Ta2 fl

Transwell assays were used to detect cell migration and invasive ability. In the migration assay, TE-1 and KYSE-150 cells (2 × 10⁵/well) were suspended in serum-free RPMI-1640 medium and seeded into the top chamber. In the invasion assay, 2 × 10⁵ cells were seeded into the top chamber pre-coated with Matrigel (Corning, USA). The lower chamber was supplemented with 800 μl of RPMI-1640 containing 10% FBS. Following a 24 h incubation period, the cells in the lower chambers were stained with 0.1% crystal violet. The migrated and invaded cells were enumerated under the microscope (Nikon, Ta2-FL, Japan).

For monolayer hepatic-differentiation, iHepLPCs were seeded and kept in TEM for 2-4 days until they reached full confluence. The medium was then changed to modified Hepatocyte Maturation Medium (mHMM) 12 (link), 13 (link), 16 (link) and cultured for 9-15 days for further maturation; medium was changed every day. The modified HMM (mHMM) comprised of DMEM/F12 supplemented with N2 and B27, 10 μM DAPT (TargetMol), 30 μM Dexamethasone (Sigma-Aldrich), 10 μM SB431542 (TargetMol), and 10 μm Forskolin (TargetMol). Light microscopic images were captured with Nikon Ta2-FL.

Paraffin-embedded tissue sections (3μm) were deparaffinized and rehydrated in graded alcohol concentrations. After antigen retrieval using sodium citrate buffer, sections were incubated with anti-CYP3A4, anti-CYP2D6, anti-GSTA2 and anti-MRP2 (All from proteintech, USA) and anti-NTCP (Aviva Biosystems, USA) antibodies overnight at 4^oC. Stained tissue sections were visualized using HRP Conjugated goat anti-Rabbit IgG (H+L) secondary antibody and 3, 3-diaminobenzidine (DAB) (Both purchased from DAKO, Denmark). For monolayer culture, differentiated and undifferentiated iHepLPCs cells were cultured in 12-well plates and then fixed with 4% Paraformaldehyde. After blocking and permeabilization, cells were incubated with anti-CYP3A4 (proteintech, USA) and anti-HNF4a (Abcam, UK) antibodies overnight at 4^oC, and then visualized using Alexa Fluor 488 Conjugated goat anti-Rabbit IgG (H+L) secondary antibody or Alexa Fluor 555 Conjugated Donkey Anti-Mouse IgG(H+L) secondary antibody and 4',6-diamidino-2-phenylindole (DAPI) (Both purchased from Beyotime Biotechnology, China). PAS staining of the monolayer culture was carried using the Glycogen Stain kit (Nanjing Jiancheng Bioengineering Institute), following the instructions provided by the manufacturer. Images were captured with Nikon Ta2-FL.