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. 2020 Aug 7;11:3984. doi: 10.1038/s41467-020-17848-4

Fig. 4. Epsin deficiency maintains calcium homeostasis in atherogenic conditions and dampens inflammation in endothelial cells.

Fig. 4

a–c MAECs from control and iDKO mice were stimulated with ATP in the absence (a) or presence (b) of 100 µg/mL oxLDL or 50 µM 7-KC for 36 h and intracellular calcium was measured and quantified (c) (n = 5 independent repeats, asterisk P < 0.001). d, e WT or iDKO MAECs were treated with 50 ng/ml TNFα for 3 h to detect P-selection or E-selectin for 16 h to detect adhesion molecules ICAM-1 and VCAM-1, as well as tubulin by western blot (d) prior to quantification (e) (n = 5 independent repeats, asterisk P < 0.05). f, g WT and iDKO mice were injected with 0.5 µg TNFα and sacrificed after 3 h (for P-selectin) or 16 h (for adhesion molecules). Heart sections were stained with antibodies for ICAM-1, VCAM-1, and P-selectin. Arrows indicate endothelial staining (f), which was quantified (g) (n = 5 mice in each group, asterisk P < 0.001). Scale bar 100 µm. h, i Neutrophil adhesion under flow (top panels) and static conditions (bottom panels) to TNF-treated MAECs isolated from WT and iDKO mice (h) and quantification (i) (n = 3 independent repeats, asterisk P < 0.001). MAECs were isolated from Epsin 1fl/fl/Epsin 2−/−: VE-cadherin-Cre (top panels) or Epsin 1fl/fl/Epsin 2−/−: iCDH5-Cre mice (bottom panels). Scale bars 10 µm (brightfield) or 200 µm (fluorescence). j, k Peritoneal macrophage migration through confluent MAECs in the absence or presence of epsin 1 and 2 using a Transwell plate. LPS (200 ng/mL) or TNFα (50 ng/mL) was added to the upper Transwell chamber to stimulate endothelial cells for 3 h, followed by the addition of WT peritoneal macrophages (j). After 3 h, macrophages that migrated to the bottom of the Transwell chambers were subjected to enumeration and quantification (LPS and TNFα WT vs. iDKO, n = 3 independent experiments, and each treatment selected 3 fields for statistical analysis, asterisk P < 0.0001) (k). All data were assessed using Student’s t-test and are presented as the mean ± SEM.