations of PF-271 (0.1 to 1.0 μM). Values are means (+/- SD) of triplicate points, (*p<0.05, *** p<0.001). (D) Protein lysates of anchorage-independent ID8-IP cells on poly-HEMA plates treated with DMSO or increasing concentrations of PF-271 (0.1 to 1.0 μM) were evaluated by immunoblotting for FAK Y397 phosphorylation (pY397), total FAK, c-Src Y416 phosphorylation (pY416 Src), total c-Src, and total actin levels.