Requirement of Erk1 and Erk2 in HSCs and immature progenitors. (A) Single SLAM-HSC from of control, Erk1^−/−, Erk2^Δ/Δ, and Erk1^−/−Erk2^Δ/Δ mice were sorted into 60-well plates (1 cell per well) and cultured in media containing SCF, Tpo, Flt3l, and IL-11. Proliferation of each clone was evaluated microscopically over 4 days. Representative data from 1 of 4 experiments with similar results are shown. The average percentages (±SEM) of surviving clones from the 4 experiments are shown at the top of each panel. *P < .05, ANOVA. (B) Lethally irradiated CD45.1⁺ recipients (6 mice per group) were transplanted with 10⁶ BM cells from control, Erk1^−/−, Erk2^Δ/Δ, and Erk1^−/−Erk2^Δ/Δ mice, along with 10⁵ wild-type CD45.1⁺ cells. After 5 weeks of engraftment, chimeric mice were treated with 5 doses of pIpC, and the percentage of donor-derived peripheral blood cells (Total), CD3⁺ (T cells), B220⁺ (B cells), and Gr1⁺ (myeloid cells) were determined flow cytometry. *P < .05, ANOVA. (C) Control, Erk1^−/−, Erk2^−/−, and Mx1-Cre Erk1^−/−Erk2^flox/flox mice were treated with pIpC for 14 days before their BM cells (5 × 10⁵) were harvested and mixed with equal numbers of wild-type CD45.1⁺ cells and intrafemorally injected into lethally irradiated CD45.1⁺ recipients (5 mice per group). After 8 weeks of engraftment, the percentage of donor-derived peripheral blood cells (Total), CD3⁺ (T cells), B220⁺ (B cells), and Gr1⁺ (myeloid cells) were determined flow cytometry. *P < .05, ANOVA. (D) Primary lin⁻ BM cells from Erk1^−/−;Erk2^flox/flox mice were transduced with the indicated retroviruses, and FACS-purified GFP⁺ cells were cultured in Iscove modified Dulbecco medium containing IL-3, IL-6, and SCF. MIG-Cre vectors coexpress the hygromycin resistance gene (hygro), Erk1, Erk2, Erk1^KR, and Erk2^KR. Relative cell proliferation in each experiment is normalized to that of Erk2. Results are shown as mean ± SEM of 4 independent experiments. *P < .05, ANOVA. (E) Lin⁻ BM cells from Erk1^−/−;Erk2^flox/flox;LSL-Kras^G12D mice were transduced with retroviruses as described in panel D, and GFP⁺ cells were cultured in IMDM medium containing no cytokines. Cells were harvested and proliferation was determined. Cell proliferation in each experiment is normalized to that of Erk2 and results are shown as mean ± SEM of 4 independent experiments. *P < .05, ANOVA.