Reproff2 medium

For sphere-forming assay, we used ReproFF2 medium (Repro cell, Kanagawa, Japan), which is a new feeder-free culture medium for human ES/iPS cells. We also used the Ultra-low attachment surface (Corning, Corning, NY, USA), which is coated with a hydrogel layer and prevents attachment of cancer cells growing in an anchorage-dependent manner.^{40 (link)} AGS and HepG2 cells were cultured in serum-free ReproFF2 medium containing 5 ng/ml bFGF with 5 μM DZNep or 1 μM SAHA in a 96-well plate with an Ultra-low attachment surface. The AGS and HepG2 cells were plated at 200 and 500 per well, respectively. Fresh culture medium was added on day 5 of the culture period. The number of spheroids was counted 10 days after drug treatment. Experiments were carried out in triplicate.

Vero cells were cultured in low-glucose DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 2% fetal calf serum (FCS). 293T cells were cultured in high-glucose DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FCS. Human iPSCs 110 (BYS0110), 112 (BYS0112), and 116 (BYS0116), were obtained from ATCC (Manassas, VA, USA). BMI and BM9 were obtained from WiCell (Madison, WI, USA). 201B7 and 409B2 were obtained from the Riken Bioresource Center (Ibaraki, Japan). Epi was obtained from Thermo Fisher Scientific. Epi, BMI, BM9, 110, 112, and 116 were maintained on Corning Matrigel hESC-Qualified Matrix (CORNING, Corning, NY, USA) in mTeSR 1 medium (STEMCELL Technologies, Vancouver, BC, Canada). 201B7 and 409B2 were cultured on vitronectin (Thermo Fisher Scientific) in ReproFF2 medium (ReproCELL, Kanagawa, Japan) supplemented with 5 ng/mL basic fibroblast growth factor (bFGF; ReproCELL). T-705, Y27632, and puromycin were purchased from Selleckchem (Houston, TX, USA), FUJIFILM Wako Pure Chemical (Osaka, Japan), and InvivoGen (San Diego, CA, USA), respectively.