Fig. 1. CIKS mediates high glucose-induced endothelial-monocyte adhesion and suppression of HAEC migration.

Primary human aortic endothelial cells (HAEC) at 50% confluency were infected with lentiviral particles expressing CIKS shRNA (MOI 0.5) for 48 hrs, treated with HG (25 mM) for 12 h, then incubated for 1 h with Calcein AM-loaded THP-1 cells. Endothelial-monocyte adhesion was quantified by measuring fluorescence at excitation and emission wavelengths of 485 and 535 nm. Wells containing HAEC without THP-1 cells served as blanks. *P < 0.001 vs. normal glucose (NG)-treated shRNA-infected cells (n=12), †P < 0.05 vs. HG + copGFP. B, CIKS knockdown in HAEC was confirmed by RT-qPCR (upper panel; n=6) and immunoblotting (lower panel; n=3). *P < 0.01 vs. copGFP. C, CIKS knockdown reverses HG-induced suppression of HAEC migration. HAEC were plated at 70,000 cells/well in a 12-well plate, infected with lentiviral CIKS shRNA (MOI 0.5 for 48 h), and scratched once with a sterile 1 ml pipette tip. The cells were then washed twice with complete media, incubated with HG for an additional 24 h, and photographed at 50X magnification. The width of the gap after 24 h was measured and subtracted from that at 0 h to quantify the distance the cells migrated (representative photomicrographs are shown). The experiments were repeated three times, and the results are summarized on the right. *P < at least 0.05 vs. NG, †P < 0.05 vs. HG + copGFP (n=3).