ESCs were treated or not (CTRL) for 4 days with BMP2 (10 ng/ml) and (A) monitored by flow cytometry using a FITC-conjugated anti–SSEA-1 antibody or (B and C) separated using the anti–SSEA-1 Miltenyi kit and the MACS columns. SSEA-1– and SSEA-1+ cDNAs were run in real-time PCR. Data are from 8 experiments, performed on the HUES-24 cell line, and reproduced in different HUESC lines (H9, HUES-9, HUES-24, HUES-26, I3, and I6). The inset in C illustrates SSEA-1+ cells immunostained with anti-Tbx6 and anti-Mesp1/2 antibody 12–24 hours after sorting. (D and E) ChIP assay using (D) anti-H3triMeK4 or anti-triMeK27 antibodies or (E) the anti-PolII or serine-phosphorylated PolII (Ser5-PPolII, left) antibodies. Q-PCR was used to amplify the chromatin-bound DNA, using primers specific for OCT4, TBX6, ISL1, MEF2C, NKX2.5, ACTC1, and PAX4 promoters (sequence within the 700 bp in 3′ of ATG; Table 2). Data (n = 5) show fold enrichment of methylated histones on promoters in the SSEA-1+ versus SSEA-1– population. The gels illustrate the specificity of PCR products (input, SSEA-1+ DNA samples from anti-triMeH3K4 IP, anti-rabbit IgG, or no antibody). OCT4IA, OCT-4 isoform A. (E) The left side shows PCR gel of DNA products after real-time PCR and indicates a complete loss of serine-phosphorylated PolII on both the NANOG and SOX2 promoters. ChiP experiments have been mostly performed using the HUES-24 cell line and were validated in 2 other experiments using the I6 cell line. *P ≤ 0.05; #P ≤ 0.01.