FIGURE 6.

NIH3T3 cells expressing the Lys-99 and Asp-101 salt bridge mutants of EN fail to form tumors in nude mice. NIH3T3 cells expressing EN, HA-EN, K99R-EN, HA-D101K-EN, and FLAG-R103A-EN constructs were injected subcutaneously into the flanks of nude mice (n = 6 for HA-D101K-EN, n = 5 for all others, two sites/mouse), and tumor formation was monitored over a period of 30 days. At day 16, EN tumors grew to ∼1000 mm³, and therefore, mice bearing these tumors were sacrificed. K99R-EN did not form any detectable tumors over the course of the study, and D101K-EN formed only very small tumors averaging below 100 mm³ at day 30. R103A-EN tumors grew very slowly in the beginning but eventually reached ∼1000 mm³ at day 27. Statistical significance was determined using a Kruskal-Wallis test comparing tumor volumes among groups represented for the indicated day of monitoring. ***, p < 0.001; **, p < 0.01.