DEK overexpression impairs cell cycle progression of HSCs and inhibits hematopoiesis in mice. (A) The violin plots show the DEK expression in HSCs cluster under the indicated cell cycle phases using published scRNA-seq data (GSE81682). (B) Representative volcano plot from RNA-seq analysis between Dek^Con and Dek^Tg HSCs (n = 4 samples per group). All genes that were significantly changed (greater than twofold change, P < 0.05) appear as red dots. (C) qRT-PCR analysis and the bar graph shows the expression of the DEK, Fos, Ifit2, and Cdk1 in HSCs from Den^Con and Dek^Tg mice (n = 4 mice per group). (D) Representative flow plots of cell cycle analysis of HSCs (CD48⁻CD150⁺LSK) by Ki-67 + DAPI staining. The right bar graph shows the percentage of the cell cycle distribution (n = 5 mice per group). (E) Representative flow plots of BrdU⁺ cell analysis of HSCs (CD48⁻CD150⁺LSK). The bar graph on the right shows the percentage of the BrdU⁺ cells in HSCs (n = 4 mice per group). (F) The representative flow plots show the EdU⁺ cells in HSCs under indicated conditions. The bar graph shows the percentage of the EdU⁺ cells in HSCs (n = 4–5 samples per group). HSCs (CD48⁻CD150⁺LSK) from Dek^Conor Dek^Tg mice were sorted and cultured with DMSO or HU (100 µM) for 24 h, followed by EdU incorporation assay. (G) Approximately 300 freshly sorted HSCs (CD48⁻CD150⁺LSK) from Dek^Con or Dek^Tg mice were cultured in M3434 medium with DMSO or HU (50 µM) for 10–12 days. The bar graph shows the colony number (n = 5 samples per group). (H) Blood cell counts of PB from Dek^Con and Dek^Tg mice at 2 mo of age (n = 6 mice per group). WBC, white blood cells; NE, neutrophils; LY, lymphocytes; MO, monocytes; EO, eosinophils; BA, basophils; PLT, platelets; and Hb, hemoglobin. (I) Representative image shows the spleen and thymus from Dek^Con and Dek^Tg mice. Scale bar: 1.0 cm (top) and 0.5 cm (bottom). The bar graphs show the weight (mg) of the spleen (left) and thymus (right) (n = 5 mice per group). (J) The bar graph shows the BM cellularity of Dek^Con and Dek^Tg mice (n = 12 mice per group). (K) The bar graph shows the number of megakaryocyte colonies (GEMM), granulocyte-macrophage colonies (GM), granulocyte colonies (G), macrophage colonies (M), and burst-forming unit-erythroid colonies (BFU-E) (n = 5 samples per group). A total of 6 × 10⁴ BM cells from Dek^Con or Dek^Tg mice were cultured in the M3434 medium for 10–12 days. (L) Flow cytometry analysis and the bar graphs show the percentage of myeloid cells (Gr-1⁺CD11b⁺) in BM (red blood cell removed) (n = 6 mice per group). (M and N) Flow cytometry analysis and the bar graphs show the percentage of immature B cells (B220⁺IgM⁻) (M) and mature B (B220⁺IgM⁺) cells (N) in BM and spleen (n = 6 mice per group). (O–Q) Flow cytometry analysis and the bar graphs show the percentage of red cells (R2:CD71^HighTer119⁺, R3: CD71^MedTer119⁺, R4: CD71⁻Ter119⁺) in BM and spleen cells (n = 6 mice per group). (R–T) Flow cytometry analysis and the bar graphs show the percentage of T cells (CD4⁺T, CD8⁺T, CD4⁺CD8⁺T) in the spleen and thymus (n = 6 mice per group). The data presented in panels C–T are representative of two independent experiments. The results shown in panel A are illustrated as violin plots, and other panels are shown as mean ± SD. Statistical analyses were performed using the Wilcoxon test for panel A and a two-tailed unpaired Student’s t test for panels C–T. P values are presented as follows: **P < 0.01, ***P < 0.001., n.s., not significant.