Cobimetinib

VLA-4 clustering assays were performed as described elsewhere, ^{11 (link)} using 7.5 mg/mL VCAM-1/Fc. AML cells were pretreated for 10 min with 10 mg/mL HA, 60 min with 1 mM midostaurin, 30 min with 10 mM PP2 and 30 min with 10 mM cobimetinib (APExBIO, Houston, USA), where indicated. Cells were allowed to adhere for 30 min at 37°C before fixation with 4% paraformaldehyde. Slides were stained with αCD49d (clone AHP1225), αCD29 (clone 12G10) primary antibodies or isotype control (not shown) followed by a secondary antibody. For CD49d cluster analysis of normal progenitor cells from patients with non-myeloid malignancies, cells were additionally stained with αCD34 antibody (clone QBEND-10). For quantification, high-resolution images were acquired on a Leica TCS SP5 II laserscanning microscope using a 63×/1.4-NA oil-immersion objective (Leica, Wetzlar, Germany). The number of clusters was analyzed using ImageJ software by particle analysis setting the size of the particle at >2 pixels.^{12 (link)}