Fig 2: p53-TP53-induced glycolysis regulator (TIGAR)-mediated glycolytic suppression attenuates reactive oxygen species (ROS), DNA damage, and chromosomal instability in Fanconi anemia (FA) HSPCs. (A): Overactivation of p53 in FA HSPCs. Whole cell lysates (WCLs) were extracted from Lineage-Sca-1+c-kit+ (LSK) cells isolated from wild-type (WT), Fanca -/-, p53 -/-, or p53 -/- Fanca -/- mice followed by Western blotting using antibodies against total p53, p53ser18, and ß-actin. (B): Lentiviral transduction and FACS sorting. LSK cells isolated from WT, Fanca -/-, p53 -/-, or p53 -/- Fanca -/- mice were transduced with lentiviral vector expressing mCherry or mCherry-TIGAR. Flow plots after FACS sorting for mCherry are shown. The sorted mCherry+ cells were then subjected to ex vivo expansion for 72 hours followed by the analyses described below. (C): Expression of TIGAR in transduced HSCs. RNA was extracted from mCherry+ SLAM cells described in (B) were subjected to quantitative polymerase chain reaction analysis for TIGAR expression. Samples were normalized to the level of GAPDH mRNA. (D): Overexpression of TIGAR reduces ROS level in HSCs. Cells described in (B) were gated for mCherry+ SLAM population and subjected to flow cytometry analysis for ROS. Representative plots (left) and quantification (right) are shown. (E): Overexpression of TIGAR reduces DNA damage in FA HSPCs. WCLs were extracted from mCherry+ cells described in (B) followed by Western blotting using antibodies against ?-H2AX and ß-actin. (F): Overexpression of TIGAR reduces spontaneous chromosomal breakage in FA HSPCs. mCherry+ cells described in (B) were subjected to chromosomal breakage assay. Representative images (left) and quantification of 50 cells in random fields (right) are shown. Arrowheads point to chromosome breaks or radial structures. (G, H): Overexpression of TIGAR enhances the pentose phosphate pathway and cellular antioxidant function in FA HSCs. Cells described in (B) were gated for mCherry+ population and subjected to assays for NADPH/NADP+ ratio (F) or intracellular GSH analysis. Results depicted in (C)–(H) are means ± SD of three independent experiments; *, p < .05; **, p < .01; ***, p < .001.

Fig 3: Regnase-1 deficiency leads to expansion of immature HSPCs. a Representative flow cytometric analysis of HSPC populations in the FL of E15.5 embryo, BM of 1-day-old neonate, BM of 2-week-old control (fl/fl) or Regnase-1-KO (?/?) mice (n = 3 per group). b The numbers of LSK-HSCs or CD34- HSCs in the total FL or BM cells assessed by flow cytometry. c Representative flow cytometric analysis of LSK cells for CD48 and CD150 expression of 8-week-old control (fl/fl) or Regnase-1-KO (?/?) (n = 3 per group). d Percentage of CD150+ CD48- cells, CD150+ CD48+ cells, and CD150- CD48+ cells of control (fl/fl) and Regnase-1-KO (?/?) mice. e Colony-forming ability of LSK-HSCs and CD34- HSC from control (fl/fl) or Regnase-1-KO (?/?) mice BM. G CFU-granulocyte; GM CFU-granulocyte/monocyte; M CFU-monocyte; E CFU-erythroid; and mix mixed CFU-granulocytes, monocyte, erythroid, and megakaryocyte. Data show the means ± SD (n = 5; 3 independent experiments). f Competitive repopulation assay of lethally irradiated (10 Gy) WT recipients (CD45.1) transplanted with BM of either control (fl/fl) or Regnase-1-KO (?/?) mice. Chimerism was analyzed in recipient mice 16 weeks after injection of 1 × 103 of CD34- HSCs from control or Regnase-1-KO mice (CD45.2) with 5 × 105 competitor BM cells (CD45.1). Donor-derived cell ratios (CD45.1-) in BM HSPC and CD34- HSC populations were determined by flow cytometry (n = 5 per group). g The number of LSK or CD34- LSK cells in the BM of mice 16 weeks after transplantation. h Hematopoietic engraftment of transplanted HSPCs for reconstitution of peripheral blood was analyzed by flow cytometry at 16 weeks after transplantation (n = 5 per group). i The contribution of transplanted donor-derived cells (CD45.1-) to B-cells (B220+), T-cells (CD3+), and myeloid cells (CD11b+) from control (fl/fl) or Regnase-1-KO (?/?) HSPCs are shown. Data summated from 3 independent experiments and an average of 6 recipient mice in each group with SD. j Gene ontology (GO) analysis of genes upregulated in the CD34-HSCs from Regnase-1-KO BM based on the GO biological process annotations. Error bars indicate mean ± SD. *p < 0.05; **p < 0.01, ***p < 0.005, two-sided t-test in b, d, g, i

Fig 4: Cholinergic-deficient niche compromises HSC quiescence, leading to increased reconstitution of primary recipients.a Scheme illustrating competitive HSC transplantation in primary recipients. 500 endosteal LSK CD48- CD150+ HSCs and 105 BM helper cells were i.v. injected into lethally irradiated congenic mice of indicated genotype. b–g Donor-derived haematopoietic chimerism (b), B cells (c), T cells (d), eosinophils (e), neutrophils (f) and monocytes (g) at specified time following primary transplantation of HSCs from donors and recipients of specified genotypes (Gfra2+/- > CD45.1, N = 5; Gfra2-/- > CD45.1, N = 6; CD45.1 > Gfra2-/-, N = 4; CD45.1 > Gfra2+/+, N = 4). Data are mean of biological replicates ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. ANOVA and Tukey’s multiple comparisons test.

Fig 5: Cholinergic signals promote HSC quiescence in endosteal BM niches.a Ratio of quiescent HSCs (G0:G1 phases of the cell cycle) in the endosteal or central BM of Gfra2-/- or WT mice (N = 6; P = 0.0006). b Representative flow cytometry diagrams showing ki67/Hoechst 33342 of lin- sca1+ ckit+ (LSK) CD48- CD150+ HSCs isolated from the endosteal or central BM of Gfra2-/- mice or control Gfra2+/- mice (N = 6). The frequencies of the gated populations are indicated. c Ratio of quiescent HSCs in the endosteal or central BM of Gfra2-/-, Adrb3-/-, compound Gfra2-/;Adrb3-/- or WT mice (N = 3; P = 0.006). a, c Each dot is a mouse. Data are mean of biological replicates ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. ANOVA and Bonferroni’s multiple comparisons test.