Fig. 5: P2rx7 as a putative novel p53-target gene in HSPC response to irradiation.

A Normalized counts per million (CPM) expression of P2rx7 gene in WT and p53KO HSPCs with and without whole-body irradiation. B Genomic snapshots of P2rx7. ChIP-Seq tracks of p53 in untreated (UT) and irradiated (IR) hematopoietic progenitor cells Ba/F3 and HPC7, and input DNA from the same cells are shown on the top five lanes. The gene feature track is shown in the middle. RNA-Seq tracks of the summation of expression in each group are shown in the bottom four lanes. The maximum data range of each track is indicated at the top-right corner of the track. Red box highlights the p53 consensus binding motif identified at the promoter of the P2rx7 gene. C Enrichment of p53 at the promoter of P2rx7 gene, validated with ChIP-qPCR in Ba/F3 hematopoietic progenitor cells. p53-regulated genes Bbc3/PUMA and Cdkn1a/p21 served as positive controls. Data presented are from one experiment and was reproduced in three independent experiments. D, E P2X7 expression on HSCs and hematopoietic progenitor cells of WT and p53KO mice, with and without whole-body irradiation (3 Gy, 6 h), analyzed by flow cytometry; mean fluorescence intensity of the P2X7 staining is plotted for each cell population. Data are from 3 to 4 mice per genotype per condition, consolidated from 2 to 3 independent experiments; bars represent means and standard errors on the mean (SEM); statistical analysis by ANOVA followed by Sidak’s multiple comparison post hoc test to compare WT versus WT-IR and p53KO versus p53KO-IR datasets; *p < 0.05, **p < 0.01, or ns not significant. Cells are gated as: D Lin⁻cKit⁺Sca1⁺ followed by CD150⁺CD48⁻CD34⁻Flt3⁻ for long-term HSCs (LT-HSCs), CD150⁺CD48⁻CD34⁺Flt3⁻ for MPP1, CD150⁺CD48⁺CD34⁺Flt3⁻ for MPP2, CD150⁻CD48⁺CD34⁺Flt3⁻ for MPP3, and CD150⁻CD48⁺CD34⁺Flt3⁺ for MPP4; E Lin⁻cKit⁺Sca1⁻ followed by CD41⁻CD16/32⁻CD150⁺CD105⁻ for pre-megakaryocyte erythroid progenitors (pre-MkE), CD34⁻CD16/32⁻ megakaryocyte erythroid progenitors (MEP), CD41⁻CD16/32⁻CD150⁺CD105⁺ for pre-CFU-E erythroid progenitors, and CD41⁻CD16/32⁻CD150⁻CD105⁺ for CFU-E erythroid progenitors; or as Lin⁻CD71⁺ for the more mature erythroid precursor cells.