Dnmt1

ChIP analysis was performed as described previously 20 (link). Briefly, after being crosslinked by 1% formaldehyde for 10 minutes at 37 °C, the cells were resuspended in 300 mL of lysis buffer and sonicated for 10 minutes. The supernatants were incubated with specific antibodies against UHRF1 (sc-373750, Santa Cruz biotechnology, USA), KLF6 (sc-7158, Santa Cruz biotechnology, USA), DNMT1 (sc-271729, Santa Cruz biotechnology, USA), H3K9me2 (A2359, ABclonal, USA), or immunoglobulin G control (Millipore, USA). The immunoprecipitated DNA was then purified using a DNA purification kit (QIAGEN, Germany) and subjected to PCR amplification. For re-ChIP assays, after being combined with protein A agarose beads and the indicated primary antibodies, the complexes were washed and sequentially eluted from the first ChIP by incubation with 10 mM DTT in 1× TE for 30 minutes at 37 °C. The DNA-protein-antibody complexes were then diluted 20 times with dilution buffer and subjected to a second round of immunoprecipitation with the indicated antibodies. After elution and DNA purification, extracted DNA was analyzed by PCR using primers spanning the proximal promoter regions of target genes. The PCR products were normalized to the input. The specific primers are listed in the Table S2 (Supplymentary Information).

Tamoxifen (4-hydroxyTamoxifen, 4-OHT/TAM) and 5-aza (5-aza-2′-deoxycytidine, 5-aza-dC) were purchased from Sigma-Aldrich (St Louis, Missouri, USA). Indicated cells were treated with 5 μmol/L TAM for 48 h. E-cadherin, vimentin, ER-α, PR, and Her-2 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Actin, p-gp, DNMT1 and DNMT3a antibodies were purchased from Santa Cruz Biotechnology (USA).

Total cell proteins were extracted using the mammalian protein extraction reagent including halt protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The protein concentrations were measured by BCA protein assay kit (Thermo Fisher Scientific) and equal amount of proteins were subjected to SDS-PAGE, then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocked with 5% nonfat milk, the membranes were incubated with primary antibodies against DNMT1, BCAT1, and GAPDH (Santa Cruz), respectively, and followed by incubation with the HRP-conjugated secondary antibodies. The signals were detected using an enhanced chemiluminescence kit (GE Healthcare Life Sciences, Pittsburgh, PA, USA).

Cells were washed with ice-cold PBS and lysed on ice with a protease inhibitor cocktail. Protein concentrations were measured by BCA method. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. The membranes were probed with NRF2 (1:500), HO1 (1:1000), SOD1 (1:1000), GST (1:1000), DNMT1 (1:500), DNMT3a (1:500), DNMT3b (1:500), MeCP2 (1:500), MBD2 (1:500), GAPDH (1:10000) antibodies (Santa Cruz Biotechnology, Inc., Texas, United States) at 4°C overnight. The bands were visualized after incubation with a chemiluminescent substrate. Quantification of the band density was determined by densitometric analysis.

Frozen blocks containing the hearts, aortic arches, and carotid arteries were prepared in TissueTek and stored at −80 °C. Sections from the blocks were stained with Oil red O by fixing of the frozen tissues with 10% formalin for 10 minutes, rinsing twice with distilled water for 5 minutes, orbital shaking in 60% propanediol for 10 minutes, and incubation in 0.2% Oil red O solution for 5 minutes at room temperature. For hematoxylin staining, the previous method was followed by 2 rinses with distilled water, a hematoxylin dip for 20 seconds, 2 rinses with distilled water and then mounting. Immunofluorescence was performed on sections from the frozen blocks using the following antibodies at a 1:150 dilution: DNMT1 (Santa Cruz Biotech), PCNA (Santa Cruz Biotech), VCAM1 (Santa Cruz Biotech), ICAM1 (Santa Cruz Biotech), cyclin A (Santa Cruz Biotech),CTGF (Santa Cruz Biotech), CD31 (Santa Cruz Biotech).