Matrigel

Undifferentiated iPSCs (1 × 10⁶) were mixed with Matrigel and injected subcutaneously into the dorsolateral area of specific pathogen-free/viral antibody-free immunodeficient mice (Orient Bio, Seoul, Korea). The mice were maintained under specific pathogen free (SPF) conditions and fed a sterilized pelleted diet and water. At 8 weeks after injection, the tumor tissues were dissected and fixed in 4% paraformaldehyde. Paraffin-embedded tissues were sectioned and then stained with hematoxylin and eosin.

All experiments were preapproved by the Institutional Animal Care and Use Committee of Sookmyung Women’s University. To explore the efficiency of specific siRNAs against target genes on tumor growth, CSLCs were reverse transfected under CSLC sphere culture condition for 3 d as described above. The indicated number of CSLC spheres cells was mixed with 40% Matrigel (Thermo Fisher Scientific) with EGF and basic fibroblast growth factor. The cells were subcutaneously injected into NOD/SCID (Koatech, Pyeongtaek, Korea) or BALB/c Nude (Orient Bio, Seongnam, Korea) mice, and the Matrigel-formed tumor sizes were measured every 2 to 3 d after injection.
To identify the efficiency of atorvastatin treatment, 5 × 10⁵ CSLC sphere cells were mixed with 40% Matrigel (Thermo Fisher Scientific) with EGF, basic fibroblast growth factor, and atorvastatin (10 nM) if needed. Atorvastatin in PBS was administered orally at 20 mg/kg once daily.
Tumor growth was assessed; the size of the tumor was measured with calipers 3 times per week, and tumor volume was estimated by width × width × length × ½, where width is the short axis and length is long axis. The diets of the mice were as follows: high-fat diet, 60% of calories from fat; low-fat diet, 10% of calories from fat; and high-fat diet + cholesterol (1.25%). All diets were obtained from DooYeol Biotech (Seoul, Korea).