The cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 0.1 mM phenylmethylsulfonyl fluoride (no. ST505; Beyotime, Shanghai, China) and 1 mM protease inhibitor cocktail (no. B14001; Bimake, Houston, TX). The cytoplasmic and nuclear protein extraction was performed according to the instructions described in the Nuclear and Cytoplasmic Protein Extraction Kit (no. P0027; Beyotime, Shanghai, China). For IP, 1 mg of total protein was incubated overnight with 3 to 5 μg of antibody at 4°C and then with Protein A/G PLUS-Agarose (no. sc-2003; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C for an additional 4 hours; then, the precipitates were washed once with RIPA and three times with PBS and resuspended in 40 μl of 2× SDS loading buffer, and immunoblotting was performed via standard protocols. The following antibodies were used for immunoblotting and/or IP: Nanog (1E6C4) (no. sc-293121; Santa Cruz Biotechnology, Santa Cruz, CA), Brachyury (T) (no. AF2085; R&D Systems; Minneapolis, MN), Gata6 (no. 5851; Cell Signaling Technology, Danvers, MA), SUZ12 (D39F6) XP (no. 3737; Cell Signaling Technology, Danvers, MA), EED (no. 16818-1-AP; Proteintech, Rosemont, IL), Bach1 (no. sc-271211; Santa Cruz Biotechnology, Santa Cruz, CA), Bach1(no. AF5776; R&D Systems, Minneapolis, MN), Sox2 (no. sc-17320; Santa Cruz Biotechnology, Santa Cruz, CA), Oct4 (no. sc-5279; Santa Cruz Biotechnology, Santa Cruz, CA), Usp7 (no. A300-033A-M; Bethyl Laboratories, Montgomery, TX), β-actin (no. sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA), H3K27me3 (no. 07-449; Millipore, Billerica, MA), H3K9me2 (no. 07-212; Millipore, Billerica, MA), H3K27ac (no. MABE670; Millipore, Billerica, MA), H3K9ac (no. 06-942; Millipore, Billerica, MA), EZH2 (no. 5246; Cell Signaling Technology, Danvers, MA), H3 (no. 4620S; Cell Signaling Technology, Danvers, MA), Nodal (no. ab109317; Abcam, Cambridge, UK), Wnt3 (no. ab172612; Abcam, Cambridge, UK), HA (no. sc-7392; Santa Cruz Biotechnology, Santa Cruz, CA), Flag (no. F1804; Sigma-Aldrich, St. Louis, MO), Fzd1 (no. sc-398082; Santa Cruz Biotechnology, Santa Cruz, CA), active β-catenin (no. 8814S; Cell Signaling Technology, Danvers, MA), total β-catenin (no. sc-7963; Santa Cruz Biotechnology, Santa Cruz, CA), ACVRIIB (no. sc-376593; Santa Cruz Biotechnology, Santa Cruz, CA), p-Smad2 (no. CY5859; Abways, Shanghai, China), p-Smad3 (no. CY5140; Abways, Shanghai, China), and Smad2/3 (no. 8685S; Cell Signaling Technology, Danvers, MA).

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