Fig. 2. ENU Treatment of 5G2^del/+ Mice.

a Survival of ENU-treated WT (n = 18) and 5G2^+/del (n = 13) littermates. Percent survival (time to euthanasia of moribund animals) is plotted vs time in days after ENU treatment. The cumulative probability of survival was computed by the Kaplan-Meier method and is significantly less in 5G2^+/del mice (p = 0.0197 by log rank). Thymic lymphoma was the major cause of death in mice of both genotypes. b Chimeras were generated by transplanting WT or 5G2^+/del BM with WT competitor cells at a ratio of 3:1 into irradiated congenic WT recipients. Recipient mice received control vehicle or ENU at a dose of 100 mg/kg intraperitoneally 5 and 6 weeks post-transplant. The fold change in the percentage of total leukocytes derived from WT or 5G2^+/del cells 4 weeks after the second ENU treatment is shown. Data represent the mean ± standard error of the mean (s.e.m.) of 4-5 mice. Chimerism was analyzed using Student’s t-test. c Experimental design for assessing the effects of the 5G2 deletion on the competitive fitness of Nras^G12D BM cells in the presence and absence of ENU treatment. Three-week-old Mx1-Cre; Nras^G12D and Mx1-Cre; Nras^G12D; 5G2^+/del mice were injected with a single dose of pIpC to induce Nras^G12D expression from the endogenous locus followed by bone marrow harvest at 8-12 weeks for competitive transplantation with WT competitor cells at a 1:1 ratio. Recipient mice were treated with a control vehicle or ENU at 5 and 6 weeks post-transplant as above and were monitored for 5 months post-transplant. d Contribution of donor Nras^G12D (blue circles) and Nras^G12D; 5G2^+/del (red squares) cells to HSC, KLS, myeloid progenitor (MP), Myeloid, T cell and B cell populations in the BM of recipient mice that did not receive ENU post-transplant. Note that the 5G2 mutation impaired the ability of donor Nras^G12D cells to repopulated the HSC, KLS, and MP compartments. Asterisks denote significant differences between Nras^G12D (blue circles) and Nras^G12D; 5G2^+/del donor cells (*p < 0.05; ***p < 0.005 by Student’s t-test). Comparable competitive fitness data are not available from recipients that received ENU because most died prematurely from lymphoid malignancies.