Piperlongumine-Induced Cellular Apoptosis

The cells (1 × 10⁶ cells/dish) were seeded in a 10 cm cell culture dish and placed in a 37 °C incubator overnight. The cells were cultured with control medium or piperlongumine, and cellular apoptosis was measured with Annexin V (Sigma-Aldrich) and PI staining. Flow cytometry was used to measure apoptosis as previously reported [62 (link),63 (link)]. Proteins involved in piperlongumine-mediated cellular apoptosis were verified with Western blots. Primary antibodies, including caspase-9, caspase-3, and PARP, were purchased from Cell Signaling Inc. (Danvers, MA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control, and the primary antibody was purchased from GeneTex (San Antonio, TX, USA). Three independent assays were performed.