Apoptosis and Mitochondrial Membrane Potential Assay

To assess apoptosis, cells were treated with TPL and ABT-199 alone or in combination for 24 and 48 h, followed by staining with Annexin-V-FITC/PI (eBioscience, San Diego, California, USA) for 15 min at room temperature in the dark according to the manufacturer’s instructions. Cells were then analyzed by NovoCyte flow cytometry with NovoExpress software (ACEA Biosciences, Inc., USA) to determine the percentage of Annexin-V positive (apoptotic) cells. Loss of the mitochondrial membrane potential (Δψm) was measured using JC-1 Fluorescent Probe Kit (Beyotime Company, Shanghai, China) by following the manufacturer’s instructions as described previously^{15 (link)}. To assess the interaction between TPL and ABT-199, two separate approaches (i.e., CompuSyn and CalcuSyn) were used to calculate the combination index (CI). CI < 1.0 indicates synergism, while CI = 1.0 and CI > 1.0 indicate an additive or antagonistic effect, respectively.