Fumagillin

Hepatoma cell lines; including Huh-7 and SK-HEP-1, were bought from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in RPMI1640 supplemented with fetal bovine serum (FBS, Lonza, Basel, Switzerland), 100 μg/mL streptomycin and 100 units/mL penicillin at 37°C in a humidified, 5% (v/v) CO₂ atmosphere. Cells were passaged every three days.
To enrich CSCs, cells were cultured in DMEM/F12 medium without FBS, and supplemented with 100 μg/mL streptomycin and 100 units/mL penicillin, 20 ng/ml human recombinant epidermal growth factor (EGF), 20 ng/ml human recombinant basic fibroblast growth factor (bFGF), and 2% B27 supplement (Invitrogen, USA) and seeded in 6-well cell culture plate with ultra-low attachment surface (BioFLOAT, USA). Medium was half-refreshed every three days.
To evaluate the effects of Fumagillin on cell viability, 1, 2.5, 5, 7.5 and 10 μmol/L of Fumagillin, bought from Sigma-Aldrich (USA) in stock at final concentration of 20 mmol/L, was added for 24-hour incubation. To evaluate the effects of Fumagillin on sphere formation, Fumagillin was added for 24–96 h. To evaluate the effects of Fumagillin in maintenance of stemness, Fumagillin was added into spheres.

The C. reinhardtii wild-type strain was obtained from the Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB-collection, Chinese Academy of Science, Wuhan, China). C. reinhardtii cells were cultured in liquid tris-acetate phosphate media (TAP) under 100 μmol (photons) m⁻² s⁻¹ white light with a 12 h photoperiod at 25 °C. Three-day-old cells in the logarithmic growth phase were collected for further experiments [26 (link)].
Fumagillin, mevastatin, radicicol, wortmannin, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), dimethyl sulfoxide (DMSO), and other chemical agents were purchased from Sigma-Aldrich (Shanghai, China). Four mycotoxins and DCMU stock solutions were dissolved in 100% DMSO and further diluted with sterile water. The final concentration of DMSO in the working solutions of all chemical agents was less than 1% (v/v).

The IPL-LD 65Y cell line (Lymantria dispar) was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany, No. ACC 181) and was maintained for routine culture in TC-100 medium (Lonza, Basel, Switzerland) with 11% fetal calf serum (FCS, PromoCell, Heidelberg, Germany). The cells were seeded with an initial concentration of 2E+05 cells per ml in tissue culture flasks (Greiner bio-one, Frickenhausen, Germany) and incubated at 27°C in a cooling incubator (Thermo Fisher Scientific, Schwerte, Germany). Cells were routinely passaged every seventh day.
Several commercially available substances were used in the screening assay. Fumagillin, surfactin, paromomycin, and metronidazole were obtained from Sigma-Aldrich (Taufkirchen, Germany); quinine, ornidazole, albendazole, tinidazole, clioquinol, and dimethylsulfoxid (DMSO) were obtained from VWR International (Darmstadt, Germany).