Indicated restriction enzymes

Genomic DNA was prepared from 2 × 10⁸ cells according to standard protocols and digested with the indicated restriction enzymes (New England Biolabs). The digested DNA was resolved in a 1.2% agarose gel and blotted onto a Hybond-XL membrane (GE Healthcare). After transfer, the membrane was cross-linked with UV and hybridized with different probes. ³²P labeling of DNA probes was performed by random priming using Klenow fragment exonuclease- (New England Biolabs), in presence of [α-³²P]CTP and hybridizations were performed in Church buffer at 65°C for Telo/STE1 and STE1 (STE1 = Subtelomeric Element 1) probes and 55°C for chromosomal probe. For spotting assays, genomic DNA were directly spotted onto a Hybond-XL membrane (GE Healthcare). Radioactive signal was detected using a Biorad molecular imager FX.

We constructed a plasmid to overexpress CD70 linked to the fluorescent protein mCherry. The coding sequence of CD70 was amplified from HUVEC cDNA by PCR using a Q5 High-Fidelity DNA Polymerase (New England Biolabs Inc., NEB, Ipswich, MA) with gene specific primers including recognition sites for NheI and EcoRI as follows: forward 5’-AAAGCTAGCATGCCGGAGGAGGGTTCGGG-3’ and reverse 5’-AAAGAATTCGGGGCGCACCCACTGCACTCC-3’. The PCR product was digested with indicated restriction enzymes (NEB) and N-terminally fused to a mCherry-myc-HIS tag into a pcDNA3.1(-) vector using the Quick Ligation Kit (NEB). HPAECs were transfected with this construct versus a control plasmid of the same vector backbone using Lipofectamine 3000. The manufacturer’s protocol was modified by performing transfection overnight in regular media followed by replacement with fresh media.