Caspase glo 3 7

Endometrial carcinoma cell lines AN3CA and KLE were obtained from ATCC and maintained in the DMEM/F12 supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. All experiments were performed prior to passage 10. Proliferation assays (3T5) and colony formation assays were performed as previously described [39 (link), 40 (link)]. Cell viability and caspase activation were performed using Cell Titer-Glo and Caspase 3/7-Glo (Promega). The same vehicle (0.3% DMSO) was used for both HCI2509 and MPA in all in vitro treatments.

SH-SY5Y cells were seeded with 4 × 10⁵ cells per well in six-well plates. The next day, cells were transfected with hsa-miR-34a-5p miScript miRNA Mimic or AllStars Negative Control (ANC) using HiPerFect Transfection Reagent (Qiagen, Hilden, Germany). At 24 h after transfection, cells were re-plated with 1.8–2.5 × 10⁴ cells per well in opaque-walled 96-well plates. After an additional 24 h, cells were treated with 1 µM tunicamycin or DMSO as control for 8 h, 24 h and 48 h. Luciferase-based CellTiterGlo 2.0, CytotoxGlo and Caspase3/7 Glo assays (Promega, Mannheim, Germany) were applied according to the manufacturer’s instructions to determine cell viability, cytotoxicity and caspase activity. Luciferase activity was measured with a GloMax navigator microplate luminometer (Promega, Mannheim, Germany). The experiments were done in triplicate in two independent experiments. As control, ANC and miR-34a-5p-transfected cells were treated with DMSO. Cell viability and caspase activity were calculated by normalizing the received luminescence values to the control transfected cells treated with DMSO for each time point. The relative number of dead cells was calculated by normalizing the luminescence derived from the dead cell protease activity to the protease activity of total cells after cell lysis. Statistical significance was calculated with a two-tailed paired t-test.

Huh7 and BAX/BAKDKO (#29 and #47) cells were infected with JEV (MOI = 5) and incubated at 37°C for 2 days. Caspase 3/7 activity was determined by using Caspase 3/7Glo (Promega) according to the manufacturer’s protocol.

Caspase activity was evaluated at the end of the apoptosis assay performed with H₂O₂ or Thapsigargin. In detail, after treatment, the cells were detached with 0.25% (w/v) trypsin in 5 mM EDTA, washed with PBS, and incubated in a 1:1 ratio (v/v) with Caspase 3/7 Glo^® (Promega, Madison, WI, USA) in a 96-well black plate (PerkinElmer, Milano, Italy) for 1 h at RT in the dark. Finally, the luminescence signal was detected with Promega/Glomax multi-detection system.

96-well plates were seeded with 100 μL of K562 cells at 10⁵ cells/mL and treated with different concentrations of translational inhibitor for 72 hr. For longer experiments, cells were diluted in a new plate and treated again with translational inhibitor in order to remain under the maximum cell density (<10⁶ cells/mL). Cell viability was determined by ATP level measurements (CellTiterGlo2.0, Promega) as an indicator of cell titer and metabolic activity. To determine the level of caspase induction, caspase3/7 levels were measured from 50 μL of cell suspension and ATP levels were measured from the remaining volume (Caspase3/7Glo, Promega). The apoptosis index for each well was calculated as the ratio of Caspase and ATP luminescence signals.