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. 2019 Jun 27;105(3):598–609. doi: 10.3324/haematol.2019.221804

Figure 1.

Figure 1

The effect of ELANE knock-down on the impaired myeloid differentiation of HL60 cells expressing mutant neutrophil elastase. (A) CRISPR/Cas9 edited human promyelocytic HL60 cells expressing p.P139L and p.C151Y mutant neutrophil elastase (NE) were electroporated with scrambled and anti-ELANE siRNA and maintained in the complete medium for five days. Western blot (WB) analysis at day 4 shows complete knock-down of NE detected with an anti-NE monoclonal antibody. For loading control, the membrane was stripped and re-probed with a β-actin-specific antibody. Representative WB membranes are depicted. (B) Myeloid differentiation was induced with 2 μM ATRA (all-trans retinoic acid). After five days, cells were labeled with CD11b myeloid differentiation surface marker and examined using Fluorescence-activated cell sorting (FACS) analysis. The proportion of CD11b-PE positive cells is indicated. Data represent means ± SD from four independent experiments. Two-sided, unpaired Student’s t-test P-values are shown, ***P<0.0001 and ***p=0.0043 for p.P139L and p.C151Y respectively compared to wild-type (WT).