Figure 9: The critical role of GATA2 SUMOylation in EC activation.

(a) GATA2 SUMOylation regulates the expression of endothelial adhesion molecules. HUVECs were transfected by GATA2 siRNA or control siRNA for 48 h followed by infection with Ad-GATA2-WT, Ad-GATA2-KR, Ad-SUMO-GATA2 or their vector control (GFP) as indicated. HUVECs were treated with TNF or vehicle control for 24 h, and the cell lysates were then subjected to western blotting with anti-ICAM-1, anti-VCAM-1, anti-E-selectin or anti-GATA2 antibodies. Actin was used as a loading control. (b) GATA2 SUMOylation site mutation increases endothelial adhesion molecule induction by TNF at the early phase. The expression of endothelial adhesion molecules was determined by quantitative real-time PCR in HUVECs with GATA2-WT or GATA2-2KR reconstitution as described in (a). Data are presented as the mean±s.e.m. from at least three independent experiments. *P<0.05 and **P<0.01; two-way ANOVA followed by Bonferroni post-test. (c,e) GATA2 SUMOylation inhibits leukocyte–endothelial adhesion. HUVECs were gene modified and treated as described in (a). Calcein green-labelled THP-1 monocytes were loaded onto a confluent HUVEC monolayer for 1 h. Non-adherent cells were then washed away, and the adherent cells were visualized using fluorescence microscopy. Representative images are shown in (c), and the normalized ratio of adherent THP-1 cells in each field is shown in (e). Bar represents 200 μm. Data are shown as the mean±s.e.m. from three independent experiments. **P<0.01 compared with GATA2 knockdown. ^#P<0.05 compared with GATA2 KD with restored GATA2-WT; one-way ANOVA followed by Bonferroni test. (d,f) GATA2 SUMOylation inhibits leukocyte–endothelial transmigration. HUVECs were transfected with GATA2 siRNA or control siRNA for 48 h and then infected with various GATA2 viruses or their vector control as indicated. HUVECs with or without GATA2 restoration were cultured on the upper chamber of the Transwell insert that was coated with 0.1% gelatin and then treated with TNF or IL-1β cytokines for 24 h. Subsequently, calcein red-labelled THP-1 monocytes were seeded onto the activated HUVEC monolayers for 24 h. Transendothelial migrated monocytes were determined by visualizing cells in the lower compartment of the insert using fluorescence microscopy. Representative images are presented in (d) with normalized quantification of migratory THP-1 cells in (f). Bar represents 50 μm. Data are shown as the mean±s.e.m. from three independent experiments. **P<0.01 compared with GATA2 knockdown. ^#P<0.05 compared with GATA2 knockdown with restored GATA2-WT; one-way ANOVA followed by Bonferroni test.