FIG 8.

Signal integration as a consequence of spatial cross talk. (a) BHK cells stably expressing GFP–K-RasG12V alone, GFP–K-RasG12V with RFP–H-RasG12V, or GFP–K-RasG12V with RFP-CTH were grown in serum-free media in the presence of the MEK inhibitor U0126 for 24 h, followed by growth for 30 min in the absence of U0126, and then analyzed by quantitative immunoblotting for pS338-CRAF, pMEK, pERK, and pAkt. The results were normalized against those for control cells expressing K-RasG12V alone. The graph shows means ± SEM from 3 independent experiments. The statistical significance of differences was evaluated using one-way ANOVA (*, P < 0.05). (b) Survival of cancer cell lines 3 days after lentiviral transduction of GFP-tH, GFP–K-RasG12V, GFP–H-RasG12V, or GFP-CTH. Cell counts of cells expressing GFP–K-RasG12V (+KG12V), GFP–H-RasG12V (+HG12V), or GFP-CTH (+CTH) are shown relative to cell counts for a cognate GFP-tH control. Cell lines are connected across each lentiviral expression column. Cancer cell lines expressing wild-type K-Ras (K-WT) were HES, ESS1, EFE184, AN3CA, KLE, and RL-95-2 (endometrial) and Bx-PC3 (pancreatic). Cancer cell lines expressing oncogenic K-RasG12D were HEC1a, HEC1b, and HEC50 (endometrial) and MoH, mPanc96, and HPAC (pancreatic). AN3CA cells (dotted blue line) behave like K-RasG12D-expressing cells in this assay. (c) Wild-type BHK cells transiently expressing GFP–K-RasG12V or a combination of GFP–K-RasG12V and RFP–H-RasG12V were serum starved for 1 h and then treated with 1 μM latrunculin for 5 min; pS338-CRAF levels were measured by quantitative immunoblotting (left panel). BHK-Cav-1KD cells transiently expressing GFP–K-RasG12V or a combination of GFP–K-RasG12V and RFP–H-RasG12V were serum starved for 1 h; pS338-CRAF levels were then measured by quantitative immunoblotting (right panel). The graphs show means ± SEM from 3 independent experiments. The statistical significance of differences was evaluated using one-way ANOVA (*, P < 0.05).