Fig. 4: Purine biosynthesis is required for LSC maintenance in vivo.

a Schematic flowchart depicting colony-forming and transplantation assays in LSCs treated with or without MMF. b Representative images of colonies from 6-well plates and individual colonies derived from control and MMF (0.125-0.25 μM)-treated LSCs. Scale bar, 200μm. c Number of colonies derived from LSCs after exposure to MMF treatment (0.125-0.25 μM) during serial plating (n = 3). d–f Cell number (d), flow cytometry histograms (e), and MFI of myeloid differentiation marker CD11b (f) from the LSC-derived colonies treated with or without MMF (0.125-0.25 μM) after the first plating (n = 3, biologically independent samples). g Kaplan-Meier analysis of survival of recipient mice transplanted with different numbers of LSCs exposed to MMF (0.125 μM) for 12 h ex vivo (n = 5). h Frequency of GFP⁺ AML cells in the peripheral blood (PB) of recipient mice receiving 10⁴ LSCs after exposure to MMF (0.125 μM) at 16 days post-transplantation (n = 5). i, l Kaplan-Meier analysis of survival of mice transplanted with MLL-AF9-induced AML and treated with either vehicle or MMF (100 mg/kg/day) at days 1 to 14 post-transplantation (i, n = 7) or days 15 to 28 post-transplantation (l, n = 8). j, k, m, n Frequency of GFP⁺ AML cells (j, m) and MFI of CD11b (k, n) in the PB of AML recipient mice treated with either vehicle or MMF as in (i) (j, k, n = 7) or as in (l) (m, n, n = 8). o, p Kaplan-Meier analysis of survival (o) and estimated frequency of LSCs (p) of MLL-AF9-induced AML mice that were treated with MMF as in (i) (n = 5). q, r Frequency of GFP⁺ AML cells in the PB (q) and survival analysis (r) of recipient mice transplanted with MLL-AF9⁺ Mx1-Cre; Impdh2^fl/fl AML cells and treated with poly(I:C) at days 5 or 15 after transplantation. s, t Kaplan-Meier analysis of survival (s) and frequency of GFP⁺ AML cells in PB (t) of secondary recipient mice transplanted with AML cells from moribund mice in (r). u, v Representative flow cytometry dot plots (u) and frequencies of Lin^- and HSPCs (v) in the bone marrow of Impdh2 WT (n = 6) and KO (n = 7) mice. All data are represented as mean ± SD. p values in this figure were calculated by unpaired, two-tailed Student’s t-test (h, j, k, m, n, t and v), ANOVA with multiple comparisons analysis using Dunnett’s post hoc analyses (c, d, and f), or log-rank test (g, i, l, o, r and s). See also Supplementary Fig. 4. Source data are provided as a Source Data file.