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. 2022 Sep 1;6(17):5100–5112. doi: 10.1182/bloodadvances.2021006945

Figure 6.

Figure 6.

Effect on VWF trafficking in HEL cells of RUNX1 knockdown by siRNA and of RAB31 knockout by CRISPR/Cas targeting, and in iMKs differentiated from WT iPSCs and iPSCs generated from a subject with a RUNX1 mutation. (A) RUNX1-knockdown in HEL cells. PMA-treated HEL cells were nucleofected with control or RUNX1 siRNA and with RAB31 plasmid along with RUNX1 siRNA. They were stained for VWF (green). Cells with control siRNA showed VWF in the perinuclear regions and a band along the plasma membrane, which was lost on RUNX1 knockdown. RAB31 reconstitution did not restore the plasma membrane VWF. (B) RAB31-CRISPR/Cas knockout (KO) in HEL cells. Immunostaining for VWF (red), CD63 (green, late endosome/MVB), and their overlap (yellow). In WT cells. VWF is present in the perinuclear regions and at the plasma membrane as a rim of red dots, with an overlap between VWF and CD63 (yellow). In RAB31 KO cells, VWF was absent at the plasma membrane with an overlap of VWF and CD63-positive areas, suggesting retention of VWF in late endosomes/MVBs and defective VWF trafficking to plasma membrane. (C) iMKs differentiated from WT iPSCs and RHD-iPSCs with RUNX1 mutation were stained for VWF (green). The RHD-iMKs showed a loss of the plasma membrane VWF. Scale bar = 10 µm. DAPI, 4′,6-diamidino-2-phenylindole.