Chemotherapy-Resistance Sphere Formation

The chemotherapy drug-treated or -untreated cells were collected for sphere formation assays. GANT61 at a final concentration of 5 μM was added simultaneously into the culture medium as the cells were seeded in the relative groups. Sphere formation assays were performed as previously described with minor modifications.^{24 (link),25 (link)} Single-cell suspensions (5 × 10³ cells per well) were seeded into serum-free DMEM/F12 medium (Cat# C11320033, Gibco, USA) supplemented with 20 ng/mL Animal-Free Recombinant Human EGF (EGF, Cat#AF-100-15, Peprotech, USA), 10 ng/mL Recombinant Human fibroblast growth factor-basic (bFGF, Cat#100-18B, Peprotech, USA) and B-27 supplement (Cat# 17504044, Gibco, USA) in ultra-low-attachment plates (Cat#3471, Corning, USA) and cultured for 10 days. The number of spheres formed under three different fields were evaluated under microscopy. Colony diameters >50 μm were counted as a single positive colony. For all sphere formation experiments, three wells were run for each group. All experiments were performed in triplicate.