Figure 5.

Transformation by a constitutively active mutant of PI3K and suppression of v-Crk-induced transformation by a PI3K inhibitor. (A) Soft-agar colony formation by the PI3K constitutively active mutant BD110. CEF transduced with BD110 or with vector were subjected to soft-agar colony formation assay. Photomicrographs of colonies were taken 3 weeks after plating. (×40.) (B) Suppression of v-Crk-induced soft-agar colony formation by a PI3K inhibitor. CEF expressing v-Crk WT were subjected to soft-agar colony formation assay with (+LY) or without (−LY) LY294002. LY294002 was included in top agar layer at 10 μM final concentration, and 0.5 ml of medium containing the same final concentration of LY294002 was added onto the top agar every 4 days. We confirmed that long-term treatment with this concentration of LY294002 showed no apparent toxicity to CEF cultured in monolayer (data not shown). CEF transduced with vector without such treatment also were examined. At 3 weeks after plating, colonies were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as described (37) and photographs of the stained colonies were taken. (×1.)