Liensinine-induced Apoptosis and Autophagy

Cells were seeded into 6-well plates with 1^∗10⁵ cells each well. Liensinine was prepared for different concentrations (0, 2.5, 5, 10, or 20 μM) and added into the wells for 48h. After washing PBS for 3 times, cells were lysed with lysis buffer (P0013 J, Beyotime, China) supplemented with protease inhibitor (P1005, Beyotime, China) and phosphatase inhibitor (P1081, Beyotime, China). After centrifuging at 12000 × g for 15 min at 4°C, the concentration of the supernatant was detected by a BCA kit (23225, Thermo Fisher Scientific, USA). And then, Western blot assay was carried out in a routine process [22 (link)]. The results were visualized using ECL substrate reagent kit (32209, Thermo Fisher Scientific, USA) or detected by exposure to a film. The primary antibody dilution is as follows: cleaved-PARP (1 : 1000), caspase 3 (1 : 1000), cleaved-caspase 3 (1 : 500), cleaved-caspase 9 (1 : 500), BAX (1 : 1000), cytochrome c (1 : 1000), LC3B (1 : 500), SQSTM1 (1 : 1000), mTOR (1 : 1000), phospho-mTOR (1 : 1000), AMPK (1 : 1000), phospho-AMPK (1 : 1000), Beclin1 (1 : 1000), ULK1 (1 : 1000), LAMP2 (1 : 1000), LAMP1 (1 : 1000), and ACTB (1 : 1000). Anti-rabbit IgG was diluted at a ratio of 1 : 2000. Primary antibody was incubated overnight at 4°C, and secondary antibody was incubated for 1 hour.