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. 2022 May 12;17(6):1428–1441. doi: 10.1016/j.stemcr.2022.04.009

Figure 2.

Figure 2

Ripk3 selectively regulates number and function of HSCs during serial transplantation in Mlkl-dependent and -independent manners, respectively

(A) Serial transplantation and analysis schedule. TP0 represents non-transplanted control. TP1, TP2, and TP3 indicate first, second, and third transplantations, respectively. “4ms” represents 4 months post-transplantation.

(B and C) BM MNCs were collected from recipient mice 4 months post-transplantation for each transplantation cycle. Activation of Ripk3-Mlkl signaling in BM MNCs (B) and LSK cells (C) was examined by western blotting and flow cytometric assays, respectively. For flow cytometric analysis, Ripk3−/− LSK cells were always used as a negative control to set up the flow cytometer in order to make the data consistent in all of the experiments.

(D) Representative flow cytometric data for HSCs and MPPs among CD45.2+ BM MNCs from second and third transplantation recipients of the indicated genotypes of donors.

(E and F) Absolute numbers of LK cells and LSK cells (E) as well as HSCs and MPPs (F) in the BM from third transplantation recipients of the indicated donor genotypes.

(G–L) BM MNCs were collected from third transplantation recipients of the indicated genotypes of donors. The cells were seeded into methylcellulose medium for CFU-C assay. The numbers of colonies were counted after 7 days of culturing (G). Cells were mixed with equal numbers of competitor BM cells for competitive transplantation study. The CHRC of the donor cells was analyzed 4 months after transplantation by examining the percentage of donor-derived cells (CD45.2+) in PB (H). ROS levels (I), p-p38 Mapk levels (J), and senescence (K) in LSK cells were examined by dichlorofluores cin diacetate (DCFDA), p-p38 antibody, and 5-Dodecanoylaminofluorescein Di-β-D-Galactopyranoside (C12GFDG) staining, respectively, followed by flow cytometric analysis. Data in (C), (I), (J), and (K) show one of the three biological triplicate experiments. “Iso” stands for isoform control. The expression of the indicated genes in LSK cells of the indicated genotypes was examined by qRT-PCR assay and normalized to the levels of the same gene in LSK cells isolated from non-transplanted WT mice (L). ∗p < 0.05 and ∗∗p < 0.01, compared with WT group or TP0 group. &p < 0.05, compared with Ripk3−/− group.