Figure 6.

Inhibition of eIF2α phosphorylation affects the rate of tumor growth in vivo. (A) Nu/Nu mice were injected on each side with 1 × 10⁶ cells/site, with either WT.Ha-RasV12 MEFs (left panel) or S51A.Ha-RasV12 MEFs (right panel). Necrotic areas on the skin and opaque morphology are evident in the S51A.Ha-RasV12 tumors. (B) Tumor growth after 3 weeks. At the end of the experiment, tumors were excised and weighed (mean±s.e., N=7, ^*P<0.05, one-tailed Student's t-test). (C) VEGF levels following hypoxia in WT and S51A MEFs. Ras-transformed MEFs were treated as in Figure 5A and secreted VEGF levels in the media were analyzed at the times indicated. Experiments were performed in triplicate and error bars represent s.e. values. (D) ATF4 upregulation in hypoxic areas is dependent on eIF2α phosphorylation. Tumor xenografts from Ha-RasV12-transformed WT and S51A cells were stained for ATF4 expression. Staining for ATF4 was observed only in WT tumors and localized within hypoxic regions. No significant staining was seen in 6/6 sections from three different S51A tumors. Bar=15 μm.