Introducing a C118S mutation into wild-type KRAS impairs HRAS^G12V-driven tumor growth.

<p>(<b>A</b>) RT-PCR amplification of <i>Flag-KRAS</i>, <i>Flag-HRAS</i>, <i>p110CAAX</i> and <i>GAPDH</i> mRNA isolated from oncogenic Flag-HRAS^G12V-transformed HEK-TtH cells infected with retroviruses encoding p110-CAAX and either scramble (scram) control shRNA or <i>KRAS</i> shRNA in the absence (-) or presence of shRNA-resistant wild-type (WT) or C118S mutant Flag-tagged KRAS. One of two replicate experiments. (<b>B</b>) Immunoblot detection of endogenous KRAS, Flag-tagged HRAS^G12V and tubulin in oncogenic Flag-HRAS^G12V-transformed HEK-TtH cells infected with retroviruses encoding p110-CAAX and either KRAS shRNA or a scramble control (scram) shRNA. One of three replicate experiments. (<b>C</b>) Mean ± SEM tumor volume over time, (<b>D</b>) photographs of excised tumors at end point and (<b>E</b>) mean ± SEM tumor weight at end point of tumors developing in immunocompromised mice (n = 5) injected with oncogenic Flag-HRAS^G12V-transformed HEK-TtH cells infected with retroviruses encoding p110-CAAX and either scramble control shRNA (pink circles, scram) or <i>KRAS</i> shRNA without (orange squares, <i>KRASi</i>) or in conjunction with shRNA-resistant and Flag-tagged wild-type (light green triangles, <i>KRASi</i>+KRAS) or C118S-mutant (dark green reverse triangles, <i>KRASi</i>+KRAS^C118S) <i>KRAS</i>. ns: non-significant, *: <i>P</i><0.05, **: <i>P</i><0.01 and ***: <i>P</i><0.001, as determined by one-way ANOVA plus post-hoc Bonferroni’s multiple comparison test 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">S1A and S1B</a> Fig</b>.