Cellular thermal shift assay (CETSA)

The potential interaction between LSS-11 and topoisomerases was assessed by CETSA as previously described [48]. Briefly, cells were harvested in RIPA buffer with complete protease inhibitors and freeze-thawed three times using liquid nitrogen. The lysate was centrifuged at 12000 rpm for 20 minutes at 4°C, and the supernatant was divided into two equal parts to incubate with LSS-11 or DMSO as vehicle for 30 min at room temperature. The resulted aliquots were then divided into smaller parts and heated at indicated temperatures using a gradient thermal cycler (T-100, Bio-Rad) for 3 minutes. The heated lysates were centrifuged at 12000 rpm for 20 minutes at 4°C to separate the soluble fractions and boiled at 95°C with 5× SDS loading buffer for 5 min and analyzed by western blotting.