Ab24684

The staining procedure was performed using a Leica Bond III Stainer (Leica,). Slides were subjected exposed to 10 mM sodium citrate buffer, pH 6.0 for 20 min at 37°C. Slides were incubated with the appropriate primary antibody for 15 min, followed by Polymer Refine Detection System (Leica) processing, including hydrogen peroxidase block, secondary antibody polymer, 3,3’ diaminobenzidine and hematoxylin stain. Specimens were then rinsed for 5 min in tap water. Slides were dehydrated in increasing concentrations of ethyl alcohol and xylene prior to permanent coverslipping in xylene-based media. Primary antibodies were as follows: mouse anti-Hif1α (1:400, Novus Biological), mouse anti-HIF2α (Novus Biologicals, NB100-132, rabbit polyclonal, 1:700) mouse anti-H3K9me2 (1:750, Abcam), rabbit anti-H3K27me2 (ab24684, Abcam, 1:500), rabbit anti-5hmC (39769, Active Motif, 1:4000), and mouse anti-SDHB (ab14714, Abcam, 1:1000).

The following antibodies were used for immunoprecipitation and Western blot analyses: C-terminal anti-ERα (F-10 sc-8002, Santa Cruz Biotechnology, Dallas, Texas), rabbit anti-estrogen Receptor Alpha (ab32063, Abcam, Cambridge, UK), rabbit polyclonal anti-DOT1L (A300-953A, Bethyl Laboratories, Montgomery, Alabama), β-actin (A1978, Sigma Aldrich, Milan, Italy), Rabbit anti-KMT4/DOT1L (ab72454), anti-Histone H3, total, (ab1791), anti-H3K79me1 (ab2886), anti-H3k79me2 (ab3594), anti-H3K79me3 (ab2621), anti-H3K4me3 (ab7766), anti-H3K27me3 (ab24684) from Abcam, anti-Rabbit IgG Isotype Control (31235, Thermo-Fisher), and the anti-Mouse IgG antibody (RM104, Aurogene, Rome, Italy).
Cells were treated with the following compounds: DOT1L inhibitors EPZ004777 (S7353), Pinometostat (EPZ5676)(S7062), SGC0946 (S7079), all from Selleckchem, and with 4-hydroxytamoxifen (4-OHT) (H7904, Sigma-Aldrich), Fulvestrant (ICI 182,780) (I4409, Sigma-Aldrich), and β-estradiol (E887-5G, Sigma-Aldrich) or vehicles (DMSO and/or EtOH), according to different experimental settings.

For Western blot analysis, histone-enriched fractions were extracted from wild type (WT), mutant, and transgenic leaves as described previously (Huang et al., 2007 (link)). Antibodies used in Western blot are: anti-H3K27me3 (07-449, Millipore), anti-H3K27me2 (ab24684, Abcam), anti-H3K27me1 (ab113671, Abcam), anti-H3 (ab1791, Abcam), anti-H3K4me3 (07-473, Millipore), anti-H3K4me2 (07-430, Millipore), anti-H3K4me1 (07-436, Millipore), anti-H3K9me3 (ab8898, Abcam), anti-H3K9me2 (07-441, Millipore), anti-H3K9me1 (ab9045, Abcam) H3K36me1 (ab9048, Abcam), anti-H3K36me2 (ab9049, Abcam), and anti-H3K36me3 (ab9050, Abcam). Anti-SDG711 was prepared by immunizing rabbits with SDG711 protein produced in Escherichia coli (in pET-28a vector) and purified with His-tag protein purification beads (V8550, GE Healthcare). The anti-serum was affinity-purified with protein-A agarose beads purchased from Millipore (16-157).

Cells were seeded at 0.25 million per mL and IC₅₀ concentrations of compounds for cells (20 μm JDM‐7 and 40 μm tadalafil for MV4‐11; 20 μm JDM‐7 and 100 μm tadalafil for MOLM‐13) were added followed by incubation for 72 h. Cells were then collected for protein extraction and run on 10–15% SDS/PAGE gels for electrophoresis. Anti‐histone H3 (#06‐755; Millipore, Darmstadt, Germany), anti‐H3K9‐me1 (#07‐450; Millipore), anti‐H3K9‐me2 (ab1220; Abcam, Shanghai, China), anti‐H3K27‐me2 (ab24684; Abcam), anti‐HOXA9 (ab140631; Abcam) and anti‐CEBPE (ab172616; Abcam) antibodies were used for primary detection. As secondary antibodies, either anti‐rabbit or anti‐mouse IgG conjugated with horseradish peroxidase (GE Healthcare, Braunschweig, Germany) were used. Western Lightning Plus ECL (Perkin Elmer, Waltham, MA, USA) reagents were used for fluorescence production and Amersham Imager 600 (GE Healthcare, China) was used for fluorescence detection to visualize the proteins detected. The optical densities of the protein bands were analyzed using Amersham Imager 600 software. Three independent repeats were performed. Relative optical densities were calculated for the indicated bands by dividing the corresponding control bands. The control groups were set as 1 and treatment groups were calculated accordingly.

Tissue was prepared as described above. Proteins were separated by using 7.5% SDS-PAGE under reducing conditions. After the proteins were transferred to a nitrocellulose membrane, the membranes were probed with either monoclonal mouse anti-dimethylated H3K9 antibody (1:1000, AB1220, Abcam, Austin, TX, United States) or the polyclonal rabbit anti-dimethylated H3K27 (1:1000, ab24684 Abcam, Austin, TX, United States). Secondary anti-mouse IgG horse-radish peroxidase-conjugate secondary antibody (1:5000, Chemicon) or anti-rabbit IgG (1:2500–1:5000, Upstate, Lake Placid, NY, United States) was used for detection.

ChIP assays were performed using a magnetic ChIP kit according to the manufacturer's instructions (53009; Active Motif, Carlsbad, CA, USA). In brief, cells were fixed by 1% formaldehyde; chromatins were fragmented by enzymatic digestion. Antibodies against PHF8 (ab36068; Abcam), H3K9me1 (ab8896; Abcam), H3K9me2 (ab1220; Abcam), H3K27me2 (ab24684; Abcam), and H4K20me1 (ab9051; Abcam) were used for immunoprecipitation. After washing and reverse‐crosslinking, the precipitated DNA was purified and amplified by qPCR. Extracted RNA was reverse‐transcribed into cDNA and qPCR was performed using the SYBR Green Master Mix (Invitrogen, Carlsbad, CA, USA) in the iCycle System (Bio‐Rad, Hercules, CA, USA). PCR data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). The sequences of the PCR primers used are listed in supplementary material, Table S3.