Tet in c35

MEFs and 293T cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin. iHepSCs were cultured in SCM-A medium as previously reported [2 (link)]. Hepatic differentiation and cholangiocytic differentiation of iHepSCs were induced as described in our previously reported procedures [2 (link), 3 (link)]. TET-IN-C35 (AOB11121) was purchased from AOBIOUS, Inc. and was dissolved in DMSO to prepare a 5 mM stock solution. For the low-density lipoprotein (LDL) uptake assay, hepatic differentiated cells were incubated with 10 µg/mL DiI AcLDL (Invitrogen) for 4 h at 37 °C and then observed by fluorescence microscopy. Glycogen storage was detected by a periodic acid Schiff (PAS) staining kit (SJ1269, ShuangJian Biotech, Shanghai) according to the manufacturer’s user manual.

The heme inhibitor succinylacetone (SA) (Sigma, D1415) is used at a concentration of 0.5 mM. Hemin (Sigma, 51280) is used at a concentration of 10 μM in 0.1 N NaOH. Diethyl butylmalonate (BM) (Sigma, 112038) is used at a concentration of 1 mM. Atpenin A5 (Santa Cruz biotechnology, sc-202475) is used at 250 nM. SIS3 (Selleck chemicals, S7959) is used at 5 μM. NF-56-EJ40 (Axon Medchem 3056) is used at 1 μM. AZD3965 (Selleckchem S7339) is used at 1 μM. Exogenous succinate is provided as either 40 mM Sodium succinate (Sigma) or 16 mM dimethylsuccinate (Sigma) supplementation. HDM inhibition was achieved with 250 nM JIB04 (Medchem express HY-13953) and TET inhibition with 5 μM TETin-C35 (Aobious AOB11121). For retinoic acid experiments, RA, HY-14649, HX531 (HY-108521) and AGN193109 (HY-U00449) were used at 0.5 µM, 10 µM and 100 nM, respectively (all from Medchemexpress). Inhibition of Sirt7 is achieved using 5 µM of the Sirt7 inhibitor 97491 (MedChemExpress).