Fig 2: p53-Dependent suppression of glycolysis in Fanconi anemia (FA) hematopoietic stem cells (HSCs). (A): Increased p53 and TP53-induced glycolysis regulator (TIGAR) proteins in FA HSPCs. Whole-cell lysates were extracted from 30,000 Lineage-Sca-1+c-kit+ isolated from wild-type (WT), Fanca -/-, p53 -/-, or p53 -/- Fanca -/- mice followed by immunoblotting using antibodies against p53, TIGAR, or ß-actin. (B): Deletion of p53 increases F2,6BP level in Fanca -/- HSCs. SLAM cells isolated from WT, Fanca -/-, p53 -/-, or p53 -/- Fanca -/- mice were subjected to MicroAssay for F26BP. (C, D): Deletion of p53 increases glucose consumption and lactate production in FA HSCs. SLAM cells isolated from WT, Fanca -/-, p53 -/-, or p53 -/- Fanca -/- mice were subjected to glucose uptake (C) and lactate production analysis (D). Results depicted in (B)–(D) are means ± SD of three independent experiments (n = 6–9 per group); *, p < .05; **, p < .01.

Fig 3: p53-Dependent cell-cycle control is not required for TP53-induced glycolysis regulator (TIGAR)-mediated metabolic reprograming. (A): Overexpression of TIGAR reduces glucose consumption in p53 R172P hematopoietic stem cells (HSCs). Bone marrow Lineage-Sca-1+c-kit+ cells from wild-type, Fanca -/-, p53 R172P, and Fanca -/- p53 R172P mice were transduced with lentivirus expressing mCherry-TIGAR or mCherry alone. The sorted mCherry+ cells were expanded for 72 hours. The transduced cells were gated for mCherry+ SLAM population and assayed for glucose consumption. (B): Overexpression of TIGAR reduces PFK1 activity in p53 R172P HSPCs. Cells described in (A) were gated for mCherry+ population and assayed for PFK1 activity. (C): Overexpression of TIGAR reduces ROS production in p53 R172P HSPCs. Cells described in (A) were gated for mCherry+ SLAM population and analyzed for intracellular ROS by flow cytometry (n = 6 per group). (D): Overexpression of TIGAR reduces DNA damage in p53 R172P HSPCs. WCLs were extracted from mCherry+ cells described in (A) followed by Western blotting using antibodies against ?-H2AX and ß-actin. Results depicted in (A) and (B) are means ± SD of three independent experiments; *, p < .05; **, p < .01.

Fig 4: Deregulated Canonical NF-?B Activation in HSCs Leads to Pancytopenia and Progressive BM Failure(A) NF-?B target gene expressions in total BM of 14-day-old CA/CA and control mice. Representative data from two independent experiments.(B) NF-?B target gene expressions in HSCs (CD150+ LSK cells) of 14-day-old CA/CA and control mice. Representative data from two independent experiments.(C) Representative pictures of CA/CA and control mice at 2 weeks old (left) and 4 weeks old (right).(D) Kaplan-Meier survival curve analysis of CA/CA and control mice (n = 18). Significance (****p < 0.0001) was assessed using the log-rank test.(E) Complete blood count (CBC) analysis of CA/CA and control mice (n = 3–6 at each time point). Hb, hemoglobin; Plt, platelet.(F) Cell number of hematopoietic organs from CA/CA and control mice (n = 3–6 at each time point). Sp, spleen; Thy, thymus.(G) Normalized (cell number per gram body weight) cell counts of hematopoietic organs from CA/CA and control mice (n = 8 at each time point).All of the data represent means ± SEMs. Two-tailed Student’s t tests were used to assess statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig 5: Reduced quiescence and self-renewing programme in endosteal HSCs from cholinergic-neural-deficient mice.Gene sets enrichment analysis (GSEA) comparing endosteal and central lin-CD48-sca1+ckit+CD150+ HSCs of WT mice and Gfra2-/- mice (N = 4 biological replicates). a Enrichment map showing upregulated pathways in central (compared with endosteal) BM HSCs. b Downregulation of RET target genes in HSCs from Gfra2-/- mice. c Enriched Notch1-Myc target gene expression in endosteal WT HSCs. d Datasets from the Reactome, Pathway Interaction Database and Hallmark database as well as published HSC datasets were used for comparison. The normalised enrichment score (NES) of significantly enriched datasets (False discovery rate (FDR) < 0.1) is depicted as heatmap. Red, blue, and white colours indicate upregulation, downregulation, and no change, respectively.