Machine learning-based prediction reveals kinase MAP4K4 regulates neutrophil differentiation through phosphorylating apoptosis-related proteins
Fig 3
Loss of MAP4K4 expression causes a decrease in the number of neutrophils.
(A) Scheme of all-trans-retinoic acid (ATRA)-induced neutrophil differentiation of the hematopoietic HL-60 cell models (left); flow cytometry analysis of the neutrophil marker CD11b has validated that ATAR can induce differentiation of HL-60 cells into neutrophils. This is demonstrated by histogram plots showing variations in mean fluorescence intensity (MFI) across different conditions: cells untreated with ATAR, cells treated with ATAR for 72 hours, and cells treated with ATAR for 96 hours (right). (B) Percentage of CD11b+ cells in HL-60 cells were treated with 0-20 µM MAP4K4 inhibitor (PF-06260933, PF) for 48 hours, followed by a subsequent 96-hour treatment with both PF and all-trans-retinoic acid (ATRA). (C) The number of granulocyte-macrophage progenitor cells (CFU-GM, CFU-G, CFU-M) colonies formed by 25,000 whole bone marrow cells from control (Ctrl) or Map4k4-cKO (cKO) mice; BM, bone marrow (n=4; mean ± SD). (D) Numbers of neutrophils in the bone marrow of control (Ctrl) or Map4k4-cKO (cKO) mice; BM, bone marrow; Neu, neutrophil. (Ctrl n=7, cKO n=6; mean ± SD). (E) PB WBC numbers of control (Ctrl) or Map4k4-cKO (cKO) mice (Ctrl n=7, cKO n=6; mean ± SD). PB, peripheral blood; WBC, white blood cell; (Ctrl n=7, cKO n=6; mean ± SD). (F) PB Mon, and GR numbers of control (Ctrl) or Map4k4-cKO (cKO) mice; PB, peripheral blood; Mon, monocyte; GR, granulocyte; (Ctrl n=7, cKO n=6; mean ± SD). (G) Numbers of eosinophils in PB of control (Ctrl) or Map4k4-cKO (cKO) mice; PB, peripheral blood; Eso, eosinophils; (Ctrl n=7, cKO n=6; mean ± SD). (H) Numbers of monocytes in PB of control (Ctrl) or Map4k4-cKO (cKO) mice; PB, peripheral blood; Mon, monocytes; (Ctrl n=7, cKO n=6; mean ± SD). (I) Numbers of neutrophils in PB of control (Ctrl) or Map4k4-cKO (cKO) mice; PB, peripheral blood; Neu, neutrophil; (Ctrl n=7, cKO n=6; mean ± SD). (J) Spleen cell numbers control (Ctrl) or Map4k4-cKO (cKO) mice (Ctrl n=7, cKO n=6; mean ± SD). (K) Numbers of eosinophils in the spleen of control (Ctrl) or Map4k4-cKO (cKO) mice; SP, spleen; Eso, eosinophils; (Ctrl n=7, cKO n=6; mean ± SD). (L) Numbers of monocytes in the spleen of control (Ctrl) or Map4k4-cKO (cKO) mice; SP, spleen; Mon, monocytes; (Ctrl n=7, cKO n=6; mean ± SD). (M) Representative FACS analysis of spleen CD11b+ Ly6G+ neutrophils (left); percentage of neutrophils in the spleen of control (Ctrl) or Map4k4-cKO (cKO) mice (right); SP, spleen; Neu, neutrophil; (Ctrl n=7, cKO n=6; mean ± SD). (N) Numbers of neutrophils in the spleen of control (Ctrl) or Map4k4-cKO (cKO) mice; SP, spleen; Neu, neutrophil; (Ctrl n=7, cKO n=6; mean ± SD). Mann-Whitney U test.