Ant β actin

Anti-PARP, anti-caspase 3, anti-ACC, anti-p-AMPK, anti-AMPK, anti-p-mTOR, anti-mTOR, anti-p-P70S6K1, anti-P70S6K1, anti-cyclin D1, anti-cyclin E1, anti-p53 and ant-β actin antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-CDK2, anti-CDK6, anti-Bcl₂, anti-BAX, and p21 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgGs were purchased from Zymed Laboratories (San Francisco, CA, USA). The compound C was purchased from Calbiochem (San Diego, CA, USA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), propidium iodide (PI), JC-1 and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The HsA was kindly provided by Prof. Jong Rok, Lee (Daegu Haany University, Gyeongsan, Korea), and isolated as previously described [23 (link)]. The pure HsA (1.31 g) was isolated from the chloroform extract (150 g) of H. lyrata, and was stored at −20 °C. The purity (>97%) was verified by comparing retention time with standard compounds.

Western blotting (WB) was performed to explore the YAP and EMT marker protein expression changes in H146 and H446 cells after STAT3 overexpression. Total cell lysates from the SCLC cell lines were extracted with lysis buffer (M-PER Mammalian Protein Extraction Reagent; Thermo Fisher Scientific Inc., Rockford, IL, USA). The proteins were run on 4–20% gradient SDS–polyacrylamide gels (Bio–Rad Laboratories, Inc., Hercules, CA, USA) and then transferred to Immobilon-Pnitrocellulose membranes (Millipore, Bellerica, MA, USA). The membranes were probed with primary antibodies purchased from Cell Signaling Technology Inc., Danvers, MA, USA, including anti-stat3 (#30835), anti-p-STAT3 (#52075), anti-YAP (#8418), anti-LATS (#3477), 1anti-E-cadherin (#3195), anti-N-cadherin (#13116), ant-vimentin (#5741), and ant-β-actin (#5057). Horseradish peroxidase-conjugated goat anti-rabbit antibody was used as the secondary antibody (Santa Cruz Biotechnology). Chemiluminescence detection was carried out using ECL Plus (GE Healthcare, Piscataway, NJ, USA) and executed according to the manufacturer’s instructions.