Fig. 4

RHEB Y35N Transforms Cells Similar to KRAS G12V and RHEB WT Does Not. (a) Growth curves of NIH 3T3 cell lines stably expressing RHEB WT, RHEB Y35N, or KRAS G12 V grown in media containing 10% FBS (left), or serum-free media (right), for 7 days. Fold Growth was calculated as follows: (OD at day X) / (OD at day 0), where OD was read according to Cell Counting Kit-8 (Dojindo Mol. Tech.) protocol. Error bars are the standard deviation measured from three separate experiments. b NIH 3T3 stably expressing cell lines were grown for 2 days with serum (left) or without serum (right). Cells were then fixed, treated with RNase A to remove RNA, and incubated with propidium iodide (PI) to dye DNA. Cells were grouped into cell cycle stage based on PI intensity measured using flow cytometry. Graphs show the percentages of cells at each stage in the cell cycle. c Foci Formation Assay. NIH 3T3 stably expressing cell lines were grown under low serum conditions for 3 weeks. Cells were fixed with methanol and stained with crystal violet dye for easy visualization. d Soft Agar Colony Formation Assay. NIH 3T3 stably expressing cell lines were grown in agar-media suspension for 3 weeks. Plates were incubated overnight with Nitroblue Tetrazolium Chloride (NBT) in order to visualize colonies on a gel imager