Anti 5hmc

The MCF‐7 cells were grown on coverslips in 35‐mm dishes. After treatment with SFN, the cells were washed in PBS, permeabilized with 0.5% Triton X‐100 in PBS for 15 min, and then fixed with 4% paraformaldehyde for 10 min. The fixed cells were blocked for 30 min in blocking reagent buffer (Beyotime, Shanghai, China) and incubated with primary antibodies in blocking reagent buffer. The primary antibodies and the dilutions used for immunostaining were anti‐5hmC (Active Motif, Carlsbad, CA, USA, no. 39770) antibodies at 1 : 200. After three 5 min washes in PBS, the cells were incubated with Alexa‐488‐labeled secondary antibody (Servicebio, Wuhan, China, GB25301) diluted 1 : 300 in blocking buffer. The nuclei were stained with bisBenzimide H33342 trihydrochloride. Images were captured using a Zeiss (Jena, Germany) LSM 700B confocal microscope. The integrated optical density (IOD) of the annotated nuclei was measured in Image‐Pro Plus 6.0 (Media Cybermetrics, Inc., Rockville, MD, USA). We selected three fields to calculate the IOD value as the following formula: total optical intensity/number of cells in each field.

Primary skin fibroblasts stably expressing TET1 were seeded on coverslips placed in a 24-well dish (0.1 × 10⁵ cells/well). Cells attached to the coverslip were fixed in 4% paraformaldehyde, washed, permeabilized with 0.5% Triton X-100, incubated in 4 N HCl, neutralized, blocked with 10% goat serum, 3% BSA, and 0.1% Triton X-100, then incubated overnight at 4 °C with anti-TET1 (1:100, Thermo Fisher Scientific, Waltham, MA, USA) or anti-5hmC (1:300, Active Motif, La Hulpe, Belgium) antibodies, washed and then incubated with the appropriate secondary antibodies. Fluorescence was analyzed using the LSM 780/ELYRA PS.1 confocal microscope (Zeiss, Jena, Germany). Laser power and acquisition parameters were similar for each image. The “Range Indicator” option was used, which helps to set the image lighting objectively. There was no post-capture image manipulation. For each sample, 50 nuclei were quantified.

Cells were washed, fixed, and incubated with each monoclonal antibody at 48 h after plasmid transfection as described previously 18. Dilution of primary antibodies was 1:1000 for anti‐FLAG M2 (Sigma) and 1:500 for anti‐5hmC (ActiveMotif, Carlsbad, CA, USA). Indirect detection of primary antibodies was achieved by 60 min‐incubation with 1:100 diluted secondary antibodies: FITC‐labeled goat anti‐mouse IgG (Zymed 62‐6511, Invitrogen, Carlsbad, CA, USA) and Rhodamine‐labeled goat anti‐rabbit IgG (Chemicon AP‐187R, EMD Millipore, Darmstadt, Germany). Cells were stained for 10 min with 0.5 μg/mL 4', 6'‐diamidino‐2‐phenylindole (DAPI). Images were acquired on an Olympus BX60 fluorescence microscope fitted with a charge‐coupled device camera (Photometrics, Tucson, AZ, USA) and controlled by a Macintosh computer running the Quips mFISH software (Vysis Inc., Downers Grove, IL, USA).

Total genomic DNA was extracted using the DNeasy Kit (QIAGEN), denatured (0.4M sodium hydroxide, 10mM EDTA at 100°C for 10 min), and then neutralized (6.6M cold ammonium acetate, pH 7). We applied 200 ng of DNA to a prewet Amersham Hybond‐N+ membrane (GE Healthcare Life Sciences). The membrane was blocked and incubated in primary antibody overnight (anti‐5hmC and anti‐5mC 1/200; Active Motif, Inc., Carlsbad, CA, USA), followed by the appropriate peroxidase‐labeled secondary antibody (1/5000; Santa Cruz Biotechnology). Blots were visualized using Luminata Forte HRP substrate (Millipore), and quantified using ImageJ software (NIH; https://imagej.nih.gov/ij/).