Dnmt1

Harvested cells and frozen tissues were treated with RIPA lysis buffer (Solarbio, China) for protein extraction. Protein lysate was loaded for SDS-PAGE, and was transferred from the gel to the membrane. Unspecific binding was blocked with 5% skimmed milk. Proteins were incubated with β-actin (Santa Cruz), tubulin, FPN (Sigma), DNMT1, SOSTDC1 (Abcam), Lamin B, p-SMAD, SMAD, G9a (Cell signaling), and E4BP4 (Abcam) antibodies at 4 °C overnight. Membranes were then incubated for 1 h with secondary antibodies. Protein detection was performed with the ECL substrate (Millipore, USA), and β-actin was used as the internal control. ELISA kits (R&D Systems) were used to detect levels of BMP4, BMP7, IL-6, and hepcidin.

We homogenized mouse tissues in buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P‐40, 0.5% deoxycholate, 0.1% SDS, and 1 mM 2‐mercaptoethanol with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, NJ, USA) and then centrifuged them at 2,500 × g for 15 min. Equal amounts of protein were separated by 5–20% SDS–PAGE and transferred to Hybond‐P membranes (GE Healthcare, Piscataway, NJ, USA). The primary antibodies and their dilutions were as follows: AR (N20, 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Dnmt1 (1:1,000; Abcam, Cambridge, MA, USA), Dnmt3a (1:1,000; Abcam), Dnmt3b (1:1,000; Abcam), Hes5 (1:1,000; Santa Cruz), and ChAT (1:1,000; Millipore, Billerica, MA, USA). Primary antibody binding was probed with horseradish peroxidase‐conjugated secondary antibodies at a dilution of 1:5,000, and bands were detected by using an immunoreaction enhancing solution (Can Get Signal; Toyobo, Osaka, Japan) and enhanced chemiluminescence (ECL Prime; GE Healthcare). An LAS‐3000 imaging system (Fujifilm, Tokyo, Japan) was used to produce digital images. The signal intensities of these independent blots were quantified using IMAGE GAUGE software version 4.22 (Fuji) and expressed in arbitrary units (n = 3 for each group). The membranes were reprobed, or the same samples were examined with an anti‐GAPDH (1:5,000; Santa Cruz) antibody for normalization.

ChIP assay was performed by using EZ-ChIP Kit (Millipore, USA) according to the manufacturer’s instructions. In brief, differentiated ESCs were collected at d10. The cells were cross-link with 1% formaldehyde for 10 min and then quenched with glycine. Cell lysates were sonicated to generate chromatin fragments and then immunoprecipitated with DNMT1, DNMT2, and DNMT3 antibodies (Abcam, USA) separately. IgG antibody (Abcam, USA) was used as the negative control. Before the immunoprecipitation, input samples were obtained as the positive control. Then, qRT-PCR was used to detect the level of RCPCD in input and immunoprecipitation samples.

Western blot analysis was performed as previously described^{22 (link)}. Placental samples were lysed in RIPA buffer in the presence of protease and phosphatase inhibitor cocktail (Pierce Biotechnology, Rockford, IL). Protein concentrations were determined by BCA assay (Pierce, Rockford, IL). Thirty µg of protein lysate was reduced (Dithiothreitol, DTT), and subjected to sodium dodecyl sulfated polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane. The blots were incubated with antibodies specific for Acel-K27-His3, DNMT1, DNMT3a, PGC-1α (Abcam, Cambridge, MA), TFAM, P(S97)-ACC, or β-actin (Cell Signaling Technology, Danvers, MA), and detected by enhanced chemiluminescence (Pierce, Rockford, IL). The blots were quantified and analyzed by imaging densitometry with Image Lab Software (Bio-Rad, Hercules, CA).

The analysis of western blot was described previously [28 (link)]. Briefly, total protein was extracted and applied to SDS-polyacrylamide gels for electrophoresis (22 mA per gel). Protein was transferred onto 0.45 μm polyvinylidene difluoride (PVDF) membranes for 1.5 h with 400 mA. The transferred membranes were incubated with blocking buffers for 10 min and then incubated with specific primary antibody against E-cadherin (BD), N-cadherin (Genetex), Vimentin (BD), DNMT 1 (Abcam), DNMT 3A (Abcam), DNMT 3B (Cell signaling), or β-Actin (Cell signaling) at appropriate dilutions at 4 °C overnight. The membranes were washed and incubated with horseradish peroxidase-linked secondary antibody for 1 h at room temperature. Bands were visualization by chemiluminescent reagent and record by photographic film and the intensity of the band was quantified and calculated. The results were conducted independently in triplicate.