HCMV secretomes decrease caspase-3 and -7 activity. HUVECs were starved overnight and then stimulated with mock- or HCMV-secretomes during a time course analysis. (A) Cytosolic extracts from HUVECs incubated with controls, mock- and HCMV VR1814-secretome for 24, 48, 72, and 96 hours were prepared in hypotonic extraction buffer. An equal volume of a proluminescent substrate (DEVD), and cytosolic proteins were added to a white-walled 96-well plate and incubated at room temperature for 1 hour. The luminescence of each sample run in triplicate was measured in a plate-reading luminometer. The graphic shows the mean ± SD of 3 independent experiments. (B) Cell lysates from HUVECs incubated with mock-secretome (MS) and HCMV VR1814 (VRS) were subjected to SDS-PAGE followed by Western blotting to evaluate the expression of activated/cleaved caspase-3 and -7. αTubulin served as an internal control. (C) HUVECs were stimulated as above, and nuclear DNA fragmentation as a sign of apoptosis was determined over the time using a TUNEL assay. Fragmented DNA is indicated by green fluorescence. Nuclei were stained with propidium iodide (red fluorescence).