Fig. 5: ADP-heptose induces pre-leukaemic cell proliferation and self-renewal through ALPK1-mediated NF-κB–UBE2N activation.
From: Microbial metabolite drives ageing-related clonal haematopoiesis via ALPK1
a, The proportion of EdU-positive HSCs (Lin−KIT+SCA1+CD150+CD48−) within the BM of mice treated with EdU in vivo (after 2 weeks). n = 5 independent mice per group. b, The experimental design to assess the effect of ADP-heptose on HSC competition in vitro. c, The number of HSCs in H2O- or ADP-heptose-treated wells at day 0 and day 14 after treatment. n = 7 biological replicates per group from three independent experiments. d, Representative flow cytometry profile of HSCs (Lin−KIT+SCA1+CD150+) after 14 days. e, Serial colony replating of ADP-heptose- or H2O-treated BM HSPCs (Lin−SCA1+KIT+). Colonies were scored at day 14. n = 3 biological replicates per group. f, Overview of the screen using inhibitors targeting inflammatory-specific effectors in an NF-kB reporter cell line. g, THP1 NF-κB reporter cells were stimulated with either ADP-heptose or IL-1β in the presence of the indicated inhibitors for 24 h. n = 3 independent biological replicates. h, TIFA-TdT THP1 cells were treated with ADP-heptose (100 µg ml−1) in the presence of the indicated inhibitors for 24 h. n = 3 independent biological replicates. i, Prioritized hits in the NF-κB and TIFAsome assays. j, Colony-formation analysis of wild-type and knockout Dnmt3a HSPCs (Lin−KIT+SCA1+) after treatment with vehicle, ADP-heptose, or ADP-heptose and UBE2N inhibitor. n = 4 biological replicates per group. k, Colony-formation analysis of BM CD34+ cells from healthy individuals (normal; n = 2 technical replicates) and two individuals with MDS (n = 2 biological replicates) treated with vehicle, ADP-heptose, or ADP-heptose and UBE2N inhibitor. Data are mean ± s.e.m. The hash symbols (#) indicate greater than 50% inhibition. P values were calculated for the indicated comparisons using two-tailed unpaired Student’s t-tests (a) and two-way ANOVA (c,e,j,k). FACS-gating schemes are shown in Supplementary Fig. 2. The diagrams in b and f were created using BioRender.
