Setdb1

CD271, SETDB1, SETDB2, p-mTOR and Actin antibodies were purchased from Abcam, (CBG, UK). Phosphorylated AKT-Ser473 (pAKT), total AKT (T-AKT), phosphorylated ERK1,2-Thr202/Tyr204 (p-ERK1,2), total ERK (ERK1,2) antibodies were obtained from Cell Signalling Technology, (MA, USA). Antibodies for H3K4me3, H3K9me3 and H3K27me3 were purchased from Active Motif (CA, USA). Alexa Fluor 594 antibody and Hoechst 33342 were obtained from Thermo Fisher Scientific (MA, USA).

The cells were cross-linked with 1% formaldehyde for 10 min at 37 °C, and crude nuclei were recovered. The crude nuclei were sonicated to produce 500 bp chromatin fragments. The following antibodies were used in the ChIP assay: MeCP2, H3K9 di-methylation, HDAC1, Dnmt1, Dnmt3a, SETDB1, MLL2 (Abcam), H3K9/K14 acetylation, H4K12 acetylation, H3K4 tri-methylation, H3K27 tri-methylation (Upstate), HAT1 (GeneTex), CBP (Abcam), p300 (Abcam) and Ezh2 (Cell Signaling Technology, Danvers, MA, USA). IgG (Sigma) was used as a negative control. For each ChIP assay, 5 μg antibodies were added, and the samples were incubated overnight at 4 °C. The ChIP and input DNA samples were quantified using quantitative PCR. Primer sequences used in ChIP-qPCR: Region1: 5′-TTTTCACACCAAAGAATCCC-3′ (forward), 5′-CTTATTTACCAAACATGGTGT-3′ (reverse); Region2: 5′-CAGGTGAAGAAAGTGGCAGA-3′ (forward), 5′-AAGATCGGACAATTAGACCAG-3′ (reverse); Region3: 5′-TCCTTAGCCCTGGAACTGCC-3′ (forward), 5′-AGGCAACACCAGGAGCAGCCCC-3′ (reverse).

ASF1a/b (11 (link)), CAF-1/p150 (Novus Biologicals #NB500–207A1), DAXX (Santa Cruz #sc-7152), HAT1 (Abcam #ab12164), HIRA (Abcam #ab20655), Histone H3 (Abcam #ab7834), H3K9me1 (Millipore #07–450), H3K9me2 (Millipore, #07–212), H4K12ac (Millipore #07–595), Hsc70 (Abcam #ab19136), Hsp90 (Santa Cruz #sc-7947), Importin4 (Abcam #ab28387), MCM2 (BD Transduction Lab #610700), MCM5 (Bethyl A300–195A), NASP (donated by Dr Almouzni), RPL5 (Abcam #ab74744), RPS3a (Abcam, ab171742), SetDB1 (Abcam #ab12317). For Western blot analysis the primary antibodies were detected with a horseradish peroxidase-conjugated secondary antibody, developed with enhanced chemiluminescence (ECL, Pierce) and exposed onto an X-ray film.

The cells were cross-linked with 1% formaldehyde for 10 min at 37 °C, and crude nuclei were recovered. The crude nuclei were sonicated to produce 500 bp chromatin fragments. The following antibodies were used in the ChIP assay: MeCP2, H3K9 di-methylation, HDAC1, Dnmt1, Dnmt3a, SETDB1, MLL2 (Abcam), H3K9/K14 acetylation, H4K12 acetylation, H3K4 tri-methylation, H3K27 tri-methylation (Upstate), HAT1 (GeneTex), CBP (Abcam), p300 (Abcam) and Ezh2 (Cell Signaling Technology, Danvers, MA, USA). IgG (Sigma) was used as a negative control. For each ChIP assay, 5 μg antibodies were added, and the samples were incubated overnight at 4 °C. The ChIP and input DNA samples were quantified using quantitative PCR. Primer sequences used in ChIP-qPCR: 5’-ATTAAATCCCGATAGTATACC-3’ (forward), 5’-ATCATAGTTAGCAATTCAATGCAGTAT-3’ (reverse);

Asf1 (Dr. Almouzni), β-actin (Sigma Aldrich A5316), Flag (Sigma Aldrich F3165), γH2AX (Abcam ab2893), HA (Sigma clone 12CA5), HAT1 (Abcam ab12164), HIRA (Abcam ab20655), Histone H3 (Abcam ab7834), H3ac (Merck Millipore, 06-599), Histone H4 (Abcam ab10158), H4K12ac (Merck Millipore 07-595), Hsc70 (Abcam ab19136), Hsp90 (Santa Cruz sc-7947), Importin4 (Abcam ab283887), JMJD1B (Cell Signaling Technology 2621S), NASP (Dr. Almouzni), Topo I (Santa Cruz sc-32736), SetDB1 (Abcam ab12317).

Protein fractions were obtained from cells using ice-cold RIPA buffer (50 mM Tris–HCl, pH = 7.5, 150 mM NaCl, 1% TritonX-100, 1 mM EDTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate) supplemented with protease and phosphatase inhibitor cocktails (Roche Diagnostics, Mannheim, Germany). About 50 µg of protein extractive was subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel (10% SDS-PAGE) and transferred to polyvinyldene fluoride (PVDF) membrance (Bio-Rad). Followed by blocking with 5% non-fat milk at room temperature for 1 h, the membrances were incubated with SETDB1 (1:2000, Abcam) and β-actin (1:5000, Abcam) antibodies at 4 °C overnight. Then, the membrances were further probed with horseradish peroxidase-conjugated secondary antibodies (1:5000, Cell Signaing Technology, Danvers, MA, USA) for 1 h at room temperature. All protein bands were analyzed using an Image-Pro plus 4.5 software (Media Cybernetics, Silver Spring, MD, USA).