Ldn 193189

Cells were cultured in iMEM with 1% P/S, 0.5× B27, 1% Pluronic® F-68, 0.25 μM SANT-1 (Merck Millipore), 50 nM retinoic acid (Merck Millipore), 10 μM ALK5 inhibitor II (ALK5iII, Santa Cruz) or the alternative candidates, 100 nM LDN-193189 (MedChemExpress, Monmouth Junction, NJ, USA), 1 μM L-3,3′,5-triiodothyronine (T₃, Merck Millipore), 50 ng/mL basic fibroblast growth factor (bFGF, PeproTech), 1 μM XAV939 (Merck Millipore), and 10 μM Y-27632 for 2 days.

Neural induced differentiation Media (NGD) and neural induction medium (NIM) were prepared according to previous reports [43 (link), 44 (link)]. Compound C (Medchem Express LLC, Monmouth, UK, 2.5 μM), FGF2 (Sino Biological Inc., Beijing, China, 20 ng/mL), EGF (Sino Biological Inc., 20 ng/mL), SB431542 (Medchem Express LLC., 10 μM), DMH-1 (Medchem Express LLC., 2 μM), LIF (Sino Biological Inc., 10 ng/mL), LDN193189 (Medchem Express LLC., 0.25 μM) and insulin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China, 10 ng/mL) were added to medium as described previously [6 , 43 (link)–48 (link)]. iPSCs^TLX were seeded onto a matrigel-coated 6-well plate at a density of 2.5 × 10⁴ cells/cm². The medium was changed to a differentiation medium on the next day. On day 6, cells were digested by Accutase™ for 7 min at 37 °C. Then iNSCs^TLX were seeded into a vitronectin-coated 6-well plate at a density of 2.5 × 10⁴ cells/cm² and passaged every five days.

For cell migration assay, 2 × 10⁵ Hep-G2 or Huh-7 cells suspended in serum-free medium were directly planted in the Transwell upper chamber after transfection of target plasmids, and the chamber was kept in cell culture medium supplemented with 20% serum. For the cell invasion assay, cells were seeded in Matrigel basement membrane matrix in the Transwell chamber. After 48 h of incubation, cells were fixed with 4% neutral formaldehyde for 20 min, followed by staining with 0.1% crystal violet for 10 min and washing with PBS. Migration and invasion were observed via an inverted microscope. For RBS6KB1 or SMAD1 blocking, PF-4708671 (MedChemExpress, Shanghai, China) or LDN-193189 (MedChemExpress) was added to the culture medium at a final concentration of 10 μM and 0.5 μM, respectively.

KGN (Selleck Chemicals, TX, USA) was dissolved with dimethylsulfoxide (DMSO) as the stock solution. Cells treated with 0.005% DMSO were used as controls. Other reagents applied in the study are listed as follows: 3-methyladenine (3-MA; Selleck Chemicals, TX, USA) (5 mM for 24 h) was used as autophagy inhibitor. Both the signal inhibitor LDN-193189 (LDN; 0.5 μM for 12 h) and the activator SB4 (0.1 μM for 24 h) were purchased from MedChemExpress (MCE, NJ, USA).