American Society of Hematology

Figure 2.

$SRGN–/– and NBEAL2–/– MKs fail to retain PF4 at early stages of development. (A) Representative maximum intensity projection images of isolated, mature (day 5) MKs adhered to fibrinogen-coated slips overnight and treated with bafilomycin (50 nM) for 4 hours. Actin (red), DAPI (4′,6-diamidino-2-phenylindole)/DNA (blue), and PF4 (green). The scale bar represents 10 μm. (B) Quantification of PF4 fluorescence intensity normalized to background fluorescence within each image. Data represent an average of 15 individual cells from each time point. (C) PF4 mRNA during MK differentiation from lineage-depleted cells. Shown as a ratio to WT day 2 average; n = 4. (D) SRGN mRNA during MK differentiation from lineage-depleted cells. Shown as a ratio to WT day 2 average; n = 2. (E) Total PF4 production in lineage-depleted MK cultures until day 5, determined by the addition of the total mass of PF4 in the lysate and media. PF4 concentrations in each fraction were measured by enzyme-linked immunosorbent assay (ELISA), and mass was calculated based on volume; n = 5. (F) Retained PF4 concentrations, determined from lysate fraction as described in panel E; n = 5. (G) Released PF4 concentrations, determined from media fraction as described in panel E; n = 5. (H) Data show the percentage of total PF4 mass (E) found in the media (G) throughout the experiment. Data represent an average of 5 independent experiments. All significance is relative to the corresponding WT time point. Significance: ns is indicated by P > .05; ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001. All error bars represent the SEM.$

SRGN^–/– and NBEAL2^–/– MKs fail to retain PF4 at early stages of development. (A) Representative maximum intensity projection images of isolated, mature (day 5) MKs adhered to fibrinogen-coated slips overnight and treated with bafilomycin (50 nM) for 4 hours. Actin (red), DAPI (4′,6-diamidino-2-phenylindole)/DNA (blue), and PF4 (green). The scale bar represents 10 μm. (B) Quantification of PF4 fluorescence intensity normalized to background fluorescence within each image. Data represent an average of 15 individual cells from each time point. (C) PF4 mRNA during MK differentiation from lineage-depleted cells. Shown as a ratio to WT day 2 average; n = 4. (D) SRGN mRNA during MK differentiation from lineage-depleted cells. Shown as a ratio to WT day 2 average; n = 2. (E) Total PF4 production in lineage-depleted MK cultures until day 5, determined by the addition of the total mass of PF4 in the lysate and media. PF4 concentrations in each fraction were measured by enzyme-linked immunosorbent assay (ELISA), and mass was calculated based on volume; n = 5. (F) Retained PF4 concentrations, determined from lysate fraction as described in panel E; n = 5. (G) Released PF4 concentrations, determined from media fraction as described in panel E; n = 5. (H) Data show the percentage of total PF4 mass (E) found in the media (G) throughout the experiment. Data represent an average of 5 independent experiments. All significance is relative to the corresponding WT time point. Significance: ns is indicated by P > .05; ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001. All error bars represent the SEM.

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