Z lehd fmk

Cardiomyocytes were seeded in 96-well tissue culture plates as previously described [21] (link). The cells were then exposed to eleven different conditions, namely 1 – media alone, 2 – conditioned media, 3 – HIV-1, 4 –100 µM of preferential caspase 8 inhibitor Z-IETD-FMK (casp8i) (Promega, USA), 5 –100 µM of preferential caspase 9 inhibitor Z-LEHD-FMK (casp9i) (Promega, USA), 6 – conditioned media and 100 µM Z-LEHD-FMK, 7 – conditioned media and 100 µM Z-IETD-FMK, 8 – conditioned media and 100 nM UCLA1 aptamer, 9 – HIV pre-incubated for 1 h with 100 nM of UCLA1 aptamer, 10 – HIV and 100 µM Z-LEHD-FMK, 11 – HIV-1 and 100 µM Z-IETD-FMK. The cells were harvested daily for 7 days followed by quantification of the caspases and cellular ATP levels, using the Caspase-Glo8, Caspase-Glo9 and the CellTiter-Glo luminescence-based detection kits as instructed by the supplier (Promega, USA). Wells containing media alone were used as controls for background luminescence and subtracted from the test values. The results were expressed as relative light units (RLUs) and plotted in a graph relating RLU to number of days.

Caspase 3, 8 and 9 activities in cells were measured at 72 h after lentiviral shRNAs transduction using a CaspaseGlo kit (Promega, Madison, WI, USA) according to the manufacturer's protocol. Caspase 3, 8, 9 or pan-caspase inhibitor (Z-DEVD-FMK, Z-IETD-FMK, Z-LEHD-FMK and Z-VAD-FMK, respectively; Promega) was used to inhibit caspase activity. The optimum concentration of all inhibitors was determined to be 20 μM. Inhibitors were added to cells 24 h after lentiviral shRNA transduction. Apoptosis was measured by Annexin V-PE assay at 72 h.