Fig 2: Zkscan3 is essential for erythropoiesis. A Immunofluorescence staining of CD71 (Green) and Ter119 (Red) in spleen sections from Zkscan3−/− and wild-type mice (n = 4). B–C Imaging flow cytometry analysis for CD71 and Ter119 expression in erythroblasts present in spleens and bone marrows of Zkscan3 knockout and wild mice (n = 6, replicates per group, non-parametric statistical and Student’s t tests). (***p < 0.001, **p < 0.01, *p < 0.05)

Fig 3: EC confers erythroblast survival. (A) Representative flow plots (left) and quantitation (right) of AnnexinV-DRAQ7-based flow cytometric apoptosis analysis in CD71intTer119− erythroid precursor R1 cells (n = 7). (B) Representative flow plots (left) and quantitation of AnnexinV-DRAQ7-based flow cytometric apoptosis analysis in CD71+Ter119− proerythroblast R2 cells (n = 7). (C) Representative flow plots (left) and quantitation of AnnexinV-DRAQ7-based flow cytometric apoptosis analysis with CD71+Ter119+ late basophilic and orthochromatic R3 erythroblasts (n = 7). (D) Cell surface c-Kit levels were quantitated in erythroid precursors using flow cytometry (n = 6). (E) Correlation plot between percent of GFP+ cells in late apoptosis vs. surface c-Kit levels created using linear regression (Graphpad Prism software). For the box-and-whisker plots, the box depicts the 25–75th percentiles of data, the line represents median, and whiskers ranging from minimum to maximum values. Statistically significant differences are indicated *P < 0.05, **P < 0.01, ***P < 0.001 using two-tailed unpaired Student's t-test. Values were compared to shLuc unless indicated.

Fig 4: Identical leukemogenic system generates histiocytic sarcoma in the hind limb of NOD.SCID mice when injected into the gastrocnemius muscle.(A) Kaplan-Meier curve showing the fraction tumor-free mice at the indicated time after transplant of p16p19−/−; Kras(G12V)-GFP with or without cardiotoxin (CTX) pre-injury, or of p16p19−/− ;Ctrl or WT; Kras(G12V) cells, with CTX pre-injury, into the muscles of NOD.SCID mice. (B, C) Body weight and spleen weight analyses at the time of sacrifice for NOD.SCID mice receiving p16p19−/−; Kras(G12V) or p16p19−/− ;Ctrl intra-muscular transplantation. Mice were sacrificed approximately 2 weeks after initial tumor detection. (D) Flow cytometry analysis of a representative tumor sample from a NOD.SCID mouse bearing an p16p19−/−; Kras(G12V)-GFP muscle tumor, revealing that most GFP+ tumor cells (gating shown on leftmost plot) are CD48hi, CD47hi, Mac1hi, but Gr1− /lo, B220− /lo, CD4− /lo, CD8− /lo, CD71− /lo and Ter119− /lo.

Fig 5: Vav‐Cre deletion of Brd4 blocks HSC development A schematic representation of targeted deletion of mouse Brd4. The exon–intron organization of the Brd4 locus (top). Targeting vector contained exon 3 flanked by LoxP sites (green) and a neomycin resistance gene flanked by FRT (white). Cre‐mediated recombination should cause deletion of exon 3 resulting in loss of BRD4 expression (bottom).Genotyping of Brd4 fl/fl mice with ERT2‐Cre (Cre) or without ERT2 ‐Cre (+/+). Tamoxifen treatment deleted 2.2 kb Brd4 fl/fl fragment from Cre/+ and Cre/Cre mice (top). Cre/+, Cre/Cre and +/+ mice were confirmed by the presence of ERT2 ‐Cre band (Cre) or its absence (WT) (bottom).WT and Brd4 KO cells were tested for mRNA (left) and the protein (right) by qRT–PCR and immunoblot assays. Values represent the average of three experiments ± SD.Vav‐Cre‐mediated Brd4 depletion was verified for cells from Brd4 fl/fl Vav‐Cre mice (fetal liver or spleen) by qRT–PCR and immunoblot (left and right). Values represent the average of three experiments ± SD.Photographic images of E14.5 embryos from WT and KO mice.Corresponding flow cytometric profile of Lin− fetal liver cells stained with cKit and Sca‐1 antibodies examining early hematopoietic progenitor population (LSK) (left). LSK populations were further divided by the Flt3 marker to detect HSCs and MPPs (middle). The number of total cells, e.g., LSKs, HSCs, MPPs, in a fetal liver was estimated on indicated days of embryonic development (E13.5, E14.5, and E17.5). Values are the average of 3–5 embryos ± SD (right).A schematic of hematopoietic stem cell and early progenitor cell pathways.Representative flow cytometric profile of CD71− cells stained for the myeloid and lymphoid markers. Values are the average of 3 E17.5 embryos ± SD.Data information: Statistical significance in all figures was determined by two‐tailed unpaired t‐test and is indicated by: *P < 0.05, **P < 0.01, ***P < 0.001, NS: no significance.