Anti dnmt1

After rinsing twice with PBS, cells were fixed for 30 min in 4% paraformaldehyde, permeabilized using 0.2% Triton X-100, and blocked using 0.5% bovine serum albumin (BSA) at room temperature. Thereafter, cells were probed overnight with anti-DNMT1 (#ab188453), anti-VDR (#ab89626), anti-PTEN (#ab137337) (Abcam, Cambridge, MA, UK), anti-DNMT1 (#sc-271,729), anti-SNAI1 (#sc-271,977), anti-nephrin (#sc-376,522) (Santa Cruz, CA, USA), anti-E-cadherin (#14,472), and anti-p-NF-κB P65 (#3033) (Cell Signaling Technology, Danvers, MA, USA), anti-HBx (#MA1-81021) (Thermo Fisher Scientific, Waltham, MA, USA) antibodies at 4 °C. This was followed by a 60 min incubation with fluorescence-conjugated secondary antibodies (Yeasen, Shanghai, China) in the dark at room temperature. Then, cells were stained with DAPI (Beyotime, Shanghai, China) for 10 min at room temperature.

Chromatin immunoprecipitation (ChIP) assays were performed essentially as described before [67 (link), 68 (link)]. Briefly, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mMTris pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ~200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-MRTF-A (Santa Cruz, sc-10768), anti-DNMT1 (Santa Cruz, sc-20701), anti-DNMT3a (Santa Cruz, sc-20703), or anti-DNMT3b (Santa Cruz, sc-20704). Precipitated genomic DNA was amplified by real-time PCR with the following primers: ICAM-1 proximal promoter, 5'-CCCTGCCACCGCCGCC-3' and 5'-AGGGGCGGTGCTGCTTTCC-3'; ICAM-1 intronic region, 5'-AATTCCAGAGCTGACTTATCC-3' and 5'-ATCTCAGGCTTTGTTGAGC-3'; SIRT6 promoter, 5'-AACTCTGCGTGGCATTCAAA-3' and 5'-AAATGCGGGACACAGGCTAT-3'. A total of 10% of the starting material is also included as the input. Data are then normalized to the input and expressed as % recovery relative to the input as previously described [56 (link), 69 (link)]. All experiments were performed in triplicate wells and repeated three times.

Cells were cultured for 24 h on cover slip. Cells were then fixed with 4% paraformaldehyde in PBS pH 7.4 for 15 min at room temperature. Permeabilization is performed with PBS containing 0.5% Triton X-100 for 20 min at room temperature. Blocking, staining, hybridization, ligation, amplification, and detection steps were realized according to manufacturer’s instructions (Olink Bioscience, Sweden). All incubations were performed in a humidity chamber. Amplification and detection steps were performed in dark room. Fluorescence was visualized by using the Axiovert 200 M microscopy system (Zeiss, Le Pecq, France) with ApoTome module (X63 and numerial aperture 1.4). Preparations were mounted by using ProLong® Gold antifade reagent with DAPI (Life Technologies, France). Pictures acquisition was realized in structured illumination microscopy. After decovolving (3.5 Huygens Essential software (SVI)), 3D view was obtained by using Amira.4.1.1 program. Finally, images were analyzed by using the freeware “BlobFinder” available for download from www.cb.uu.se/~amin/BlobFinder. Thus, we obtained either number of signals per nuclei since nuclei can be automatically identified. DNMT1 and UHRF1 were detected with anti-DNMT1 (Santa Cruz, sc10221, France) and anti-UHRF1 (Santa Cruz, sc98817, France).