After the HUVECs were treated with plasmids and 5.5 or 30 mM glucose, the viability of the HUVECs cultured in 96-well plates was measured by MTS assay, as previously described (24). Following the addition of glucose 1-5 days later or transfection 24 later, the ECM medium of cultured HUVECs was replaced with 100 μl ECM medium containing 10 μl of CellTiter 96 AQueous One Solution (Promega Corporation). The plates were then incubated at 37°C for 4 h. The 96-well plate was then examined at 490 nm using a Multiskan spectrum spectrophotometer (Thermo Fisher Scientific, Inc.).

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