Wnt3a

For in vitro studies, stock solutions of MLN8237 (20 mmol/L), FH535 (10 mmol/L) and LY294002 (20 mmol/L) were prepared in DMSO. Stock solutions of recombinant human Wnt3a (100 μg/ml), EGF (100 μg/ml) and TGF-β1(100 μg/ml) were diluted in phosphate-buffered saline (PBS).
Dimethyl sulfoxide (DMSO), LY294002 and EGF were purchased from Sigma. FH535 was purchased from Merck. Wnt3a was purchased from abnova. MLN8237 was purchased from selleck.
AURKA, β-catenin, AKT1, p-AKT1, GSK-3β, p- GSK-3β, Twist, H3K4 me3/AC and H3K27 me2/me3/AC antibodies were purchased from Abcam. E-cadherin, N-cadherin, H3K4 me1/me2 and H3K27 me1 antibodies were purchased from Cell Signaling Technology. The Ki67 antibody was purchased from Santa Cruz Biotechnology. The H3 antibody was purchased from Ray Antibody. The GAPDH antibody was purchased from Zhongshan Bio Corp.

FACS-sorted single endometrial cells were seeded in growth factor-reduced Matrigel (Corning, 356231) and cultured in Advanced DMEM/F-12 media supplemented with 10 mM HEPES, 2 mM Glutamax, 1x N2, 1x B27 (all Invitrogen), N-acetyl-cysteine (Sigma, A9165), Primocin (Invivogen, ant-pm1), 50 ng/ml EGF (Invitrogen, PHG0311), 100 ng/ml FGF10 (Peprotech, 100-18B), 100 ng/ml Noggin (Peprotech, 250-38-50), 100 ng/ml Wnt3a (Millipore, GF-160), 1 μg/ml R-spondin (Peprotech,120-38-500), 50 ng/ml HGF (Peprotech, 100-39H), 2 μM A83-01 (Tocris, 2939) and 10 mM nicotinamide (Sigma, N0636). 10 μM Rock-inhibitor Y27632 (Sigma, Y0503) was also added to single-cell cultures. Culture medium was changed every other day. After 7–10 days of culture, organoids were harvested for analysis. DT was added at a concentration of 0.25 ng/μl to the culture medium to induce Lgr5⁺ cell ablation at the indicated time point. 20 μM CHIR 99021 (Tocris, 4423) and 100 μM ETC159 were, respectively, added for differentiation experiments. Wnt4 (R&D, 6076) and Wnt7b (Abnova, 7477-P) were added at 100 ng/ml to substitute for Wnt3a in indicated experiments.