Polyethyleneimine max

The expression plasmid pCA7, encoding soluble hSLAM, which has its authentic signal peptide and a C-terminal 6x His-tag, was kindly provided by Dr. Y. Yanagi (Kyushu University; Hashiguchi et al., 2007 (link)). Subconfluent (~80%) monolayers of HEK293 cells were transfected with the plasmid using polyethyleneimine max (Polysciences). After transfection, the cells were maintained for 5 days in DMEM supplemented with 2% FBS. hSLAM secreted into the culture media was harvested and purified using Ni²⁺ affinity chromatography. Approximately 6 μg of protein was separated by SDS-PAGE using 10–20% gradient gels and transferred to polyvinylidene fluoride membranes using a semi-dry blotting system. Protein bands were visualized by staining with Coomassie Brilliant Blue, cut out and the amino acid sequence analyzed by standard Edman degradation.

PGZL1 HCs and LCs were cloned using Gibson Assembly Enzyme mix (NEB) into expression vectors with the appropriate IgG1, Igκ, or Igλ constant domains^{49 (link)}. Antibodies were expressed in FreeStyle 293F cells (Life Technologies Cat#R79007). Briefly, ~ 750 µg DNA (500 µg HC and 250 µg LC plasmid) were added to 25 ml Opti-MEM (Life Technologies, 31985-070), which was mixed with Opti-MEM containing 2250 µg polyethylene imine MAX (molecular weight 40,000 kDa; Polyscience, 24765-1). After incubation for 20 min at room temperature (RT), the transfection mix was added to 1 L cells at a density of ~ 1.2 × 10⁶ cells/ml in FreeStyle293 Expression Medium (Life Technologies, 12338018). The cells were incubated at 37 °C and 8% CO₂ for 6 days. After collecting the cells, the supernatant, containing IgG or Fab, was filtered and loaded into a protein A beads column (Thermo Scientific) or HiTrap KappaSelect column (GE Healthcare Life Sciences, 17545812). The column was washed with phosphate-buffered saline (PBS) and eluted with 0.2 M citric acid pH 3.0 or 0.1 M glycine pH 2.7. The fractions were concentrated and the buffer was changed to 20 mM sodium acetate pH 5.5. The Fab was loaded into a Mono S column and was eluted with a 0–60% linear gradient of 1 M sodium chloride and20 mM sodium acetate pH 5.5 buffer. The Fabs were concentrated and stored in 20 mM sodium acetate pH 5.5 at 4 °C.

To construct retrovirus-based short hairpin RNA (shRNA) expression vectors for knockdown of epigenetic demethylases, two oligonucleotides containing shRNA sequences (Supplementary Table S1) were annealed and inserted into a mouse U6 promoter-driven vector pMXs-U6 puro (provided by Dr Toshio Kitamura) (26 (link)). Expression plasmids of individual enzymes (Supplementary Table S2) were constructed using a long terminal repeat promoter-driven expression vector pMXs-puro (from Dr. T. Kitamura), in which the original puromycin-resistant gene was replaced with the blasticidin S-resistant gene. The iron-binding site of each enzyme sequence was modified by a previously reported mutation by the PCR-based site-directed mutagenesis (27–43 (link)) (Supplementary Table S2). Using the similar method, shRNA-resistant plasmids were prepared by introducing silent mutations in the targeted sequence (Supplementary Table S3). Retroviruses were produced by transfecting each plasmid into Platinum-E packaging cells (from Dr T. Kitamura) using polyethyleneimine MAX (Polyscience, 24765-1). Retrovirus infection was performed as reported previously (25 (link)) and the infected cells were selected by 10 μg/ml puromycin (Thermo Fisher, ant-pr-1) or 10 μg/ml blasticidin S (Thermo Fisher, ant-bl-1).

HeLa and MIN6 m9 cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) supplemented with 4.5 g/L glucose, L-glutamine, sodium pyruvate, 10% (v/v) heat-inactivated foetal bovine serum (Sigma-Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich), at 37 °C in an atmosphere of 5% CO₂. The media to culture MIN6 m9 cells also contained 50 μM 2-mercaptoethanol^{24 (link)}. For imaging experiments, the cells were dissociated with trypsin and then plated onto glass coverslips coated with poly-L-lysine (Sigma-Aldrich) placed within 35-mm dishes. The cells were transfected with plasmids using Polyethyleneimine “MAX” (Polysciences, Warrington, PA, USA) or Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific, Waltham, MA, USA), 2 days after plating. The media was exchanged 4 h after transfection and the cells were cultured at 32 °C for 2or 3 days until imaging.