Page 77 - Haematologica April 2020

EPHB4 drives macrophage-erythroblasts interactions
to as links. Images were taken every hour to screen as many conditions as possible on the Incucyte and ensure comparability. The box plots in Figure 3B and C demon- strate that the addition of the EPHB4 inhibitor TNYL- RAW significantly reduced the average number of interac-
tions (50% vs. 75% macrophage with more than 1 link on average; P<0.0001) but not the mean duration of these contacts. Importantly, the movement of the macrophages was not affected by the addition of inhibitors, as +Dex macrophages still exhibit a high degree of movement with
A
BC
DE
Figure 3. EPHB4 is required for contact inhibition of locomotion in erythroblastic island formation. (A) Example of analysis performed on an Incucyte experiment with the control peptide, DYP. Dexamethasone (+Dex) macrophages (labeled with Cell Tracker green) were grown from the peripheral blood mononuclear cells (PBMC) selected by adherence. The erythroblasts were added at a ratio of 10:1. The excess erythroblasts were gently removed by washing with media after 16 hours (h) incu- bation. The cells were imaged every hour. Macrophages are individually identified by the program due to their green color. The program then recognizes at each frame how many erythroblasts (red) are in contact (link) with the identified macrophage. Scale bar is 20 μm. (B) Min to max boxplot showing the mean duration of links between macrophages and erythroblasts. Kruskal-Wallis test was performed on 2,038 macrophages for +Dex EPHB4 inhibitor, 2,444 for +Dex Control peptide, and 1,841 for +Dex VLA-4 inhibitor (n=3). The y-axis is a log2 scale. (C) Min to max boxplot showing the average number of links between macrophages and erythroblasts. Kruskal-Wallis test was performed on 2,038 macrophages for +Dex EPHB4 inhibitor, 2,444 for +Dex Control peptide, and 1,841 for +Dex VLA-4 inhibitor (n=3). (D) Scaled cell-displacement vector diagrams of macrophage movement for 18 randomly selected macrophages from control peptide and 13 from the EPHB4 inhibitor condition. (E) Mean plot of total path length for 82 randomly selected macrophages from control peptide and 46 from EPHB4 inhibitor conditions. Two-tailed t-test was run on these samples. ns: not significant (P≥0.05); **** P≤0.0001.
haematologica | 2020; 105(4)
919