4.10. Cellular Thermal Shift Assay (CETSA)

MDA-MB-231 cells (3 × 10⁵ cells/well) were seeded in 6-well plates and the cells were allowed to attach for 24 h. After 24 h, cells were treated with PNSA at different concentrations and incubated for 3 h. After incubation, cells were resuspended in PBS and divided into several aliquots. Four aliquots were treated with DMSO and other four aliquots were treated with PNSA. Then, cells were heated at different temperatures by Biometra TOne PCR (Analytikjena, Jena, Germany). The heated cells were freeze-thawed three times with liquid nitrogen for 4 min every time and followed by centrifugation at 20,000× g for 20 min at 4 °C. The supernatants were harvested and loading buffer was added before boiling for 15min. Protein levels were assessed by Western blotting.