Chir99021

Purified human endometrial EpCAM⁺ and EpCAM⁻ cells were seeded onto 12-well plates and cultured in TEM. This was based on DMEM/F12 (Invitrogen) supplemented with ITS or N2 (both from Invitrogen), and the following factors: 20 ng/ml EGF (Peprotech), 10 μM Y27632, 3 μM CHIR99021, 1 μM A8301 (all from TargetMol), 1 μM S1P and 5 μM LPA (both from Santa Cruz). TEM was changed every 2 days. When the cells reaching 80–90% confluence, clonal cells were passaged at a ratio of 1:3 after dissociation with 0.25% trypsin/1 mM EDTA. To assess population doubling time, 10,000 cells were seeded on 24-well plates and counted on designated days, following dissociation with 0.25% trypsin/1 mM EDTA to obtain single cells. Population doubling time was calculated as following: log2/ (logNt – logN0), t = time period (h), Nt = number of cells at time t, N0 = initial cell number.

The TβRI inhibitor LY2157299 (Interchim, LSK040) was added to the cells at a final concentration of 10 µM. The SMAD3 inhibitor (E)-SIS3 (MedChemExpress, HY-13013) was used at a final concentration of 10 µM. Cells were treated with 0.5 µM PD184352 (Sigma Aldrich, PZ0181), a MEK inhibitor, 10 µM SP600125 (Sigma Aldrich, S5567), a JNK inhibitor, 10 µM SB203580 (Sigma Aldrich, S8307), a p38 inhibitor and 0.1 µM wortmannin (Sigma Aldrich, W1628), a PI3K inhibitor. All treatments with the inhibitors were performed 1 h prior the addition of TGFβ1. The GSK3 inhibitor CHIR-99021 (TargetMol, T2310) and the tankyrase inhibitor XAV-939 (TargetMol, T1878) were used at 10 µM final concentration. The SMARCA2/4 bromodomain inhibitor PFI-3 (MedChemExpress, HY-12409) and the allosteric dual SMARCA2 and brahma-related gene 1 (BRG1)/SMARCA4 ATPase activity inhibitor BRM/BRG1 ATP Inhibitor-1 (MedChemExpress, HY-119374) were administrated to cells at 10 and 5 µM final concentrations, respectively. Dimethyl sulfoxide (DMSO) was used as a vehicle control treatment.

Six-week-old male BALB/c nude mice were obtained from Shanghai SLAC Laboratory Animal Center, Shanghai, China. All mice were maintained according to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, USA. SMMC7721 cells (1 × 10⁶ cells) were injected subcutaneously into the armpit area of each mouse. When the tumor volume reached 20–80 mm³, 100 mg/kg day 3-HAA and/or 10 mg/kg day sorafenib was administered intraperitoneally daily for 16 days. CHIR99021 (TargetMol, Cat: T2310) was administered orally at 40 mg/kg day. The tumor volume was determined at the indicated time points using digital caliper measurements and calculated by the following formula: $\mathrm{Tumor}\mathrm{volume}\left({\mathrm{mm}}^3\right)=1/2\times \mathrm{longest}{\mathrm{diameter}}^2\times \mathrm{shortest}\mathrm{diameter}$ At the indicated time points, the mice were sacrificed, the tumors were excised, and tumor weight was measured.
The study protocol was approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine and animal study was carried out in accordance with established national and institutional guidelines on the use of experimental animals.

Viability of purified hepatocytes was around 90% as determined by Trypan blue (Sigma-Aldrich). The cells were plated on a Matrigel-coated (Corning) culture dish at 0.5-2 × 10⁴ cells per cm² and cultured in modified TEM.^{8 (link)} This was based on Advanced DMEM/F12 (Invitrogen) supplemented with N2 and B27 (Both from Invitrogen), 1 mM sodium pyruvate (Invitrogen), 10 μg/ml ascorbic acid (Sigma-Aldrich) and the following factors: 20 ng/ml HGF, 20 ng/ml EGF (both from Peprotech), 10 μM Y27632, 3 μM CHIR99021, 1 μM A8301 (all from TargetMol), 1 μM S1P and 5 μM LPA (both from Santa Cruz). 6-12 days after seeding, clonal cells were passaged at a ratio of 1:3-6 after dissociation with Accutase (eBioscience). Medium was changed every other day. To assess population growth and doubling time, 1000 cells were seeded on 6-well Matrigel-coated plates and counted on the designated days, following dissociation with TrypLE Express (Invitrogen) to give single cells. In each experiment, the cell number was determined based on 3 technical replicates. Population doubling time was calculated using the web tool provided by http://www.doubling-time.com/compute.php.

For neural ectoderm differentiation, mESCs were adapted on gelatin-coated plate in N2B27 medium (50% DMEM/F12 (Hyclone, SH30023.01), 50% Neurobasal (Gibco, 21103049), N2 (Gibco, 17502048), B27 (Gibco, 17504044), NEAA (Gibco, 11140050), GlutaMAX (Gibco, 35050061), Sodium Pyruvate (Gibco, 11360070), 100 μM β-mercaptoethanol (MERCK, M6250)) + 2iL (1 μM PD0325901 (TargetMol, T6189-1 mL), 3 μM CHIR99021 (TargetMol, T2310-2 mg), 1000 units/mL mLIF (Millipore, ESG1107)) for a minimum of 4 days, and then the cells were seeded on gelatin-coated 6-well plate in N2B27 medium for 48 h. Afterwards, cells were treated with 500 nM retinoic acid (RA) in N2B27 medium for 4 days before subjected for subsequent analysis. Medium was changed every 2 days.

PRDM14 (#83527, Cell Signaling Technology), ACTB (AC026, ABclonal), β-catenin (ab32572, Abcam), GATA3 (ab199428, Abcam), GATA3 (ab282110, Abcam), KRT7 (ab68459, Abcam), TFAP2C (ab218107, Abcam), HAND1 (D263110, Sangon Biotech), total H3 (AF0009, Beyotime), H3K27me3 (#9733, CST), SUZ12 (#3737, CST), TACSTD2-PE (363803, BioLegend), SIGLEC6-APC (FAB2859A, R&D), goat anti-mouse IgG (H + L) (115-005-003, Jackson ImmunoResearch), goat anti-rabbit IgG (H + L) (111-005-003, Jackson ImmunoResearch), goat anti-mouse IgG H&L (Alexa Fluor® 488) (ab150113, Abcam), goat anti-rabbit IgG H&L (Alexa Fluor® 647) (ab150079, Abcam), GSK126 (T2079, TargetMol), LGK974 (T2618, TargetMol), A83-01 (HY-10432A, MCE), CHIR99021 (T2310L, TargetMol), EGF(AF-100-15, Peprotech), Y27632 (T1725, TargetMol), BMP4 (120-05, Peprotech), TSA (T6270, TargetMol), and Vitamin C (Vc) (HY-B0166, MCE) were obtained.

For DKK1 Inhibitors, Gallocyanine was from Santa Cruz, WAY262611 was from TargetMol. STAT3 inhibitor III (WP1066) was from Santa Cruz. EGF and bFGF recombinant proteins were from PeproTech. The DKK1 recombinant protein was purchased from Abcam. Insulin was purchased from Sigma-Aldrich. The Wnt/β-catenin pathway inhibitor XAV939 and activator CHIR99021 were from TargetMol. The ferroptosis inducer Erastin and RSL3 were from MedChem Express. The liproxstatin-1 was from TargetMol. BODIPY-C11 (D3861) was from ThermoFisher. ALDEFLUOR™ Kit (Cat#01700) was from Stem Cell. ChIP Assay Kit (Cat#P2078) was from Beyotime. Human DKK1 Quantikine ELISA Kit (Cat#DKK100B) were from R&D System.