Morusin

Span 60 (sorbitan monostearate) and cholesterol were procured from Tokyo chemical industry Co. Ltd. Morusin was purchased from Chemfaces. Chloroform was purchased from Kanto chemicals, Japan. Human triple negative breast cancer (MDA-MB-453), colorectal adenocarcinoma (HT-29), ovarian cancer (SKOV-3), pancreatic cancer (PANC-1) cell lines and Mc-coy medium were purchased from American Type Culture Collection (ATCC), USA. Mouse fibroblast cell lines (L929) was purchased from Riken BioResource Center, Japan. Dulbecco's modified Eagle's medium (DMEM), trypsin (0.25%), fetal bovine serum (FBS), penicillin (5000 μg ml⁻¹)/streptomycin (5000 μg ml⁻¹) and phosphate buffer saline (PBS) were procured from Gibco (Life Technologies). Alamar blue cell viability kit reagent was purchased from Invitrogen, USA. Live/dead viability/cytotoxicity kit, Leibovitz media, Lysotracker deep red and Nuc blue live ready probes reagent were acquired form Thermo Fisher Scientific. All other chemicals and solvents were of analytical grade.

Morusin was purchased from ChemFaces (Wuhan, China) and dissolved in dimethyl sulfoxide (DMSO) at 100 mM as a stock solution. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was bought from Duchefa (Haarlem, The Netherlands). Trypan blue was purchased from WelGENE (Daegu, Korea). Compound C, 4,6-diamidino-2-phenylindole (DAPI), DMSO, propidium iodide (PI), RNase A, and bafilomycin A1 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against autophagy-related 5 (Atg5), Beclin-1, LC3, phospho-AMPK (Thr172), AMPK, phospho-acetyl-coA carboxylase (ACC; Ser79), ACC, phospho-mTOR (Ser2448), and mTOR were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies against Atg12, phospho-p70S6 kinase (p70S6K, Ser434), p70S6K, and actin were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Goat anti-mouse (Bethyl Laboratories, Montgomery, TX, USA) and goat anti-rabbit antibodies (Enzo Life Sciences, Farmingdale, NY, USA) were used as secondary antibodies.