Total histone 3

Whole-cell or nuclear protein extracts were prepared using the M-Per Mammalian Protein Extraction Reagent (Pierce Biotechnology, Woburn, MA) or the Nuclear Extraction Kit (Chemicon International, Temecula, CA) according to the manufacturer's instructions. Total protein (30 μg) and nuclear protein (10 μg) were loaded onto 10–20% SDS-PAGE gels, electrophoresed, and then transferred to nitrocellulose membranes. Antigen-antibody complexes were detected using the enhanced chemiluminescence blotting analysis system (Amersham Pharmacia Biotech, Buckinghamshire, UK). The following antibodies were used: EZH2 (Cell Signaling; 5246), H3K27me3 (Cell Signaling; 9733), total histone 3 (Cell Signaling; 9715), E-cadherin (GenScript; A01589), Vimentin (GenScript; A01189), Twist (Abcam; ab50887), p-AKT (Santa Cruz; sc-293125), AKT (Santa Cruz; sc-1618), GAPDH (Santa Cruz; sc-47724), YY1 (Santa Cruz; sc-7341) and lamin B1 (Santa Cruz; sc-20682). GAPDH (whole cell lysate) and lamin B (nuclear protein) were applied as loading controls. Primary antibodies were used at a dilution of 1:1000.

Western blot were performed as described previously^{5 (link),54 (link)}. All of the antibodies used for the experiments were obtained from commercial sources, as follows: LSD1, mono-methylated histone 3 lysine 4, total histone 3, and GSK3β were from Cell Signaling Technology (Danvers, MA, USA); calmodulin binding peptide tag (CBP) was from Millipore (Billerica, MA, USA); halo tag was from Promega (Madison, WI, USA); actin and STIP1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All corresponding horseradish peroxidase-conjugated antibodies were obtained from Santa Cruz Biotechnology. Enhanced chemiluminescence reagents were from Millipore. The signal intensity of autoradiograms was quantified using the ImageJ software (https://imagej.nih.gov/ij/) after normalization with the corresponding actin signal intensity.