Rabbit anti h3k9me3

The cells were fixed with 4% paraformaldehyde for 15 min. After several washes, they were blocked with 0.01M phosphate-buffered saline supplemented with 1% BSA and 0.3% Triton X-100 for 30 min. Primary antibodies (mouse anti-LAP2α, 1:500, Abcam, UK; mouse anti-betaIII tubulin, 1:500, Abcam; mouse anti-NeuN, 1:1000, Abcam; rabbit anti-NeuN, 1:200, Abways; rabbit anti-Tbr1, 1:500, Abcam; mouse anti-γH2AX, 1:250, Millipore, USA; mouse anti-Lamin A/C, 1:200, Abcam; rabbit anti-H3K9me3, 1:4000, Abcam; mouse anti-6E10, 1:200, Abcam) were diluted with blocking buffer and applied overnight at 4° C. The cells were stained with suitable Alexa Fluor–labeled secondary antibodies (Invitrogen) in blocking buffer at a concentration of 1:500 for 1.5 h at room temperature. 4', 6-Diamidino-2-phenylindole (Thermo Fisher, IL, USA) was applied to counterstain cells for visualizing nuclei at 1:1000 in Milli Q water (Biocel, Millipore, USA). The images were obtained with an Olympus IX71 microscope using a Hamamatsu ORCA CCD camera and Leica TCS SP5.

The following primary antibodies were used: Mouse anti-OCT3/4 (Santa Cruz Biotechnology, Clone C-10, #sc-5279, 1:50), Rabbit anti-CDX2 (BioGenex, Clone EP25, #NU777-5UC, 1:100), Mouse anti-CDX2 (BioGenex, Clone CDX2-88, #MU392A-5UC, 1:100), Rabbit anti-EOMES (Abcam, #ab23345, 1:100), Mouse anti-SMA (SIGMA ALDRICH, Clone 1A4, #A2547, 1:200), Mouse anti-TUJ1 (Biolegend, Clone TUJ1, #801201, 1:200), Goat anti-GATA6 (R&D Systems, #AF1700, 1:100), Mouse anti-5mC (Millipore, Clone 33D3, #MABE146, 1:200), Rabbit anti-H3K27me3 (Millipore, #07-449, 1:100) and Rabbit anti-H3K9me3 (Abcam, #ab8898, 1:100). The secondary antibodies used were: Goat anti-Mouse IgG FITC-conjugated (Thermo Fisher Scientific, #62-6511, 1:50), Goat anti-Mouse IgG Alexa Fluor^R 555 (Abcam, #ab150114, 1:1000), Donkey anti-Goat IgG Alexa Fluor^TM 488 (Thermo Fisher Scientific, #A11055, 1:1000), Donkey anti-Mouse IgG Alexa Fluor^TM 555 (Thermo Fisher Scientific, #A31570, 1:1000), Donkey anti-Rabbit IgG Alexa Fluor^TM 555 (Thermo Fisher Scientific, #A31572, 1:1000), and Donkey anti-Rabbit IgG Alexa Fluor^TM 488 (Thermo Fisher Scientific, #A21206, 1:1000).

Chromatin immunoprecipitation (ChIP) was performed on native chromatin extracted from 2 × 10⁷ ES or 1 × 10⁷ TS cells using standard protocols (40 (link)). Nuclei were purified on a sucrose gradient and chromatin digested with 60 U/ml Micrococcal Nuclease (Affymetrix). Lysates were pre-cleared with Protein G Sepharose beads (GE Healthcare) and incubated with 4 μg of either rabbit anti-H3K9me3 (Abcam ab8898) or rabbit anti-H3K27me3 (Millipore 07–449) at 4°C overnight. Chromatin was immunoprecipitated with Protein G Sepharose beads at 4°C for 4 h. Mock ChIPs were performed in parallel with an isotype-matched control IgG or with beads alone. Eluted DNA from bound and input fractions was subjected to quantitative polymerase chain reaction (PCR) analysis with primer sets specific to genomic promoter regions. Enrichment values were expressed as bound:input ratios and normalized against the corresponding mock values. All ChIPs were performed on at least three biological replicates and compared by T-test. All primers are given in the Supplementary Material.

Rabbit anti-p53 (Santa Cruz Biotechnology), rabbit anti-p-JNK (Invitrogen), mouse anti-synaptophysin (Sigma), mouse anti-amyloid clone DE2B4 (Abcam), rabbit anti-amyloid clone FCA3542 (Calbiochem), mouse anti-amyloid MOAB2 (Novus; NBP2-13075)), pan anti-Tau k9JA (DAKO); mouse anti-p-Tau clone AT-8 (Thermo scientific), mouse anti-p-Tau clone PHF1 (a gift from Dr. Davies, Albert Einstein College of Medicine), mouse anti-NeuN (Chemicon), rabbit anti-cleaved caspase-3 (Cell Signaling), rabbit anti-GFAP (Dako; Z0334), Iba1 (Wako; 019-19741), mouse anti-Bmi1 clone F6 (Millipore), mouse anti-Bmi1 (Abcam), mouse anti-p-ATM (Novus), rabbit anti-p-ATR (Santa Cruz Biotechnology), rabbit anti-H3K9^me3 (Abcam), mouse anti-H2Aub clone E6C5 (Millipore), mouse anti-HP1 (Millipore), mouse anti-β-actin (Sigma), mouse anti-tubulin (Sigma), mouse anti-H3 (Upstate), and rabbit anti-mouse IgG (Upstate).