Anti h3k9me1

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.

Briefly, HCC cells were grown to 80-90% confluence on a 10 cm dish and treated with 1% formaldehyde for 10 min. Subsequently, cell lysates were sonicated to generate DNA fragments. After removing cell debris, the supernatant containing chromatin was immunoprecipitated overnight at 4 °C using anti-DDX56, anti-ZEB1, anti-MECOM, anti-H3K9me1, anti-MIST1, or anti-IgG antibodies (a negative control, Millipore). Then, DNA-protein complexes were pulled down with magnetic beads and subsequently eluted from the beads using an elution buffer. Enrichment of specific DNA fragments was examined using RT-PCR. The products were loaded onto a 1.5% agarose gel and subjected to electrophoresis. All antibodies used in this study are listed (Table S2).

Antibodies used for immunoblot experiments: anti-GFP-HRP (Milteny Biotec #130-091-833); monoclonal anti-FLAG M2 (Sigma-Aldrich #F3165); anti-HA-HRP (Roche #3F10), anti-H2B (Millipore #07–371) or kindly provided by Prof. Spiker, anti-H2Bub (Medimabs #MM-0029); anti-H3K4me2 (Millipore #07–030); anti-H3K4me3 (Millipore #05–745); anti-H3K9me1 (Millipore #07–450); anti-H3K27me3 (Millipore #07–449); anti-H3K36me3 (Millipore #07–353); anti-H3ac (Millipore #06–599); anti-H3K9ac (Millipore #06–942); anti-H3K27ac (Millipore #07–360); anti-H4ac (Millipore #06–598); anti-H3 (Millipore #05–499); anti-RPT5 (Enzo Life Sciences# BML-PW8245). Antibodies used in cytological analyses: Anti-MYC (Millipore #05–724) or anti-GFP (ThermoFisher Scientific #A11122) primary antibodies, Alexa-488 coupled anti-mouse (ThermoFisher Scientific #A11001) or anti-rabbit (ThermoFisher Scientific #A11008) secondary antibodies. Antibodies used in ChIP-seq analyses: anti-H2Bub (Medimabs #MM-0029-P).

Ten-day-old seedlings were ground in liquid nitrogen and the powder was boiled for 5 min in protein sample buffer. The proteins were resolved on a 15% SDS/PAGE gel and transferred onto nitrocellulose membranes (Bio-Rad). Then the membranes probed with anti-H3K27me3 (Millipore 07-449); anti-H3K27me2 (Millipore 07-452); anti-H3K27me1 (Millipore 07-448); anti-H3K4me3 (Millipore 07-473); anti-H3K4me2 (Millipore 07-030); anti-H3K4me1 (Millipore 07-436); anti-H3K9me2 (Millipore 07-441); anti-H3K9me1 (Millipore 07-450); anti-H3K36me3 (Abcam ab9050); anti-H3K36me2 (Millipore 07-274); anti-H3K36me1 (Millipore 07-548); anti-H3 (Abcam ab1791) or anti-GFP (Roche 11814460001) in TBST (137 mM NaCl, 20 mM Tris·HCl pH 7.6, 0.1% Tween-20). After three washes with TBST, the signals were detected with Immobilon Western Chemiluminescent HRP Substrate (Millipore) for histone antibodies or Super Signal West Dura Extended Duration Substrate (Thermo Fisher Scientific) for GFP antibody.