Introducing a C118S mutation into the endogenous wild-type <i>Kras</i> gene impairs HRAS^G12V-driven tumor growth.

<p>(<b>A</b>) PCR amplification of genomic DNA yielding a 621 bp or 517bp fragment indicative of the wild-type or C118S <i>Kras</i> alleles in three SV40-immortalized <i>Kras</i>^<i>+/+</i> or <i>Kras</i>^{<i>C118S/C118S</i>} MEF cell lines, respectively. (<b>B</b>) Immunoblot detection of endogenous Kras, HRAS and tubulin in the indicated SV40-immortalized <i>Kras</i>^<i>+/+</i> or <i>Kras</i>^{<i>C118S/C118S</i>} MEF cell lines transformed with HRAS^G12V. Relative Kras or HRAS levels normalized to tubulin are shown beneath the immunoblot. (<b>C</b>) Representative images and (<b>D</b>) the mean ± SEM number of colonies growing in soft agar by SV40-immortalized <i>Kras</i>^<i>+/+</i> versus <i>Kras</i>^{<i>C118S/C118S</i>} MEF cell lines stably infected with a retrovirus encoding no transgene (vector) or HRAS^G12V, seeded in triplicate. (<b>E</b>) Mean ± SEM tumor volume over time, (<b>F</b>) photographs of excised tumors at end point and (<b>G</b>) mean ± SEM tumor weight at end point of tumors developing in immunocompromised mice (n = 5) injected with SV40-immortalized <i>Kras</i>^<i>+/+</i> (pink boxes) versus <i>Kras</i>^{<i>C118S/C118S</i>} (orange squares) MEF cell lines transformed with HRAS^G12V. (<b>H</b>) Mean ± SEM tumor volume over time of tumors developing in immunocompromised mice (n = 5) injected with SV40-immortalized and HRAS^G12V-transformed <i>Kras</i>^<i>+/+</i> MEFs (pink circles), <i>Kras</i>^{<i>C118S/C118S</i>} MEFs (orange squares) or <i>Kras</i>^{<i>C118S/C118S</i>} MEFs stably infected with a retrovirus encoding KRAS* (light green triangles) or KRAS*^C118S (dark green reverse triangles). (<b>I</b>) Kaplan-Meier survival curves based on the time to reach end point of immunocompromised mice (n = 5) each injected with one of the three SV40-immortalized <i>Kras</i>^<i>+/+</i> (pink line) versus <i>Kras</i>^{<i>C118S/C118S</i>} (orange line) MEF cell lines transformed with HRAS^G12V. ns: non-significant, **: <i>P</i><0.01, ***: <i>P</i><0.001 and ****: <i>P</i><0.0001, as determined by one-way ANOVA plus post-hoc Bonferroni’s multiple comparison test (D, H), two-tailed unpaired <i>t</i> test (E, G) or long-rank test (I) using GraphPad Prism 5 Software. Full-length immunoblots and gels are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123918#pone.0123918.s001" target="_blank">S1F and S1G</a> Fig.</b>