Neutralization of IL-6 activity in the HCMV secretomes induces apoptosis. HUVECs were starved overnight and then stimulated with mock-secretome, HCMV-secretome, and neutralized HCMV-secretomes for 48 hours. (A) Cytosolic extracts from HUVECs incubated with controls, mock-secretome, HCMV VR1814-secretome, and HCMV VR1814-secretome neutralized with anti–IL-6, anti–GM-CSF, anti–IL-8/CXCL8, or nonrelated antibody 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 results are expressed as mean ± SD of 3 independent experiments, ***P < .001 versus mock. (B) HUVECs were starved overnight and then stimulated with mock-secretome, mock-secretome with IL-6 (10 ng/mL), and mock-secretome with IL-6 (10 ng/mL) + anti–IL-6 Ab (2 μg/mL) for 48 hours. Cytosolic extracts were prepared as above and the luminescence of each sample run in triplicate was measured in a plate-reading luminometer. The results are expressed as mean ± SD of 3 independent experiments, **P < .01 versus mock. (C) TUNEL assay performed in the same experimental conditions as above. Fragmented DNA is indicated by green fluorescence. Nuclei were stained with propidium iodide (red fluorescence).