a–d Endosteal BM was collected from mice with advanced GFP+ AML (MLL-AF9 induced, n = 5/group), or no leukemia controls (n = 4/group), stained for E-selectin cell surface expression on viable endothelial cells (7AAD− lineage−CD45− CD31+ GFP−) and analyzed by flow cytometry. a, b Gating strategy for BM endothelial cells from one representative mouse in each group: a Healthy non-leukemic control mouse, b leukemic mouse. c Overlay histogram comparing cell surface E-selectin expression on gated BM endothelial cells from mice with AML (blue line), non-leukemic mouse (black dashed line). Isotype negative control is gray filled. Bar shows E-selectin positive gate. d Histogram showing percent of endothelial cells that are positive for surface E-selectin expression. Each dot represents data from an individual mouse. Bars are mean ± S.D. Statistics: two-tailed t test. e, f BMEC-1 cells were cocultured with TNF-α (positive control for E-selectin activation), or with BM cells from healthy (non-leukemic) or leukemic mice ± TNF-α inhibitor etanercept for 16 h at 37 °C. Cocultured cells were then collected and stained for E-selectin expression on BMEC-1 cell surface and analyzed by flow cytometry. e Gating strategy for E-selectin expression on viable BMEC-1 cells. Shown are viable BMEC-1 gate (left) and surface E-selectin-APC expression (right). Representative dot plot from one well per group. f Histogram representing percentage of BMEC-1 expressing E-selectin after co-culture with medium alone, added BM cells from healthy and from leukemic AML mouse, or BMEC-1 with TNF-α, ± etanercept as indicated. Mean ± S.D. of pooled data from three independent experiments (n = 7,6,8,8,5,5,3,3,3,3 wells/group). Statistical significance calculated by one-way ANOVA with Bonferroni correction for multiple comparisons. Source data are provided as a Source Data file.