Figure 2.
Knocking out Atg5 in the eosinophil lineage results in reduced severity of FIP1L1-PDGFRα–mediated experimental eosinophilic leukemia. (A) Cell counts. Total leukocyte counts were measured in the peripheral blood of CtrlII5tg-F/P and Atg5eoΔII5tg-F/P recipient mice using the scil Vet abc Plus+ hematology analyzer (scil animal care company GmbH, Viernheim, Germany) on day 33 after transplantation. Relative numbers of neutrophils (CD45+/CD11b+/Ly6G+), B cells (CD45+/B220+), T cells (CD45+/CD3+), monocytes (CD45+/CD11b+/CD115+), and eosinophils (CD45+/CD11b+/Siglec-F+) were determined by flow cytometry, and an absolute count for each cell population was calculated (n = 3-4). (B) Light microscopy. Peripheral blood smears of CtrlII5tg-F/P and Atg5eoΔII5tg-F/P recipient mice on day 33 after transplantation were stained with the Hemacolor Rapid Staining Kit (MilliporeSigma, Burlington, MA), followed by light microscopy analysis. Scale bars, 10 µm. (C) Flow cytometry. Relative numbers of cell populations transduced with the F/P fusion gene were determined by EGFP coexpression in the peripheral blood of CtrlII5tg-F/P and Atg5eoΔII5tg-F/P recipient mice 33 days after transplantation (n = 3-4). (D) Enzyme-linked immunosorbent assay measurements. IL-5 concentrations were measured in the plasma of CtrlII5tg-F/P and Atg5eoΔII5tg-F/P recipient mice on day 33 after transplantation (n = 3-4). Confocal microscopy. Formalin-fixed, paraffin-embedded liver (E) and colon (F) tissue sections of CtrlII5tg-F/P and Atg5eoΔII5tg-F/P recipient mice were stained with monoclonal mouse anti-EPX (red) and polyclonal goat anti-myeloperoxidase (MPO) (green) antibodies. Hoechst 33342 (blue) was used for nuclear DNA visualization. Quantification of EPX+ and MPO+ cells was performed by counting cells manually in 10 randomly selected high-power fields, each covering the area of 22.5 × 10−3 mm2 using an automatic digital slide scanner (Pannoramic MIDI II, 3DHistech, Budapest, Hungary) (n = 3). Right: representative confocal microscopy images are shown. Scale bars, 10 µm. Values are means ± standard error of the mean, or single data are presented in scatter dot plots in which the medians are indicated as red lines. *P < .05; **P < .01; ***P < .001. n.s., not significant.

Knocking out Atg5 in the eosinophil lineage results in reduced severity of FIP1L1-PDGFRα–mediated experimental eosinophilic leukemia. (A) Cell counts. Total leukocyte counts were measured in the peripheral blood of CtrlII5tg-F/P and Atg5eoΔII5tg-F/P recipient mice using the scil Vet abc Plus+ hematology analyzer (scil animal care company GmbH, Viernheim, Germany) on day 33 after transplantation. Relative numbers of neutrophils (CD45+/CD11b+/Ly6G+), B cells (CD45+/B220+), T cells (CD45+/CD3+), monocytes (CD45+/CD11b+/CD115+), and eosinophils (CD45+/CD11b+/Siglec-F+) were determined by flow cytometry, and an absolute count for each cell population was calculated (n = 3-4). (B) Light microscopy. Peripheral blood smears of CtrlII5tg-F/P and Atg5eoΔII5tg-F/P recipient mice on day 33 after transplantation were stained with the Hemacolor Rapid Staining Kit (MilliporeSigma, Burlington, MA), followed by light microscopy analysis. Scale bars, 10 µm. (C) Flow cytometry. Relative numbers of cell populations transduced with the F/P fusion gene were determined by EGFP coexpression in the peripheral blood of CtrlII5tg-F/P and Atg5eoΔII5tg-F/P recipient mice 33 days after transplantation (n = 3-4). (D) Enzyme-linked immunosorbent assay measurements. IL-5 concentrations were measured in the plasma of CtrlII5tg-F/P and Atg5eoΔII5tg-F/P recipient mice on day 33 after transplantation (n = 3-4). Confocal microscopy. Formalin-fixed, paraffin-embedded liver (E) and colon (F) tissue sections of CtrlII5tg-F/P and Atg5eoΔII5tg-F/P recipient mice were stained with monoclonal mouse anti-EPX (red) and polyclonal goat anti-myeloperoxidase (MPO) (green) antibodies. Hoechst 33342 (blue) was used for nuclear DNA visualization. Quantification of EPX+ and MPO+ cells was performed by counting cells manually in 10 randomly selected high-power fields, each covering the area of 22.5 × 10−3 mm2 using an automatic digital slide scanner (Pannoramic MIDI II, 3DHistech, Budapest, Hungary) (n = 3). Right: representative confocal microscopy images are shown. Scale bars, 10 µm. Values are means ± standard error of the mean, or single data are presented in scatter dot plots in which the medians are indicated as red lines. *P < .05; **P < .01; ***P < .001. n.s., not significant.

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