Its supplement

Primary hepatocytes were isolated from male WT and KO mice (3-month-old) by using a two-step collagenase perfusion method as before [40 (link)]. 1 × 10⁶ cells/well were seeded on collagen-coated 6-well plates. Four hours after attachment, cells were switched to serum-free media containing 1% ITS supplement (Invitrogen, Carlsbad, CA, USA) and cultured overnight. Hepatocytes were treated with Tm (5 and 10 μM) with the indicated time and harvested for further experiment.

The iPSCs from a 20-year-old male donor were generated as previously described^{31 (link)}. For the step-wise neural differentiation^{32 (link)}, iPSCs were detached from feeder cells with dispase (0.5 mg/ml, Invitrogen) and transferred onto Ultra-Low adhesion plates in differentiation medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) for EB formation. After 6 days in the suspension cultures, the EBs were plated onto gelatin-coated culture dishes to allow for EB attachment. The medium was replaced for 8 weeks of neural differentiation with N2 medium consisting of DMEM/F12 medium supplemented with non-essential amino acids, L-glutamine, ITS supplement (Invitrogen), and human basic fibroblast growth factor (bFGF, 10 ng/ml). To characterize the neural progenitors, cell clusters with early neuroepithelial rosette-like structures were dissected, collected under a dissection microscope, and analyzed by RT-PCR and immunocytochemistry. For the co-cultures or transplantation experiments, the neurospheres from clone i201 were washed and resuspended in serum-free DMEM/F12 medium. The aliquots were adjusted to a concentration of 5 × 10⁴ cells/µl.

FLAG-tagged ULK3 and GLI2, Shh and His-tagged ULK3-Ubi proteins were purified as previously described [6] (link), [39] (link), [40] (link). SU6668 was dissolved in DMSO (both from Sigma-Aldrich, Steinheim, Germany) and stored at − 70 °C prior use. Human recombinant TGF-β1 and TGF-β3 were purchased from PeproTech (Rock Hill, NJ, USA). Human insulin, dexamethasone (DEX), IBMX, indomethacin and ascorbate-2-phosphate were purchased from Sigma-Aldrich, while ITS supplement was purchased from Gibco, Invitrogen (Carlsbad, CA, USA).

Human HepG2 cells were cultured in DMEM/L media (Gibco, USA) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (P/S) (Invitrogen, USA) at37°C and 5% CO2. The immortalized mouse normal hepatocyte AML12 cells were cultured in DMEM/F12 media (Gibco) containing 10% fetal bovine serum, ITS supplement (Gibco), 40 ng/ml dexamethasone, and 1% P/S (Invitrogen) at 37°C and 5% CO2. For the cell experiments, the cells were treated with or without 2 mM metformin in serum-free media containing 30 mM glucose, 100 nM insulin, and 0.25% bovine serum albumin for 24 h.