Cell Cycle Analysis Kit (no. C1052; Beyotime, Shanghai, China) was used in cell cycle assay. hESCs were harvested and fixed in 70% ethanol for 2 hours at 4°C and then stained with a solution containing propidium iodide (0.05 mg/ml), RNase A (1 mg/ml), and 0.3% Triton X-100 in the dark for 30 min. The percentage of cells in different phases of the cell cycle was examined by measuring the DNA content (propidium iodide intensity) with a flow cytometer (Beckman Coulter, Brea, CA), and populations of G1, S, and G2/M phase cells were determined with the ModFIT software. Each experiment was repeated three independent times.

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