Figure 1
Peripheral blood was obtained from healthy volunteers and JAK2-V617F-positive patients (PV, ET and PMF) who were untreated with JAK inhibitors after informed consent and upon approval by the local ethics committee (protocol no MD115108). Mononuclear cells were removed by Ficoll-Paque density gradient centrifugation, followed by lysis of erythrocytes with BD FACS Lysing solution (BD Biosciences, Franklin Lake, NJ, USA). (a) Static adhesion assay of primary granulocytes from healthy donors (n=5) and JAK2-V617F-positive patients (n=5) on immobilized VCAM1 (R&D Systems, McKinley, MN, USA, ADP5-050) was performed as described before for ICAM1.15 (b) sVCAM1 binding assay using soluble VCAM1/Fc (R&D Systems, 862-VC) in primary granulocytes from healthy donors (n=10) and JAK2-V617F-positive patients (n=10) as described previously for ICAM115 (right). Correlation of sVCAM1 binding of granulocytes isolated from JAK2-V617F-positive patients with JAK2-V617F allelic burden of peripheral blood cells (left). (c) Static adhesion of 32D JAK2-WT (WT) and JAK2-V617F (V617F) cells on immobilized VCAM1 (R&D Systems, 862-VC) in the absence and presence of JAK inhibitor I treatment (shown are results obtained upon subsequent washing steps II, III and IV) as described before for ICAM1.15 Cells were treated either with DMSO (−) or with JAK inhibitor I (200 nM) (+) for 16 h. Three independent experiments were performed. *P⩽0.05; **P⩽0.01; ***P⩽0.001 (unpaired, two-tailed Student’s t-test).
