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Cell migration and invasion assay 

The ESCs were starved for at least 24 h. After resuspending them in serum-free  DMED/F12 media, 200 µL was inoculated on the upper part of a transwell chamber to  perform a migration assay or on the Matrigengel for invasion assay (NovaMedica,  Shanghai, China.). The lower chamber was added with 700 ul of DMEM/F12  containing 10% fetal bovine serum and incubated at 37°C and 5% CO2 for 24 hours.  Subsequently, the ESCs in the lower chamber were fixed using 4% paraformaldehyde  for 30 minutes and stained with 0.5% crystal violet for 30 minutes. Each chamber was  recorded with a fluorescence microscope (Nikon, Tokyo, Japan) with three random  fields of view and quantified using ImageJ software. The average value representing  the whole chamber was calculated from the three different fields of view.