Ezh2 d2c9

The following antibodies were used: ß-actin (Sigma-Aldrich, A5441, mouse antibody, 1:5000 dilution), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Cell Signaling Technology, 5174s, rabbit antibody, 1:5000 dilution), RBM39 (Atlas Antibodies, HPA-001591, rabbit antibody, 1:2000 dilution), c-Myc (Cell Signaling Technology, 5605s, rabbit antibody, 1:2000 dilution), MYCN (Santa Cruz Biotechnology, 53993, mouse antibody, 1:200 dilution), FLAG (Sigma-Aldrich, F1804, mouse antibody, 1:2000 dilution), ATM (D2E2) (Cell Signaling Technology, 2873, rabbit antibody, 1:1000 dilution), EZH2 (D2C9) (Cell Signaling Technology, 5246, rabbit antibody, 1:1000 dilution), CDK4 (D9G3E) (Cell Signaling Technology, 12790, rabbit antibody, 1:1000 dilution), RET (C31B4) (Cell Signaling Technology, 3223, rabbit antibody, 1:1000 dilution), and EED (Millipore 09-774, rabbit antibody, 1:1000 dilution).

H3K27me³ (C36B11) and EZH2 (D2C9) antibodies were purchased from Cell Signaling Technology. Tumor tissues harvested from mice were frozen in OCT. Frozen tumor tissue was fixed in ice-cold methanol and blocked in 5% goat serum in PBS after permeabilization with 0.5%Triton-X. They were then stained at 4°C overnight with primary antibody. Subsequently tissues were washed and stained with secondary antibody goat anti-rabbit IgG (H+L), Alexa flour 488 (Thermo Fisher Scientific; A1108) at room temperature for 1h. Finally, the tissues were stained with 1x DAPI (Molecular Probes/Invitrogen; D3571) washed and mounted using fluorescent mounting agent (DAKO; S3023). For cellular staining, 8-chamber slides were used and coated with 0.1mg/mL Poly-L-lysine. 1x10⁵ cells were cultured for 24-48h and then treated with GSK126 and/or IFNγ for 24h. Cell were then washed in PBS and fixed using 4% paraformaldehyde. Permeabilization, blocking and staining with primary and secondary antibodies were carried out as described above. Once stained the cells were mounted with Vectasheild with DAPI (Vector Laboratories; H1200). All stained tissues and cells were imaged on a Zeiss LSM 880 or Zeiss LSM 780 confocal microscopes. The images were analyzed and quantified using ImageJ software.

Cells were lysed in RIPA buffer. 20 μg protein was loaded and separated on 10% SDS/PAGE gel. Samples were transferred onto PVDF membranes (Millipore). After being blocked in 5% milk for 1 h, the membranes were probed with specific primary antibodies overnight at 4 °C: Ezh2 (D2C9, Cell signaling), GAPDH (SC-32233, Santa Cruz). After 3 times extensive wash, blots were incubated with HRP-conjugated secondary antibody for 1 h at room temperature before the chemiluminescent reaction.

Cells were lysed in RIPA lysis buffer (Beyotime Biotechnology, Jiangsu, China) with protease inhibitor (Roche, 11697498001) and phosphatase inhibitor (Roche, 4906837001) for 10 min on ice and shaken every 5 min on a vortex mixer for 30 s. Lysates were centrifuged at 12,000 rpm at 4°C for 10 min and the supernatant fraction was retained. Protein concentrations were quantified with BCA Protein Assay Kit (Thermo Fisher Scientific, A53226). Protein samples were separated via 10% SDS-PAGE and then transferred onto PVDF membrane. The membrane was blocked in 5% milk containing TBST (Tris-buffered Saline with Tween 20) at room temperature for 1 h, and this was followed by incubation with indicated primary antibodies overnight at 4°C. The membrane was then washed 3 times with TBST and incubated with secondary antibody for 1 h at room temperature before its development with the ECL kit (Thermo Fisher Scientific, 34,578). Primary antibodies to the following proteins were used: BRD4 (Bethyl Laboratories, A301-985A100), BRD3 (Bethyl Laboratories, A302-368A), BRD2 (Bethyl Laboratories, A302-583A), EZH2 (D2C9) (Cell Signaling Technology Inc, #5246), c-MYC (Abcam, ab32072), caspase-3 (Cell Signaling Technology Inc, #9662) GAPDH (ABclonal, AC033), β-Actin (ABclonal, AC006).

ChIP assay was performed as described previously^{61 (link)}. Heart tissue was cut into small pieces and crosslinked with PBS with 1% formaldehyde (Thermo Fisher Scientific, 28906) at room temperature for 10 min. Fixation was terminated by addition of 0.125 M (final concentration) glycine (Sigma-Aldrich, 50046) at room temperature for 5 min. Chromatin was isolated by addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and sheared into a size range of ~200–500 bp.
An aliquot of chromatin (30 μg) was precleared with Dynabeads Protein G (Life Technologies, 10009D) at 4 ^oC for 1h. The precleared chromatin was immunoprecipitated using antibodies as follows: SUZ12 (D39F6) (Cell Signaling Technology, 3737, 1:100), EZH2 (D2C9) (Cell Signaling Technology, 5246, 1:100) and H3K27me3 (C36B11) (Cell Signaling Technology, 9733, 1:50). Chromatin were decrosslinked by incubation with proteinase K at 65 °C for overnight, and ChIPed DNA was purified by MinElute kit (Qiagen, 28004). Genomic DNA (Input) was prepared from an aliquot of precleared chromatin by decrosslinking and purification. The resulting DNA was quantified on a Qubit spectrophotometer.