Journal: iScience
Article Title: R274X-mutated Phf6 increased the self-renewal and skewed T cell differentiation of hematopoietic stem cells
doi: 10.1016/j.isci.2023.106817
Figure Lengend Snippet: Phf6 R274X mutation increases the proliferation of hematopoietic stem cells (A) Hematoxylin and eosin (H&E) staining of BM, spleen, thymus, and liver. (B) Wright-Giemsa staining of PB smears and BM cytospin. (C) White blood cell (WBC), red blood cell (RBC), platelet (PLT), and lymphocyte (Lym) counts in PB by routine blood tests from Phf6 fl-R274X/Y (n = 5) and Vav1-Cre;Phf6 fl-R274X/Y (n = 4). (D) FACS analysis of the percentage of T, B and myeloid cells in PB and BM from Phf6 fl-R274X/Y (n = 5) and Vav1-Cre;Phf6 fl-R274X/Y (n = 4) mice. (E) The absolute number of T, B, and myeloid cells in BM from Phf6 fl-R274X/Y (n = 5) and Vav1-Cre;Phf6 fl-R274X/Y (n = 4) mice. (F) Primary T cells from Phf6 fl-R274X/Y and Vav1-Cre;Phf6 fl-R274X/Y mice were treated with anti-CD3/CD28 antibodies for 24 h. The proliferation index of CD4 + and CD8 + cells were measured by CFSE-staining analysis (n = 4). (G and H) Primary T cells from Phf6 fl-R274X/Y and Vav1-Cre;Phf6 fl-R274X/Y mice were treated with anti-CD3/CD28 antibodies and analyzed by flow cytometry (n = 4). (G) Stimulated for 12h and expression of CD69 on both CD4 + and CD8 + T cells. (H) Stimulated for 72 h and expression of CD25 on both CD4 + and CD8 + T cells. (I and J) After primary T cells were treated with anti-CD3/CD28 antibodies for 24 h, cells were stimulated with the indicated concentrations of PMA, ionomycin, and Golgiplug for 6h. Flow cytometric analysis of IFN- γ and TNF-α secretion by (I) CD4 + cell; (J) CD8 + cell (n = 3). (K) Primary T cells from Phf6 fl-R274X/Y and Vav1-Cre;Phf6 fl-R274X/Y mice-mediated killing assay in primary spleen cells from MAL-AF9 mice, measured by Annexin V staining (n = 3). (L–O) The absolute number of LSK (Lin – Sca1 + c-Kit + ) cells, LT-HSC (Lin – Sca1 + c-Kit + CD34 – Flt3 low ), ST-HSC (Lin – Sca1 + c-Kit + CD34 + Flt3 low ), MPP (Lin – Sca1 + c-Kit + CD34 + Flt3 + ), MPP1 (Lin – c-Kit + Sca1 – CD135 − CD150 − CD48 − ), MPP2 (Lin – c-Kit + Sca1 – CD135 − CD150 + CD48 + ), MPP3 (Lin – c-Kit + Sca1 – CD135 − CD150 − CD48 + ), MPP4 (Lin – c-Kit + Sca1 – CD135 + CD150 − CD48 + ), LK (Lin – Sca1 − c-Kit + ) cells, CMP (Lin – c-Kit + Sca1 – CD34 + CD16/32 low ), GMP (Lin – c-Kit + Sca1 – CD34 + CD16/32 high ) and MEP (Lin – c-Kit + Sca1 – CD34 – CD16/32 low ) populations, and CLP (Lin – IL-7r + Sca1 + c-Kit + ) in BM from Phf6 fl-R274X/Y (n = 5) and Vav1-Cre;Phf6 fl-R274X/Y (n = 4) mice. Data information: All mice used here were male mice of 8–10 weeks of age. In (C–O) data are shown as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 by the Student’s t test.
Article Snippet: Anti-mouse CD150 (PE-Cy7) , eBioscience , Cat#: 25-1502-82; RRID: AB_10805742.
Techniques: Mutagenesis, Staining, Flow Cytometry, Expressing
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