Anti pan histone h3

Dex and insulin (from bovine pancreas) were purchased from Sigma-Aldrich, and STZ was from Wako. An LSD1 inhibitor, T-3775440 hydrochloride, was from MedChemExpress. The primary antibodies used for western blot, co-IP, immunohistochemistry, and immunocytochemistry experiments were as follows: anti-LSD1 (Abcam, ab17721), anti-FOXK1 (Abcam, ab18196), anti-ERRγ (Abcam, ab12893), anti-MHC type I (Developmental Studies Hybridoma Bank [DHSB], BA-F8), anti-MHC type IIA (DHSB, SC-71), anti-Laminin (Sigma-Aldrich, L9393), anti-Akt (Cell Signaling Technology [CST], #9272), anti-Phospho-Akt (Ser473) (CST, #9271), anti-4E-BP1 (CST, #9454), anti-Phospho-4E-BP1 (Thr37/46) (CST, #2855), anti-SQSTM1(p62) (Santa Cruz, sc-28359), anti-GAPDH (Santa Cruz, sc-25778), anti-LC3(MBL, PM036), and anti-Sin3A (Santa Cruz, sc-994). The secondary antibodies used were as follows: anti-mouse IgG-horseradish peroxidase (GE Healthcare, NA931V), anti-rabbit IgG-horseradish peroxidase (GE Healthcare, NA934V or CST, #7074), Alexa Fluor 488 anti-Mouse IgG1 (Thermo Fisher, A21121), Alexa Fluor 350 anti-Mouse IgG2b (Thermo Fisher, A21140), and Cy3 anti-Rabbit IgG (Jackson ImmunoResearch, 711-165-152). The antibodies used for ChIP experiments were anti-tri-methylated histone H3K4 (Millipore, 07-473), anti-pan histone H3 (Abcam, ab1791), and normal rabbit IgG (Santa Cruz, sc-2027).

The cultured cells were suspended in western lysis buffer (55 mM Tris-HCl (pH 6.8), 0.72 M 2-mercaptoethanol, 2% SDS, 0.005% bromophenol blue, and 8% glycerol) and boiled for 5 min. After centrifugation at 14,000 xg for 5 min, each supernatant was collected as a cell lysate. An EZQ Protein Quantitation Reagent (Life Technologies) was used to measure the protein concentration of each sample. Each protein sample (5 to 10 μg) was loaded onto a SDS-PAGE mini-gel; separated proteins were then blotted onto a polyvinylidene difluoride membrane (Merck Millipore). After sequential exposures to a primary antibody, a biotinylated secondary antibody (GE Healthcare), and a streptavidin-conjugated alkaline phosphatase (GE Healthcare), each membrane was developed with Western Blue (Promega). The primary antibodies were used as follows: anti-aromatase (CYP19 product), #SM2222PS, Acris; anti-β-actin, #A1978, Sigma; anti-pan-histone H3, #ab1791, Abcam; anti-H3K27me3, #9733, Cell Signaling; anti-EZH2, #5246, Cell Signaling.

Chromatin crosslinked in the two-step method [27 (link)] was subjected to a ChIP assay as described previously [25 (link)]. An aliquot containing 1 to 5 μg of DNA (estimated by using a Quant-iT PicoGreen Kit (Life Technologies)) was used for a single ChIP reaction. The primary antibodies were used as follows: anti-pan-histone H3, #ab1791, Abcam; anti-H3K27ac, #39685, Active Motif; anti-H3K27me3, #9733, Cell Signaling. Immunoprecipitation of protein-DNA complexes was performed with secondary antibody-conjugated Dynabeads M-280 (Life Technologies). To detect co-precipitated DNA, quantitative PCR was performed as described above. PCR primers used for these assays are listed in S1 Table.