Dnmt1

Endometrial cancer cells were lysed with cold lysis buffer, and proteins were extracted followed by quantification of protein concentration. Equal amounts of proteins from each group were purified with 10% SDS-polyacrylamide gels, and then transferred to a polyvinylidene difluoride (PVDF) membrane, followed by blockading of non-specific binding by incubation with 5% skim milk over 1 h. The membrane was next incubated with primary antibodies overnight at 4 °C, and then treated with horseradish peroxidase-conjugated secondary antibody for 1 h. The ECL reagent (Millipore Corp.) was used for detecting antibody-reactive bands. The housekeeping proteins α-tubulin or β-actin was used as loading control. Primary antibodies against POLD1, JNK, p-JNK, c-JUN, and FOSL1 were purchased from Santa Cruz Biotech (Dallas, USA); primary antibodies against PGK1, HSP90, ERK, p-ERK, AKT, p-AKT, DNMT1, DNMT3A, DNMT3B, and SPARC were purchased from Cell Signaling Technology (Boston, USA); primary antibodies against Bcl-xL, Mcl-1, and Bax were purchased from Abcam (Cambridge, UK).

Antibodies to H3K27me3, JMJD3, p-Smad3, Smad3, p-AKT, AKT, p-ERK1/2, ERK1/2, Notch1, Jagged-1, UTX and DNMT1 were purchased from Cell Signaling Technology (MA, USA). Antibodies to fibronectin, α-SMA, collagen III, JMJD3, TGF-β1, Notch3 were purchased from Abcam (MA, USA). An antibody to Smad7 was obtained from Santa Cruz Biotechnology (CA, USA). An antibody to β-actin, PTEN and FBXW7 were obtained from proteintech (Wuhan, China). β-tubulin was purchased from Sigma-Aldrich (MO, USA). GSKJ4 was purchased from Selleck (Houston, USA). DMSO and other chemicals were obtained from Beyotime (Shanghai, China). Lipofectamine 3000 was obtained from Invitrogen-Thermo Fisher Scientific (CA, USA).