Figure 4.

Ripk3 signaling promotes low-dose IR-induced HSC elimination in an Mlkl-dependent manner

(A and B) WT, Ripk3^−/−, and Mlkl^−/− mice were irradiated with 6 Gy X-ray. BM MNCs were collected 2 and 48 h after IR. DNA damage was examined in LSK cells by γ-H2A.X staining followed by flow cytometry (A) and microscopic analysis (B).

(C and D) WT, Ripk3^−/−, and Mlkl^−/− mice were irradiated with 1.75 Gy X-ray weekly × 4. BM MNCs were collected from the mice 1 month following the last IR. HSCs and HPCs were analyzed by flow cytometry gating on LSK and LK populations. Representative flow cytometric data (C) and absolute numbers of HSCs and HPCs (D) in the BM of the indicated genotypes of mice are presented. Five mice were studied in each group. ^$p < 0.05 compared with Ripk3^−/− or Mlkjl^−/− groups.

(E–H) WT and Ripk3^−/− mice were irradiated with X-rays, 1.75 Gy, every week for a maximum of 4 weeks. BM and PB were collected from the mice at the indicated times after the first IR. Ripk3-Mlkl signaling in BM MNC and LSK populations from WT mice was examined at the indicated times after the first IR by western blotting (E) and flow cytometry (F), respectively. Data in (F) show one of the three biological triplicate experiments, and LSK cells from Ripk3^−/− mice were always studied in parallel as controls. Levels of Tnf-α in PB were examined by ELISA (G). HSC numbers/two hindlegs were examined by flow cytometric analysis (H).

(I) WT and Tnfr^−/− mice were irradiated with 1.75 Gy X-ray weekly × 4. Mice were monitored for leukemia development. Survival curves for the mice were plotted by Kaplan-Meier graphing. ^∗p < 0.05 and ^∗∗p < 0.01, compared with non-irradiated WT mice or D0 group. ^&p < 0.05, compared with irradiated Ripk3^−/− or Mlkl^−/− mice. In (G), ^∗∗p < 0.01 compared with days 0, 1, and 7.