Figure 7.

TNFα contributes to PAM₃CSK₄-mediated spleen HSPC expansion. WT, TNFα^−/−, Ccl2^−/−, Cxcr5^−/− and IFNγ^−/− mice (6–8 weeks old) were treated with PAM₃CSK₄ (100 μg intraperitoneally (i.p.) q48 h × 3 doses, analyzed 24 h after final dose) or water alone. Shown are the KSL SLAM cells per femur (a) and spleen (b). (c) Whole spleen cells from PAM₃CSK₄-treated mice or water-treated controls were plated on complete methylcellulose, and colonies counted after 7 days of growth at 37 °C. (d) Following PAM₃CSK₄ treatment, the cell cycle status of KSL SLAM cells in the spleen of mice of the indicated genotypes were determined by flow cytometry using Ki-67 and DAPI. (n=4–12 mice per group). (e–g) WT mice were treated with PAM₃CSK₄ as described above, and in addition some mice received G-CSF and TNFα neutralizing antibodies or isotype control antibody before PAM₃CSK₄ injections. KSL SLAM cells in the bone marrow (e) and spleen (f) were determined by flow cytometry. In addition, whole spleen cells were plated on complete methylcellulose, and colonies counted after 7 days of growth at 37 °C (g); n=5–6 mice per group. For all panels, error bars represent mean±s.e.m., and P-values were determined by the two-tailed Student's t-test.