H4k20me1

Proteins were extracted from the HUVECs of the different cultures by cell lysis buffer (Cell Signaling Technology, Danvers, MA). Equal amounts of protein were separated by 10% SDS–PAGE gels and then transferred to PVDF membranes (Millipore Corporation). The PVDF membranes were blocked in 5% skim milk at room temperature for 1 h and then incubated with the corresponding primary antibody (1:1000) at room temperature for 2 h. The primary antibodies were monoclonal antibodies against β-actin (ProteinTech, Wuhan, China), PTEN (ProteinTech, Wuhan, China), SET8 (ProteinTech, Wuhan, China), H4K20me1 (Abcam, Cambridge, UK), forkhead box protein O1 (FOXO1) (Cell Signaling Technology, Danvers, MA), and e-selectin (Santa Cruz Biotechnology, Santa Cruz, CA), and polyclonal antibodies against ICAM-1 (Cell Signaling Technology, Danvers, MA) and p-p65 (Cell Signaling Technology, Danvers, MA). The membranes were incubated with the corresponding secondary antibody (1:1000) at room temperature for 1 h. The membranes were then washed, and the proteins were detected by a LAS-4000 mini CCD camera (GE Healthcare).

Cells were grown on sterile 12 mm glass coverslips, fixed in 3% formaldehyde in PBS for 15 min at room temperature, washed once in PBS, permeabilized for 5 min at room temperature in PBS supplemented with 0.2% Triton X-100 (Sigma-Aldrich), and washed twice in PBS. All primary and secondary antibodies (Alexa fluorophores; Life Technologies) were diluted in filtered DMEM containing 10% FBS and 0.02% Sodium Azide. Antibody incubations were performed for 2 h at room temperature. After antibody incubations, coverslips were washed once with PBS and incubated for 10 min with PBS containing 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, 0.5 μg/ml) at room temperature to stain DNA. After three washing steps in PBS, coverslips were briefly washed with distilled water and mounted on 6 μl Mowiol-based mounting media. The following primary antibodies were used for immunostaining: H2AX Phospho S139 (mouse, 613401, 1:1,000; BioLegend), 53BP1 (mouse, Upstate MAB3802, 1:1,000), H4K20me2 (rabbit, ab9052, 1:100; Abcam), H4K20me1 (rabbit, ab9051, 1:200; Abcam), BRCA1 (mouse, sc-6954, 1:100; Santa Cruz), Cyclin A (mouse, sc-271682, 1:100; Santa Cruz), and RAD51 (rabbit, 70-002, 1:1,000; Bioacademia).

ChIP assays were implemented with a Simple ChIP Plus Sonication Chromatin IP Kit (Cell Signaling Technology, MA) according to the manufacturer’s instructions. In brief, HUVECs were fixed with 1% formaldehyde for 10 min at room temperature to mediate DNA and protein cross-linking. Glycine was then used to terminate the DNA and protein cross-linking reaction. Chromatin was sheared with the use of a Microson Ultrasonic Cell Disruptor XL (Misonix). Ten microliters of the sonicated solution was collected from each sample as input, and the remaining sample was incubated with anti-ELF3 (NOVUS, NBP1-30873), and anti-histone H4 lysine 20 methylation (H4K20me1; Abcam, ab9051) antibodies or a negative control IgG at 4 °C overnight. Immunoprecipitants were bound to protein G magnetic beads, and the DNA-protein cross link was reversed by incubating at 65°C for 2 h. Then, the DNA was purified, and enriched DNA sequences were analysed by qPCR. MARK4 oligonucleotide sequences for PCR primers were as follows: forward 5′-CCAACTGGGGAGAGAATGGG-3′ and reverse 5′-AGACTGAGAGAGACCCCAC-3′.

Whole cells were washed with ice-cold phosphate-buffered saline, harvested, and resuspended in lysis buffer (Cell Signalling Technology, Danvers, MA). Protein samples were boiled for 10 min at 100 °C in sample loading buffer. Equal amounts of protein (50 μg) from different groups of HUVECs were separated by 8–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Billerica, USA). Membranes were blocked with 5% skimmed milk solution for 1 h, and then all membranes were incubated with primary antibodies at 4 °C for 12 h. The primary antibodies used in the present study were as follows: monoclonal antibodies against β-actin (ProteinTech, Wuhan, China, 60004-1-Ig), KMT5A (ProteinTech, Wuhan, China, 14063-1-AP), CREB (Cell Signalling Technology, Danvers, MA, #9197), PTP1B (ProteinTech, Wuhan, China, 11334-1-AP), p-p65 (Signalway antibody, Maryland, #11014) and H4K20me1 (Abcam, Cambridge, UK, ab9051). After washing the membranes with PBST 5 times, an HRP-conjugated secondary antibody was added for 1 h at room temperature. Then, the membranes were washed five times with PBST, and the protein signal was detected by an ECL system.