3D Culture of HepG2 Cells

3D culture was performed as described previously with modification [26], [27]. In brief, the stable cell lines (HepG2-Empty, LAP, and LIP) were seeded at 4,000 cells/well in a 96-well plate in 50 μl Matrigel (Corning, Corning, NY, USA). Following solidification, 500 μl of advanced DMEM/F12 (Thermo Fisher Scientific) supplemented with 1× Glutamax and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (15 mM) was added and incubated at 37 °C for 2 weeks. The growth medium was replenished every other day. The 3D spheroids were recovered by digesting the Matrigel with DispaseI (1,000 PU/ml, Wako Pure Chemical Industries), fixed overnight in 4% paraformaldehyde (Wako Pure Chemical Industries), and then embedded in 2% bacto-agar and 2.5% gelatin and subjected to histologic analyses. Hematoxylin and eosin (H&E) staining was performed as described previously [28]. Data analysis was performed with NanoZumer Digital Pathology View 2 (Hamamatsu Photonics, Hamamatsu, Japan).