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VPS18-deficient neutrophil progenitors have a late maturation defect. A, B Spectral flow cytometric analysis followed by dimensional reduction using UMAP of all single, living cells of all analyzed cell lines from all days of differentiation (day 0-4). A Cells were manually gated for the days of differentiation, namely day 0 (blue), day 1 (green), day 2 (yellow), day 3 (orange) and day 4 (red). B Cells were manually gated for the differentiation stages with granulocyte-monocyte progenitor (GMP)-like cells (yellow), neutrophil progenitors (proNeu, orange), neutrophil precursors (preNeu, pink), immature neutrophils (purple) and mature neutrophils (blue). Arrow, direction of maturation. C UMAP analysis of all single, living cells of CTRL, clone 1 and clone 2 cells at day 4. Clusters of mature (blue) and immature neutrophils (magenta) were manually gated and overlaid onto UMAP plot of all cells during neutrophil differentiation (not gated, gray). D Relative expression of CXCR2 and CD101 during differentiation (day 0-4) in indicated cell lines using spectral flow cytometry. Geometric mean as fold change of day (d) 0. n = 4. Mean ± SEM. *P < 0.05, ****P < 0.0001 compared to CTRL. Two-way ANOVA, Tukey’s multiple comparisons test. E Quantification of GMP-like cells, proNeu, preNeu, immature neutrophils, mature neutrophils and dead cells during differentiation from (A, B) of CTRL, clone 1 and clone 2 cells in % (100%, all single cells). n = 4, Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to CTRL. Two-way ANOVA, Tukey’s multiple comparisons test.
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