Fig. 7
Apabetalone prevents monocyte adhesion to endothelial cells. a Static assay. Endothelial cell monolayer was pretreated with DMSO, JQ1, or apabetalone for 1 h before the addition of TNFα (2.5 ng/ml; 4 h incubation). Monocytes (0.5 × 106cells/mL; loaded with calcein-AM) had 1 h to adhere to the monolayer before washes and fluorescence measures (plate reader). b Upper panel: fluorescent micrographs of monocyte adhesion to endothelial cells under static conditions. Lower panel: dose-response curves titrating JQ1 and apabetalone effect on monocyte /endothelial cell adhesion. JQ1 IC50 = 0.08 μM. apabetalone IC50 = 22 μM. c Flow assay. Pretreatment as in a; monocytes (0.4 × 106 cells/ml) were perfused over the treated monolayer for 3 min with a flow rate of 50 s−1 then for another 3 min with a flow rate of 25 s−1. A high flow rate of 120 s−1 was applied to remove all unbound THP-1 cells, and images were acquired for analysis. d Upper panel: phase-contrast micrographs of monocyte adhesion to endothelial cells under flow conditions. Lower panel: apabetalone pretreatment prevents monocyte adhesion to endothelial cells under flow conditions; 0.2 μM JQ1 and 5 μM apabetalone had a comparable effect on adhesion. Statistical significance was determined through 1-way ANOVA analysis followed by Dunnett’s Multiple Comparison Test using TNFα response for the comparison, where *p < 0.05, **p < 0.01, ***p < 0.001