Apoptosis assay Endothelial apoptosis assay was performed using a Cell Apoptosis DAPI DetectioKit.Ibrief, PAECs 48h immediately after transductiowere seeded into a 6 nicely plate.The cells were theundergone either serum starvatiofor three days orh2O2 treatment method for 24h.The cells had been up coming fixed with 20% ethanol at RT for 10 min, and thestained with 49, 6 diamidino two phenylindole for thirty sec.Apoptotic cells had been characterized by the irregular edges throughout the nucleus and nuclear pyknosis as previously described.Images have been takeunder a computer assisted NikoEclipse TE2000 S microscope.Apoptosis fee was assessed i10 randomly picked fields under a microscope for each sample with three independent replications.Generatioof SUMO1 transgenic mice The SUMO1 coding regiowith aterminal six xhis tag was cloned into a pHY R vector implementing thehind Iand BamhI cutting sites.
AhumaB actipromoter was implemented to drive the trans gene expression.The great post to read expressiocassette was released by Xba I digestioand themicroijected into NOD embryos.Pups resulted from foster mothers have been genotyped by PCR fol lowed by Southerblotting employing the probes fromhumaB actipromoter.Two founders, SUMO1 Tg1 and SUMO1 Tg2, had been character ized with germline transmissioafter screening a total of sixteen pups.All mice werehoused ia SPF facity imicroisolator cages supplied with autoclaved YM201636 food and acidified water which has a 12 12h light dark cycle.Experiments involving SUMO1 Tg model had been carried out iSUMO1 Tg1 mice, whe SUMO1 Tg2 mice were used for confirmation.All experiments involving mice have been finished according to a protocol reviewed and accepted through the Institutional Animal Care and Use Committee in the Tongjihospital.
Immunohistochemistry Tissues had been fixed i4% formaldehyde

at four C overnight and theembedded iparaffin.Tissue sections were deparaffinized ixylene and rehydrated igraded alcohol.Endogenous peroxidase was blocked with 3%h2O2 and nonspecific proteins were blocked with 10% goat serum or rabbit serum for 30 min.The sections were theprobed which has a rab bit anti CD31 or anti SUMO1 antibody at 4 C overnight, fol lowed by incubatiowith aHRconjugated goat anti rabbit secondary antibody at RT for 30 min.DAB substrate was utilized for 5 mifor colour advancement as reported.Matrigel plug assay Wd variety and SUMO1 Tg mice have been injected subcutaneously othe back of both sides with 0.5 ml ice cold twelve duted Matrigel cotaining 200 ng ml VEGF and 60 U mlheparin.A single week later on, the mice were sacri ficed and gel plugs wereharvested.A part of the plugs was subjected to immunohistochemical evaluation of CD 31 as over.The rest part of plugs was weighed, chopped and immersed i0.5 ml distled water at four C overnight.The volume ofhemoglobiithe plugs was thedetermined applying Drabkireagent as instructed.