Anti h4k20me1

Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing 1% phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China) on ice for 30–60 min and centrifuged at 12,000 rpm for 5 min, and the pellet was discarded. The protein samples were boiled in sodium dodecyl sulfate (SDS)-loading dye for 15 min. The proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto a 0.45-µm nitrocellulose membrane (Millipore, Bedford, MA, USA). The membranes were blocked with Protein Free Rapid Blocking Buffer (EpiZyme, Jiangsu, China) and subsequently probed with primary antibodies. The primary antibodies used were as follows: rabbit anti-human SET8 (#2996), anti-PIK3CB (#3011), anti-FOXO1 (#2880), anti-AKT (#4685), anti-p-AKT (#4060), and anti-BAK (#12105), anti-histone H4 (#13919), anti-BCL2 (#15071), and anti-GAPDH (#51332) purchased from Cell Signaling Technology (Beverly, MA, USA) and anti-H4K20me1 (Abcam; #ab177188; Cambridge, UK). Thereafter, the membranes were washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1.5 h at room temperature. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system.

ChIP assays were carried out with a Simple ChIP Plus Sonication Chromatin IP Kit (Cell Signaling Technology, MA) according to the manufacturer's instructions. Briefly, cells (1 × 10⁷) were fixed with 1% formaldehyde for 10 min at room temperature to cross-link DNA and proteins. Glycine was then added to stop the cross-linking reaction. Chromatin was sheared using a Microson Ultrasonic Cell Disruptor XL (Misonix) with 16 cycles of sonication (15 s each, 2 min rest, amplitude = 10, power = 15 W). Ten microliters of sonicate was collected from each sample as an input control, while the remaining sample was incubated with anti-SP1 (Abcam, USA), anti-H4K20me1 (Abcam, USA) antibodies, histone H3-positive control, or IgG negative control at 4°C overnight. Immunoprecipitants were bound to protein G magnetic beads, and the DNA-protein cross-link was reversed at 65°C for 2 h. DNA was purified, and enriched DNA sequences were analysed by qPCR. ESE-1 oligonucleotide sequences for PCR primers were forward 5′-TGCAATTGTGCCCTTGAGGA-3′ and reverse 5′-CCTACGGCCACACTGAACTC-3′.

ChIP assays were performed with the use of a Simple ChIP Plus Sonication Chromatin IP Kit (Cell Signaling Technology, MA). Briefly, the cells (1 × 10⁷) were fixed with 1% formaldehyde for 10 min at room temperature to cross-link the DNA and the proteins. The cross-linking reaction was then stopped with the use of glycine. A Microson XL ultrasonic cell disruptor XL (Misonix) was used to shear the chromatin. Ten microliters of the sonicated solution was gathered as an input control. The surplus sonicated solution was incubated with anti-FOXO1 antibody (Cell Signaling Technology, Danvers, MA), anti-H4K20me1 (Abcam, Cambridge, UK) antibody or a negative control IgG at 4 °C overnight. The immunoprecipitants were bound to protein G magnetic beads, and the DNA–protein cross-linking was terminated by incubation at 65 °C for 2 h. After purification, the enriched DNA sequences were detected by PCR. To confirm whether FOXO1 binds to the methylated promoter of PTEN, a re-ChIP assay was performed. In brief, after the standard ChIP assay, the beads were incubated with 10 mM dithiothreitol for 30 min at 37 °C. The eluent was then diluted with sonication buffer, followed by a second round of the ChIP assay. The PTEN oligonucleotide primer sequences were forward, 5′-TTGGATGTGGGTGCTTGTGT-3′, and reverse, 5′-CTTCTTCCTTTGCTCGGGGT-3′.

Whole-cell extracts were acquired via protein lysis buffer (Cell Signaling Technology, Danvers, MA). Same amounts of proteins (60μg) of each group were segregated by SDS-PAGE and moved to PVDF membranes (Millipore, Billerica, USA). After blocked by 5% fat-free milk at room temperature for 1 hour, the PVDF membranes were immersed in specific primary antibodies at 4 °C overnight. The primary antibodies included anti-β-actin (ProteinTech, Wuhan, China), anti-KMT5A (ProteinTech, Wuhan, China), anti-RFX1 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-ENO1 (ProteinTech, Wuhan, China), anti-CD31 (ProteinTech, Wuhan, China), anti-H4K20me1 (Abcam, Cambridge, UK), anti-vimentin (ProteinTech, Wuhan, China), anti-αSMA (Cell Signaling Technology, Danvers, MA), anti-COL Ⅰ (Abclonal, Wuhan, China) and anti-COL Ⅲ (Abclonal, Wuhan, China) antibody. Then, corresponding secondary antibodies were used to immerse the PVDF membranes at room temperature for 1 hour. Then, the PVDF membranes were washed with PBST 3 times. Subsequently, the protein signal was displayed by an ECL system.

Cells were lysed on ice in cold EBC-buffer (150 mM NaCl; 50 mM TRIS pH 7.4; 1 mM EDTA; 0.5% NP-40/Igepal) containing protease inhibitors (EDTA free-Protease inhibitor cocktail, 1 mM PMSF, 50 mM NaF) and 1 mM DTT. The lysates were sonicated using Bioruptor Plus sonication device on “High” mode for 10 cycles each of 30 s ON/30 s OFF. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Blocking and blotting with primary antibodies were performed in PBS-T supplemented with 5% skimmed milk powder. Proteins were visualized on films using secondary horseradish peroxidase-conjugated antibodies and ECL (GE Healthcare). Films were developed using an X-ray machine (Valsoe; Ferrania Technologies). The primary antibodies used in this study are anti-Vinculin (Sigma; V9131—1/25000), anti-SET8 (Millipore; 06-1304—1/1000), anti-H4K20me1 (Abcam; ab9051—1/5000), anti-H4K20me3 (Diagenode; C15410207—1/1000), anti-H4 pan (Millipore; 04-858—1/5000).