Supplementary Figure 6: Granule proteins and signaling of G-CSF and GM-CSF receptors in Jagn1-mutant neutrophils and flow cytometry analysis of JAGN1-mutant human bone marrow.

(a) MPO levels in blood neutrophils were assessed by staining with an MPO-specific antibody. Representative staining for Jagn1^fl/fl and Jagn1^Δhem blood neutrophils is shown. (b) Expression of secondary granule proteins Mmp8 and Mmp9 as assessed by intracellular staining and flow cytometry. Left, MFI ratios of either Jagn1^fl/fl/C57BL/6 or Jagn1^Δhem/C57BL/6 blood neutrophils as distinguished by staining for CD45.2 and CD45.1 in bone marrow chimeric mice. n = 4 for each genotype. Right, histogram overlays from representative MMP8 and MMP9 staining of the indicated populations of blood neutrophils of bone marrow chimeric mice. (c) MPO release from purified bone marrow neutrophils co-cultured with Candida albicans for 24 h as measured by ELISA in triplicate. (d) Jagn1^fl/fl and Jagn1^Δhem blood neutrophils were stimulated with G-CSF for 20 min and assayed for phosphorylation of STAT3 (pSTAT3) by intracellular staining. Representative histogram overlays are shown for neutrophils stimulated with the indicated concentrations of recombinant mouse G-CSF or left unstimulated. (e) Cell surface (left) and intracellular (right) expression of G-CSF-Rα in neutrophils isolated from the peripheral blood of Jagn1^fl/fl and Jagn1^Δhem mice. Plots depict average mean fluorescence intensity (MFI) ± s.d. Each data point represents an individual mouse. (f) Levels of phosphorylated STAT3 (pSTAT3) in peripheral blood neutrophils from Jagn1^fl/fl and Jagn1^Δhem mice infected intraperitoneally with Candida albicans 24 h before analysis. Representative histogram overlays are shown for mice infected with Candida albicans or left uninfected. Of note, baseline STAT3 phosphorylation was comparable between Jagn1^fl/fl and Jagn1^Δhem neutrophils in uninfected mice. (g) Peripheral blood neutrophils were stimulated with the indicated concentrations of recombinant GM-CSF for 20 min and then assayed for phosphorylation of STAT5 (pSTAT5) by intracellular staining. Histogram overlays are shown for cells stimulated with the indicated concentrations of recombinant mouse GM-CSF or left unstimulated. (h) Cell surface expression of GM-CSF-Rα on neutrophils isolated from the peripheral blood of Jagn1^fl/fl and Jagn1^Δhem mice. The plot depicts average mean fluorescence intensity (MFI) ± s.d. Each data point represents an individual mouse. **P < 0.01, ***P < 0.001, Student’s test. (i) CD34^– bone marrow cells from the indicated JAGN1-mutated patients and healthy control subjects were analyzed by flow cytometry. Granulocytes were identified on the basis of forward and side scatter characteristics. (j) CD34^– bone marrow cells were stained with the granulocyte marker CD66b. Histograms depict CD66b expression on granulocytes as gated in i.