Caspase glo 9

The Caspase-Glo^® 3/7, Caspase-Glo^® 8, and Caspase-Glo^® 9 (all Promega) kits were used as previously described (30 (link)). The caspase-1 activity assay kit (Novus Biologicals) and the mitochondrial ToxGlo™ (Promega) assay were used according to the manufacturer’s instructions. Fluorescence (488/525 nm) and luminescence were measured with the SpectraMax i3 (Molecular Devices).

Culture media, including the minimum essential media (MEM), penicillin-streptomycin-glutamine, sodium pyruvate, fetal bovine serum (FBS), and DAPI, were purchased from Thermofisher Scientific. The following reagents were acquired from Sigma (Mendota Heights, MN, USA): secondary anti-rabbit antibody, simvastatin, MeV, FPP, GGPP, cholesterol, and MTT. Rabbit anti-human Bax, Bcl-2, Bcl-xl, and Mcl-1 were purchased from Cell Signaling. Anti-mouse monoclonal GAPDH was purchased from Santa Cruz Biotechnology, and Casapase-Glo^®-3/7, Caspase-Glo^®-8, and Caspase-Glo^®-9 assays were obtained from Promega (Madison, WI, USA).

Caspase activity was analyzed using the caspase-Glo 3/7 Assay, caspase-Glo 8 Assay and caspase-Glo 9 (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, 1×10⁴ cells (treated with or without CA16 virus at the MOI of 0.2) were collected at 0, 12, 24, 36 or 48 h as indicated and lysed using the manufacturer-provided homogeneous caspase 3/7 or caspase 8 reagent. The lysates were incubated at room temperature for 1.5 h before reading in a fluorometer at 485/530 nm. The relative caspase activity was calculated as the fold-changes of samples at 12, 24, 36 and 48 h (compared with sample at 0 h).

Cells were plated in white 96-well plates at 1 x 10⁴ cells per well and were allowed to attach. Then, the cells were treated with the compound (50 μM) for different periods of time (1, 3, 6, 12, 18, 24 and 30 hours). Caspase activity was measured by adding 50 μl of Caspase-Glo® 3/7 (Z-DEVD-aminoluciferin), Caspase-Glo® 8 (Z-LETD-aminoluciferin) or Caspase-Glo® 9 (Z-LEHD-aminoluciferin) (Promega, Madison, WI) and then reading the luminescence using a microplate reader (Infinite 200, Tecan, Männedorf, Switzerland). The activities of the individual caspases were expressed as fold increases with respect to the untreated control. For the inhibitor assay, cells were pre-treated with 10 μM caspase 3 inhibitor (Z-DEVD-FMK), caspase 8 inhibitor (Z-IETD-FMK), caspase 9 inhibitor (Z-LEHD-FMK) or general caspase inhibitor (Z-VAD-FMK) for 30 minutes. Then the cells were incubated with the compound (50 μM) for 18 hours, and the caspase assays were performed as outlined above. After another 6 hours of incubation with the compound, cell viability was measured using the MTS reagent, as described in the cell culture and cytotoxicity assay section.

Propidium Iodide (PI) (BD Pharmingen™, BD Biosciences) was used to quantify non-viable cells. An amount of 1 µL of PI dye was added to the cell suspension prior to performing flow cytometry. For caspase assays, 5000 22Rv1 cells or 2000 BPH-1 cells were seeded in white 96-well plates (Corning, NY, USA). Caspase-Glo^® 8, Caspase-Glo^® 9, and Caspase-Glo^® 3/7 Assay systems (Promega, Madison, WI, USA) were carried out as per the manufacturer’s instructions. Luminescence readings were taken 90 min (min) post-addition of the reagent. Readings were normalized to that of mock-infected cells and data are expressed as fold of virus-infected cells over mock-infected cells.

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.