Figure 2. P2X7 is highly expressed in LICs and promotes AML development.

(A and B) mRNA levels of P2x1–P2x7 were determined in the immunophenotypic Mac-1⁺c-Kit⁺ LICs (A) or Lin^–Sca-1^–c-Kit⁺CD34⁺CD16/32^– L-GMPs (B) by quantitative RT-PCR (n = 3). (C) mRNA levels of P2Xs were analyzed in cells of AML patients from TCGA database (n = 179). (D) mRNA levels of P2x7 in normal mouse bone marrow cells (Normal-BM), Lin^–Sca-1⁺c-Kit⁺CD34^–Flk2^– long-term HSCs (LT-HSCs), Lin^–Sca-1⁺c-Kit⁺CD34⁺Flk2⁺ short-term HSCs (ST-HSCs), YFP⁺ leukemia cells (AML-BM), Mac-1⁺c-Kit⁺ LICs, and Lin^–Sca-1^–c-Kit⁺CD34⁺CD16/32^– L-GMPs were measured by quantitative RT-PCR (n = 3). (E) P2X7-mediated influx of ions in murine Mac-1⁺c-Kit⁺ LICs cells was measured upon sequential treatments with extracellular ATP and P2X7 antagonist A-740003 by whole-cell patch-clamp recording (n = 3). (F) Nucleotide base sequences deleted in the P2x7-knockout (P2x7-KO or P2x7-null) mouse using the CRISPR-Cas9 strategy. (G) mRNA level of P2x7 in WT and P2x7-KO BM cells was measured by quantitative RT-PCR (n = 3). ***P < 0.001 by Student’s t test. (H) P2X7 protein levels in WT and P2x7-KO BM cells were measured by Western blot. (I) Representative flow cytometric analysis of leukemia cells (YFP⁺) in the peripheral blood upon primary transplantation. (J) Quantification of data in panel I (n = 5). PB, peripheral blood. **P < 0.01 by Student’s t test. (K) The overall survival of the recipient mice transplanted with WT or P2x7-KO MLL-AF9⁺ BM cells upon primary transplantation (n = 5). ***P < 0.001 by log-rank test. (L) Representative images of the size of spleens and livers of recipients upon primary transplantation. (M) Quantification of data in panel L (n = 4). *P < 0.05, **P < 0.01 by 2-way ANOVA with Sidak’s multiple-comparison test. (N) The overall survival of recipient mice transplanted with WT or P2x7-KO MLL-AF9⁺ BM cells upon secondary transplantation (n = 5). ***P < 0.001 by log-rank test. (O) Limiting dilution assays for the frequency of the functional LICs of WT and P2x7-KO BM cells. Experiments were repeated independently 3 times.