MTT assay and synergism evaluation

Cell viability following treatment with various compounds was evaluated using MTT assay, performed in triplicates. Briefly, cells (4×10⁴/well) were incubated in a 24-well plate containing 0.5 mL of serum-containing medium. Cells were allowed to adhere for 18–24 h and washed with PBS. Solutions were always prepared fresh in culture medium by dissolving 0.2% DMSO (control) or 1.5 μg/mL Tan-IIA in the presence or absence of 2.5 or 5 μM sorafenib or 5 μM SC-1 before their addition to cells. The drug-containing medium was removed after treatment for 48 h, cells were washed with PBS, and culture medium containing 300 μg/mL MTT was added for 1 h at 37°C. After the MTT medium was removed, 0.5 mL of DMSO was added to each well. Absorbance at 570 nm was detected using a multi-well plate reader, Infinite 200 Pro Tecan™ (Tecan, Mannedorf, Zurich, Switzerland). The absorbance of DMSO-treated cells was considered as 100%. The combination index (CI) value was determined according to the Chou–Talalay method using CompuSyn software (ComboSyn, Inc., Paramus, NJ, USA), where a CI value below 1 indicated synergism.22