Anti-proliferation assay

The anti-proliferation activity of shikonin against cancer cells was tested by the MTT method in 96-well plates. First, 4 × 10³ cells were seeded per well and treated with the shikonin with serial concentrations for 48 h. Then, the cells were incubated with 10 μL of MTT solution (5 mg/mL in PBS) for an additional 2–4 h at 37 °C. The formazan crystals were dissolved with 100 μL of DMSO after removing the supernatant. The absorbance at 570 nm was measured by a BioTek Synergy 2 plate reader (BioTek Instruments, Inc., Winooski, VT, USA). Doxorubicin, a chemotherapy medication used to treat cancer and having a broad spectrum of anticancer activities [29], was used as a positive control here.