Dnmt3a

Following the guidelines set by the Research Ethics Committee of the Shanghai Ninth People's Hospital, which is affiliated to the Shanghai Jiao Tong University School of Medicine, 10 pairs of tumors, in which the pathological results revealed HNSCC, were selected for this study. Normal human oral mucosal epithelial tissues obtained from the oral mucosa during the surgical resection of HNSCC were used as a control. Paraffin-embedded samples corresponding to the most representative tumor area on H&E-stained slides were selected for performing the IF assay. Briefly, the original fresh-frozen IF sections with a thickness of 6 μm were acquired through cryosection, air-dried for 10 min, fixed with acetone, washed with phosphate buffered saline (PBS) (1 ×), and incubated at room temperature for 2 h with NSUN5 (Proteintech Group, Rosemont, IL, USA, 1:50), DNMT1 (Bioworld Technology, Inc., St. Louis Park, MN, USA, 1:50), and DNMT3A (Novus Biologicals, Littleton, CO, USA, 1:170) primary antibodies. The sections were rinsed with sterile PBS (1 ×) and then incubated with a Goat Anti-Rabbit IgG secondary antibody (Abcam, Cambridge, UK, 1:200) protected from light. Next, the nuclei of the sections were counterstained with DAPI (Beyotime, Shanghai, China). Primary antibodies were replaced with PBS as a negative control.

ChIP assays were performed as previously described (Gazin et al., 2007 (link)) using the following antibodies: ZNF304 (described above), KAP1 (Bethyl Laboratories), SETDB1 (Millipore, Billerica, MA), DNMT1, DNMT3A, and DNMT3B (all from Imgenex, San Deigo, CA), cJUN (Millipore), H3K27me3 (Cell Signaling Technology), H3K9me3 (Millipore), H3K4me3 (Abcam), EZH2 (Millipore) and BMI1 (Abcam). The CDX1 antibody (Chan et al., 2009 (link)) was kindly provided by Walter Bodmer (University of Oxford, UK). ChIP products were analyzed by qRT-PCR (see Supplementary file 1 for primers). Samples were quantified as percentage of input, and then normalized to an irrelevant region in the genome (∼3.2 kb upstream from the transcription start site of GCLC). Fold enrichment was calculated by setting the IgG control IP sample to a value of 1.

Cells were lysed in RIPA buffer (Thermo Scientific, Waltham, MA, USA) with Protease Inhibitor Cocktail Tablets (Roche, Mannheim, Germany) for 15 minutes on ice. The total protein concentrations were measured using Protein BCA Assay Kit (Bio‐Rad). Samples were denatured with 5× loading buffer at 100°C for 5 minutes. Equal amounts of protein (25 μg) were loaded on a 10% SDS‐PAGE gel. The protein in the lysates were resolved by electrophoresis and transferred onto NC membranes (Bio‐Rad). The membrane was blocked for 2 hours at room temperature in 5% nonfat milk, and subsequently incubated with primary antibody as follows: DNMT3A(1:1000, IMG268; Imgenex), MYC (1:1000, N262; Santa Cruz Biotechnology), Sex determining region Y‐box 2 (SOX2, 1:1000, ab92494; Abcam, Cambridge, MA, USA), CD133(1:200, 130105226; Miltenyi Biotec, Bergisch Gladbach, Germany), ZEB1(1:1000, ab203829; Abcam) and E‐cadherin (1:1000, 3195; Cell Signaling Technology). Then, the membranes were incubated with HRP‐conjugated secondary antibody (1:5000; Santa Cruz Biotechnology). An anti‐β‐actin antibody (1:5000, A5441; Sigma‐Aldrich Corporation) was used as internal control.