4.1. Cell Culture, Reagents and Antibodies

Human lung cancer cell lines (A549, H446 and 95-D) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and cultured in DMEM containing 10% fetal bovine serum (FBS) and a 100 U/mL mixture of penicillin and streptomycin. All cells were kept at 37 °C and 5% CO2. Cyclovirobuxine D (CVB-D) was purchased from the Chengdu Alpha Biotechnology Incorporation and dissolved in methanol to generate a 70 mM stock solution. The primary antibodies for Western blotting, immunoprecipitation and immunohistochemistry against target proteins are as follows: caspase3 (Zen Bioscience, 300968, Chengdu, China), cleaved-caspase3 (Zen Bioscience, 380169), PARP (Zen Bioscience, 380451), cleaved-PARP (Zen Bioscience, 380374), VDAC1 (Sangon Biotech, D124100, Shanghai, China), TOMM20 (Sangon Biotech, D153158), COX4I1 (Sangon Biotech, D262690), LC3 (Sangon Biotech, D163557), ATG5 (Sangon Biotech, D121650), SQSTM1/p62 (Beyotime, AF5312), BNIP3 (Zen Bioscience, 381756), BNIP3L (Zen Bioscience, 381891), β-actin (Santa Cruz, sc-47778), p65 (Cell Signaling Technology, 8242S, Danvers, MA, USA), CDC2 (Cell Signaling Technology, 9116S), cyclinB1 (Cell Signaling Technology, 4135S), p-BNIP3L (phosphoS81) (abcam, ab208190, Cambridge, UK) and phosphoserine (Millipore, AB1603, Burlington, MA, USA).