Fig 1.
Introducing a C118S mutation into wild-type KRAS impairs HRASG12V-driven tumor growth.
(A) RT-PCR amplification of Flag-KRAS, Flag-HRAS, p110CAAX and GAPDH mRNA isolated from oncogenic Flag-HRASG12V-transformed HEK-TtH cells infected with retroviruses encoding p110-CAAX and either scramble (scram) control shRNA or KRAS shRNA in the absence (-) or presence of shRNA-resistant wild-type (WT) or C118S mutant Flag-tagged KRAS. One of two replicate experiments. (B) Immunoblot detection of endogenous KRAS, Flag-tagged HRASG12V and tubulin in oncogenic Flag-HRASG12V-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. (C) Mean ± SEM tumor volume over time, (D) photographs of excised tumors at end point and (E) mean ± SEM tumor weight at end point of tumors developing in immunocompromised mice (n = 5) injected with oncogenic Flag-HRASG12V-transformed HEK-TtH cells infected with retroviruses encoding p110-CAAX and either scramble control shRNA (pink circles, scram) or KRAS shRNA without (orange squares, KRASi) or in conjunction with shRNA-resistant and Flag-tagged wild-type (light green triangles, KRASi+KRAS) or C118S-mutant (dark green reverse triangles, KRASi+KRASC118S) KRAS. ns: non-significant, *: P<0.05, **: P<0.01 and ***: P<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 S1A and S1B Fig.
Fig 2.
Introducing a C118S mutation into wild-type KRAS impairs EGF stimulation of AKT and ERK1/2 phosphorylation.
Immunoblot detection of input and GTP-bound Flag-KRAS captured by the Ras-binding domain of Raf1, as well as HA-eNOS and tubulin, in (A) HEK-TtH cells or (B) SV40-immortalized MEFs stably infected with retroviruses encoding either wild-type (WT) or C118S-mutant Flag-tagged KRAS* in conjunction with either the HA-tagged S1177D constitutively-active or the S1177A inactive mutant versions of eNOS. High molecular weigh bands were variably detected using this assay, and because they are above the size of Ras, they were considered to be non-specific. Relative mean ± SEM of GTP-bound KRAS (normalized to KRAS input) is shown beneath the immunoblot. One of three replicate experiments. (C) Immunoblot detection of total (T) and phosphorylated (P) AKT and ERK1/2 in HEK-TtH cells stably infected with retroviruses encoding a scramble control (scram) shRNA, a KRAS shRNA, or KRAS shRNA and wild-type (WT) or C118S-mutant Flag-tagged shRNA-resistant KRAS and serum starved overnight and treated without (-) or with (+) EGF for five minutes. Relative mean ± SEM of P-AKT (normalized to T-AKT) or P-ERK1/2 (normalized to T-ERK) is shown beneath the immunoblot. One of three replicate experiments. Full-length immunoblots are shown in S1C–S1E Fig.
Fig 3.
Introducing a G12V activating mutation overcomes the inability of KRASC118S to promote HRASG12V-driven tumor growth.
(A) RT-PCR amplification of Flag-KRAS, Flag-HRAS, p110CAAX and GAPDH mRNA (note that this is the same gel as in Fig 1A, except with the addition of analysis for Flag-KRASG12V,C118S, one of two replicate experiments), (B) mean ± SEM tumor volume over time, (C) photographs of excised tumors at end point and (D) mean ± SEM tumor weight at end point of tumors developing in immunocompromised mice (n = 5) injected with Flag-HRASG12V-transformed HEK-TtH cells infected with retroviruses encoding p110CAAX and KRAS shRNA alone (pink circles) or with wild-type (WT, orange squares), C118S-mutant (light green triangles) or G12V,C118S-mutant (dark green reverse triangles) Flag-tagged and shRNA-resistant KRAS. ns: non-significant, *: P<0.05, **: P<0.01 and ***: P<0.001, as determined by one-way ANOVA plus post-hoc Bonferroni’s multiple comparison test using GraphPad Prism 5 Software. The full-length gel is shown in S1A Fig.
Fig 4.
Introducing a C118S mutation into the endogenous wild-type Kras gene impairs HRASG12V-driven tumor growth.
(A) PCR amplification of genomic DNA yielding a 621 bp or 517bp fragment indicative of the wild-type or C118S Kras alleles in three SV40-immortalized Kras+/+ or KrasC118S/C118S MEF cell lines, respectively. (B) Immunoblot detection of endogenous Kras, HRAS and tubulin in the indicated SV40-immortalized Kras+/+ or KrasC118S/C118S MEF cell lines transformed with HRASG12V. Relative Kras or HRAS levels normalized to tubulin are shown beneath the immunoblot. (C) Representative images and (D) the mean ± SEM number of colonies growing in soft agar by SV40-immortalized Kras+/+ versus KrasC118S/C118S MEF cell lines stably infected with a retrovirus encoding no transgene (vector) or HRASG12V, seeded in triplicate. (E) Mean ± SEM tumor volume over time, (F) photographs of excised tumors at end point and (G) mean ± SEM tumor weight at end point of tumors developing in immunocompromised mice (n = 5) injected with SV40-immortalized Kras+/+ (pink boxes) versus KrasC118S/C118S (orange squares) MEF cell lines transformed with HRASG12V. (H) Mean ± SEM tumor volume over time of tumors developing in immunocompromised mice (n = 5) injected with SV40-immortalized and HRASG12V-transformed Kras+/+ MEFs (pink circles), KrasC118S/C118S MEFs (orange squares) or KrasC118S/C118S MEFs stably infected with a retrovirus encoding KRAS* (light green triangles) or KRAS*C118S (dark green reverse triangles). (I) 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 Kras+/+ (pink line) versus KrasC118S/C118S (orange line) MEF cell lines transformed with HRASG12V. ns: non-significant, **: P<0.01, ***: P<0.001 and ****: P<0.0001, as determined by one-way ANOVA plus post-hoc Bonferroni’s multiple comparison test (D, H), two-tailed unpaired t test (E, G) or long-rank test (I) using GraphPad Prism 5 Software. Full-length immunoblots and gels are shown in S1F and S1G Fig.