Fig. 2

Enforced neuropathy worsens pre-leukemic phenotype in NRAS-G12D⁺ primary mutant mice. (A) In vivo intraperitoneal (i.p.) administration with 6-hydroxydopamine (6-OHDA) to induce chemical sympathectomy or vehicle in NRAS-G12D⁺ (Mx1-Cre NRAS^G12D) female mice aged 25–49 weeks for a total of 16 weeks, starting 14 weeks after polyinosine-polycytosine (pI-pC) induction. (B) Fraction of CD11b⁺Gr-1⁺f4/80⁺ monocytes and B220⁺ B lymphocytes in total white blood cells (WBC) analyzed by fluorescence-activated cell sorting (FACS), after 12 weeks of treatment (n = 5–6 per group). (C) Fraction of CD11b⁺ myeloid cells, monocytes and B lymphocytes in total spleen cells (TSP), analyzed by FACS, after 16 weeks of treatment (n = 3–6). (D) Fraction of CD11b⁺ myeloid cells, monocytes and B lymphocytes in bone marrow nucleated cells (BMNC) analyzed by FACS, after 16 weeks of treatment (n = 3–6). (E) Total BM (TBM) number of Lin⁻c-Kit⁺Sca-1⁺ (LSK) cell subsets: LSK CD34⁻Flt3⁻CD48⁻CD150⁺, hematopoietic stem cells (HSC); LSK CD34⁺Flt3⁻CD48⁻CD150⁺, multipotent progenitors 1 (MPP1); LSK CD34⁺Flt3⁻CD48⁺CD150⁺ (MPP2); LSK CD34⁺Flt3⁻CD48⁺CD150⁻ (MPP3); LSK CD34⁺Flt3⁺CD48⁺CD150⁻ (MPP4); LSK CD34⁺Flt3⁻CD48⁻CD150⁻ (MPP5); LSK CD34⁻Flt3⁻CD48⁻CD150⁻ (MPP6) analyzed by FACS, after 16 weeks of treatment (n = 3–6). (F) Gene expression analysis of Cebpa, Mki67, Bcl2 and p53 relative to Gapdh by digital droplet PCR after reverse transcription of total mRNA extracted from FACS-sorted LSK, after 16 weeks of treatment (n = 3–6). (G) TBM number of CD45⁻CD31⁻Ter-119⁻CD63⁺ mesenchymal stromal cells analyzed by FACS, after 16 weeks of treatment (n = 3–5). (H) Representative immunostaining of endomucin (red) to visualize sinusoids and arterioles in BM, 16 weeks after treatment; scale bar, 500 µm. Magnification is shown for better appreciation of blood vessels; scale bar, 50 µm (n = 2–4). *, sinusoids; arrows, arterioles. *p < 0.05, **p < 0.01 unpaired two-tailed t test