Transcriptomic analysis of Runx1^KTAMK/KTAMKmice shows that Runx1^KTAMK/KTAMKLT-HSCs exhibit progenitor cell gene priming. (A) A volcano plot showing the difference of mRNA expression between Runx1^+/+ and Runx1^KTAMK/KTAMK LT-HSCs. Each dot represents one gene. Genes with |log₂ fold change| > 1 and false discovery rate (FDR) < 0.05 are shown in red (n = 3 mice, 8 to 10 weeks old). (B) GSEA plots comparing Runx1^+/+ and Runx1^KTAMK/KTAMK LT-HSCs using published data of control and Runx1^flox/flox; Vav1-Cre Flt3^– LSK cells. Each solid bar represents one gene within the gene set. (C) GSEA plots comparing Runx1^+/+ and Runx1^KTAMK/KTAMK LT-HSCs using published data of HSC and MPP1 signatures. (D) mRNA expression of HSC-related genes in Runx1^+/+ (blue) and Runx1^KTAMK/KTAMK (red) LT-HSCs as detected by RNA-sequencing (left) and quantitative polymerase chain reaction (qPCR) (right) (n = 3 mice). (E) Surface ALCAM expression detected by flow cytometry. The upper panels show representative flow cytometry plots of Runx1^+/+ (blue) and Runx1^KTAMK/KTAMK (red) CD34^–Flt3^– LSK cells (left) and Flt3^– LSK cells (right). The mean fluorescence intensity of Runx1^+/+ (blue) and Runx1^KTAMK/KTAMK (red) cells is shown in the lower graph (n = 4 mice, 8 to 10 weeks old). (F) ALDH activity detected by Aldefluor. The left panel shows a representative flow cytometry plot of Runx1^+/+ (blue) and Runx1^KTAMK/KTAMK (red) CD34^–Flt3^– LSK cells. The fluorescence minus one control is shown in gray. The percentages of Aldefluor^high cells in Runx1^+/+ (blue) and Runx1^KTAMK/KTAMK (red) LT-HSCs are shown in the right graph (n = 3 mice; 7-9 weeks old). NES, normalized enrichment score.