G8090

To quantify cellular apoptosis after therapy with ONC201, the MEK inhibitor trametinib, a Caspase-Glo 3/7 assay (G8090; Promega), or both were performed according to the manufacturer’s instructions. In brief, TNBC cells were seeded into a 96-well plate at 1 × 10⁴ cells/well and incubated overnight. Cells were then treated with ONC201 (2.5 µM), trametinib (1 µM), or both for 24 h. Equal volumes of Caspase-Glo 3/7 assay buffer were added to the wells with cells and incubated for 1 h at 37 °C. The luminescent intensity of each well was measured using a Victor X3 microplate reader (PerkinElmer, Waltham, MA, USA).

Caspase 3/7 assays (G8090, Promega) were performed according to the manufacturer's instructions. Briefly, 10,000 cells/well were plated overnight in a 96-well plate. Cells were treated as described in the figure legends for the indicated time period. To measure caspase 3/7 activity, 50 μL of caspase Glo 3/7 reagent was added to each well for 2 h with constant shaking at room temperature. Luminescence was measured using a BMG Labtech FLUOstar Optimi plate reader. Cytotoxicity was evaluated by CellTox Green® cytotoxicity assay (Promega). Briefly, 10,000 cells/well were plated overnight in a 96-well plate. Cells were treated as described in the figure legends for the indicated time period. CellTox green dye was diluted 1/500 in test media and applied to cells for the times indicated in the figure. Fluorescence was measured at 485–500 nm_Ex/520–530 nm_Em using a BMG Labtech FLUOstar Optimi plate reader.

Promega’s caspase kits (G8090, G8200 and G8210) were used to run the assay and the methods follow the protocol provided in the kits.
The cells were seeded at a density of 10,000 cells per well in a white-walled 96-well luminometer plate and treated with IC₅₀ value of PU for 72 h. Before starting the assay, Caspase-Glo Reagent was prepared by mixing Caspase Glo substrate with Caspase Glo Buffer provided in the kit. The plate was removed from the incubator and it was allowed to equilibrate to room temperature. Then, 100 µL of Caspase Glo reagent was carefully added to each well and the plate was sealed with a lid. The contents were mixed on a plate shaker 300–500 rpm for 30 s. The contents were incubated for 30 min at room temperature. The luminescence of each sample was measured using a spectrophotometer.

To perform growth curves, 20,000 cells were plated in 12-well plates and appropriate samples were treated with inhibitors and doxycycline. Cells were counted every 24 h over 4 days using the hemocytometer. Cell death following inhibitor treatment was assessed by the caspase glow assay according to the manufacturer’s instructions (G8090, Promega).