HSPCs in stressed BM receive strong GM-CSF signals. (A–C) LSK cells, which were sorted from CAG-EGFP mice, were transplanted into steady-state control (Ctrl) or 5-FU–treated WT mice. Then, EGFP⁺ cells in the recipient BM were sorted from two groups, and RNA-seq analyses were conducted. (A) Schematic of the procedure. (B) Flow cytometry plots of BMMNCs of untreated (control) or 5-FU–treated recipient WT mice that were transplanted with LSK cells from CAG-EGFP mice. Results shown are representative of two independent experiments. (C) Heat map of differentially expressed genes in RNA-seq analyses of HSPCs transplanted and homed into recipient BM (5-FU versus control); n = 2 per group. (D–F) Phenotypical changes of WT mice through a single 5-FU (200 mg/kg) injection. Each experiment was performed on day 0 (control) and day 2. (D) qRT-PCR analyses for the expression of indicated genes of total BM cells and ECs; n = 3, representative of two independent experiments. (E) The numbers of total BM cells in femurs and tibias; n = 3, representative of two independent experiments. (F) GM-CSF protein levels in femur analyzed by ELISA; n = 3, representative of two independent experiments. (G) qRT-PCR analyses for the expression of Csf2ra and Csf2rb genes in Lin⁻ cells, CAR cells, and ECs that were collected from BM of WT mice 2 d following 200 mg/kg 5-FU injection; n = 3 or 4, representative of two independent experiments. In the bar charts, the results are shown as mean ± SEM, and each dot represents an individual mouse. Statistical significance was determined by unpaired Student’s t test. ND, not detectable.