Ultra low attachment 24 well culture plates

The spheroid formation assay was performed following the published protocol (Dontu et al., 2003 (link)) with minor modifications. Briefly, single cells were plated in ultra-low attachment 24-well culture plates (Corning, NY) at a density of 2 × 10³ cells per well suspended in serum-free DMEM containing human recombinant basic fibroblast growth factor (bFGF, 20 ng/mL) (R&D, Minneapolis, MN), human recombinant epidermal growth factor (EGF, 20 ng/mL) (R&D, Minneapolis, MN), B27 (Invitrogen, Carlsbad, CA) and heparin (4 μg/mL, Sigma-Aldrich). Plates were incubated at 37°C in cell incubator with 5% CO₂. Spheres > 50 μm were viewed, photographed and counted under a phase-contrast microscope after 10-day culture.

The spheroid formation assay reflecting the stem cell property was performed following the published protocol (Shaheen et al., 2016 (link); Qiu et al., 2012 (link); Dontu et al., 2003 (link)) with minor modifications. Briefly, single cells were plated in ultralow attachment 24-well culture plates (Corning, Corning, NY) at a density of 2.5 × 10³ cells per well suspended in serum-free DMEM or MEM containing human recombinant basic fibroblast growth factor (bFGF, 20 ng/ml), human recombinant epidermal growth factor (EGF, 20 ng/ml) (R&D, Minneapolis, MN), B27 (50 times diluted from the original 50x stock solution, Invitrogen, Carlsbad, CA) and heparin (4 μg/ml, Sigma). Plates were incubated at 37°C in a humidified 5% CO2 atmosphere. For spheroid formation assays with G9a inhibitor BX01294 and EZh2 inhibitor DZNeP, Cr(VI)-transformed cells were treated with a vehicle control or BIX01294 (2.5 μM) or DZNeP (0.25 μM) for 72h, then collected by trypsinization and neutralization with culture media and used for spheroid formation assays. The same concentration of vehicle control or inhibitors was included in the sphere-forming culture media. Spheres were viewed, photographed and counted (if > 100 μm) under a phase-contrast microscope after 10-day culture.