Total histone h3

To extract total protein, cells or tissues were lysed in radioimmunoprecipitation assay buffer. To extract nuclear protein, nuclear lysates from cells or tissues were prepared as described previously (38 ). Tissues were homogenized using a 5 mm stainless steel bead (Qiagen) and TissueLyser LT (Qiagen). Cell or tissue lysates were briefly sonicated to shear DNA. Lysates were mixed in 4× LDS loading buffer containing DTT (ThermoFisher) and heated to 70 °C for 10 min. Proteins were separated by SDS-PAGE and resolved using semidry transfer. Polyvinylidene difluoride membranes were blocked in 5% skim milk. Antibodies used were as follows: HIF-1α (NB100-479, 1:500, Novus), Tri-Methyl-Histone H3 Lys9 (13969, 1:1000, Cell Signaling Technologies), Mono-Methyl-Histone H3 Lys9 (14186S, 1:1000, Cell Signaling Technologies), Di-Methyl-Histone H3 Lys9 (4658S, 1:1000, Cell Signaling Technologies), Total Histone H3 (ab1791, 1:2000, Abcam), Hexokinase II (TA325030, 1:500, Origene), Glut1 (ab115730, 1:1000, Abcam), LDHA (3582T, 1:1000, Cell Signaling Technologies), JMJD2A (5328, 1:1000, Cell Signaling Technologies), JMJD2B (8639, 1:1000, Cell Signaling Technologies), JMJD2C (ab226480, 1:500, Abcam).

Chromatin immunoprecipitation (ChIP) was performed as previously described (Schiavone et al, 2014). Sonicated chromatin samples were incubated overnight at 4°C with the following antibodies: total histone H3 (Abcam, ab1791), histone H3.3 (Cell Signaling, 09‐838), H3K4me3 (Cell Signaling, 9727), H3K36me3 (Abcam, ab9050). Normal rabbit IgG (Millipore) was used as a negative control. PCR primers for ChIP qPCR are listed in Table EV2.

Suberoyl anilide hydroxamic acid (SAHA, a pan-HDAC inhibitor) and 3-Deazaneplanocin A (DZNep, a polycomb EZH2 subunit inhibitor) were purchased from Millipore, Temecula, CA. The DNA methylation inhibitor 5-AZA-Cytidine (AZA) was from Sigma, St. Louis, MO. Cells were treated with indicated concentration and incubated for 72 hours before harvesting. Agarose A/G plus is from Santa Cruz. The following antibodies were used as primary antibodies: SOX4 (Santa Cruz; 1:1000); EZH2 (BD; 1:4000); HDAC3 (Millipore; 1:4000); total histone H3 (Abcam; 1:5000); Tri-methylated histone H3 H3K27me3 (Millipore; 1:3000); SUZ12 (Santa Cruz; 1:1,000); EZH1 (Santa Cruz; 1:1,000); β-tubulin (Sigma-Aldrich; 1:5,000).

MM cell lines, MCF7 cells and 5T33MM bone marrow cells were harvested and washed in ice-cold PBS (Gibco; cat. no 70011044) and collected post centrifugation at 100 RCF for 5 min. Histone proteins were extracted by utilizing a histone extraction kit (Abcam plc.; Cambridge, UK; cat. no 113476) following the manufacturer’s instructions. Protein extraction was conducted using RIPA extraction buffer (Millipore; Merck; cat. no 20188) with protease inhibitors (cOmplete™, EDTA-free Protease Inhibitor Cocktail; Roche; Merck; cat. no 11873580001). Western blot was performed as previously described^{4 (link)}, using anti-H3K27me3 (1:1000) (Active Motif; Waterloo, Belgium; cat. no 39155), anti-γH2AX (1:500) (Millipore; Darmstadt, Germany; cat. no 05-636-I), oxidative stress defence cocktail (1:250) (Abcam; cat. no ab179843) and total histone H3 (1:3000) (Abcam; Cambridge, UK; cat. no 1791) antibodies. Total histone H4 (1:1000) (Active Motif; Waterloo, Belgium; cat. no 91295) and actin (1:500) (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA; cat. no sc-47778) were used as loading controls.
Western blot image analysis and quantification was performed in ImageJ V.1.52a^{65 (link)} and normalized against Total histone H4. Cell line western blot quantifications were analysed by multiple t-test correcting for multiple testing by the Holm-Sidak method.

From FHC, HCT116 cells and isolated colonocytes, the proteins are extracted with RIPA buffer (Cell Signaling, #9806), 1mM PMSF (Cell Signaling, #8553) and phosphatase inhibitor cocktail (Cell Signaling, #5872). Quantifications of protein were measured by Bradford assay. Gel electrophoresis and transfer were performed using standard protocol for Western blotting. Antibodies that were used included pan-acetylated-histone H3 (Active motif, Cat# 39139), total Histone H3 (Active motif, Cat# 39763), total PDH (Abcam, Cat# ab110330), ACL (Cell Signaling, Cat # 4332), SCAD (Abcam, Cat# 154823), HDAC1 (Cell signaling, Cat# 34589), HDAC2 (Cell signaling, Cat# 57156), HDAC3 (Cell signaling, Cat# 85057) and β-actin (Sigma, Cat# A1978). Chemiluminescence detection was conducted with the Odyssey Fc and bands were quantified with Image Studio Software (LI-COR Biosciences, Lincoln, NE).