Cellular thermal shift assay (CETSA)

CETSA was developed as a method to directly detect the binding capacity of compounds with targets at the cellular level, based on the principle of ligand-induced target protein stabilization. Briefly, SW620 cells cultured with 90% confluent in 100 × 20 mm tissue culture dishes were treated with media containing DMSO or compound 1 (30 μM) for 12 hours. After treatment, the cells were isolated with trypsin, collected by centrifugation, and then resuspended in PBS. The cell suspension was divided equally into 4 PCR tubes and heated to 44, 46, 48, 50, 52, and 54°C for 3 minutes, respectively. Subsequently, cells were analysed by Western blot assay [45–46].