8/13/2019 IMG_20130329_0009

1/1

BLOOD, 6 DECEMBER 2012. VOLUME 120. NUMBEFI 24

PLT-rich plasma (PRP). The PRP was centriluged twice more, each timeretaining the supematant and discarding the leukocyte-rich pellet togetherwith the 0.5-mL PRP layer immediately above it. The pRp was furtherleukol,te-depleted by mixing with anti-CD45 nrlgnetic beads (DynabeadsCD:15, Invitrogen; 33 pL beads/ml of PRP) and rotating at room tempera-ture lbr 20 rninutes. "l'he beads rvere renloveil by Dynal MPC-L magnet,usiug 2 cycles of 2-minute lnagnetization steps and transfer:ring the pRp tofresh tube-s after each step. The leukocyte-deplcted PRP was centrifuged for10 nrinutes at 15003. The supernatant was discarded and the PLT pelletresuspendeiJ in 2 mL Trizol (Invitrogen) until a plirticle-free solution rvasobtained: l-ml- aliquots were frozen on dr1, ice and stored at -80.C.Startin,' PRP volunres ranged from 5 to 15 mL, ltnd corresponding yieldsrangecl ' . onr 0.-5 to 2.5 x 10e PUfs.BNA preparation and cDNA synthesisItNA tiom cultLrred cells and PLTs was prepared using Trizol essentiallyaccording Lo the nianufacturer's instructiorls, except that 2-rnl- PhaseI-ock Gel tubes (5') u'cre used for the phase separation. RNA pelletsu'ere air dried for no rr)ore than 7 rninutes and theri resuspended in t5 pLnuclease free water (Antbion, Invitrogen). RNA yields averaged 9,10 ng/l0e PLTS u'ith A260/280 rarios between 1.33 and 1.66. The 25-ng pLTRNA rvlrs processed bl the ABI TaqiVlan Rer,erse Transcription Reagents(Applied Biosl-stems) in a 25-pL volume, accorcling to the manufactur-er's protocoi. At the end of the reaction. RNA u,as diluted to 250 pLqith nuclease-free water and stored at -20"C. In addition, total RNAfronr hunan brain (P/N #5.+005, 5:1007. and 5401 17) rvas obrainecl fromStratase're (Agilent Technologies).Gene expression quantitative real-time PCFI

GWAS SEOUENCE VARIANT FOR PLATELET VOLUME 4861MK suspension culturesMouse f'etal liver cells were collected frorn wr cD1 mice (Charles RiverLaboratories) on day E13._5 and cultured at 37"C and 5% CO2 in thepresence of 0.1 pg/ml purified recombinant mouse c_Mpl ligand fcrr5 days. Fetal Iivcr cell cultures rvere layered on a single_step gradier.rt(1.5Vc-3.070 BSA) on culture da1,4, and MKs were allowed ro sediment for30 minutes.2-i The N,lK pellet u,as then resuspcnded in tiesh media andcultured for an additional 24 hours alone or in the presence of either0.3clc DMSO or 100pM Dl,naSore in DIvISO. NIK cultures were exarnineclon day 5 by phase-contrast microscopv using a Nikon eclipse TSl00benchtop rnicroscope (Nikon) at 20x magnilicationl digital irrages u,erecollccted on a l{antarnatsu C2400 CICD carrera and analyzcri using IrnageJ.tr'lature N,lKs rvcre identified by size (> lOpm diar-neter) and drsrin,suishedfrom pro-PLT ploducing IVIKs because ol'thc presence or absence of longpro PLT elongations (circularitl > 0.7). Samples were exar.nined in tripli_cate. and at least 78 cells u,er-e counted for each condition tested. All studiescomplicil uith institutional guicielincs appr.oved by the Children,s Hospitalanimal carc and use committee, and the lnstitutional Aninral Care and IIseCommittee.

Statistical analysisResults are expressed as ntcan + SD rvith number of experiments.Statistical cornparisons between groups \rere performed bv 2-tailed I testusing Prisnr unlcss stirted olheru.ise. Statistical comparisons of Lucil'erasedata rveru perfornred on log transformcd signal intensities using mixedliuear moclels to acc(,Llnt lor tlic hierarchical n:iturc of tlte clata.

Absolute quxntiiication of Dn'NI3 transcript abunclance in RNA sanrplestl'orn huuran PI-Ts and other cells was carried out on aABI Prism 7900HTSequencc Dctection Svste-m (Applied Bioslstenrs) using the followingprorocol: -50"C I rnrnutes. 9-5oC I0 minutos. 40 x (95"C l5 seconds.60'C I nrinutc). Ploduct nuntbers of the uscd Tatli\4an gr'nc cxpres\iunassal,s (Appliecl Biosvstems) can be lbunri in suppiemental Tnble l(availeble on the B/o.r.1 Web site; see the Supplemenrai Nlatcrials link at thetop of the onlino arricle).'lb test lbr abundance of the novel DNMJtrattscril]t variant. a custonr ThqMan assay was dcsi_sned ri,ith the probespanninr the boLrndary betrveen the novel exon 28 and e:,cn 3. Transcriptlelels rrcrc 1l(rrnr.lized Io Gi\PDH. Reactiorrs uere rncasurecl in tr.iplicateanil prinrt'r el'liciencies obtained through cDNA staudard dilution series.RACE-5'-Rapid anrpliiication of cDNA ends (RACti) orr RNA sanrples fionrCHRF and \lK cells and PI-Ts rvas pertbrmecl usinc rhe 5'l3'RACE Kir,2nd Generation (Roche Dia.snostics) fblJouing the manuticturer's proto-col. For the PCR artrpliiicarion steps. recombinanr Tecl poll-rncrase anddNTPs tiom Fernteitllis uere used (Fcrnentast. DNA fragmcnts lveresnbclonecl iuto pGent T erisl' (Prornega) and sequenced. Prirner and cloneseqlrences can be found in supplemental T.rble 2.Dual Luciferase reporter assaysDNA ll ti-qrncrrrs u ere clonecl into rhe fire111, Lucitcrlse vector p6i.l. i 0(Promegrl.'ltn nritlion cells uete transiectetl *.irh I0 p.r lirefll Luciferlser':clor lrrai -ii]il nS llcnilh L-ueiferase yector pGI-1.7-1 (PlonreglL.l. 'l'ranstec-ti )D 1\11s trrrl-rIntttl iirt elerrloooration using a BiLr,liad GencPLriser.\cellt::1ttittetiti:Lr -:r,,j. ll{) \'. l;tl)lf pF. resisupce u,. -}-itrrrr cu\ctteS). i_ri-ciferrse ae tiviri u as ineasured rn a LUMIstar Optimr iuminonteter (BlvIGl-ubrech r using tirc Dual-I-uciiirase Reporter Assar, kiL (Proniega.l. RandomDNA sequenccs lor use ls negative cotltrols \1ere generate(l b1,the RanclonrDNA Sequc'nce Generator (htqr;11wwu,.facult1'.ucr.edu,/- mnracluro/randont.htnr) and prociucecl b1' artiticia) gene st,nthesis (GencAr.r). I-irr: sequences ofcloninl prinrers anil ccinstruct inserts. sec supplernentiLl Table 3.Electrophoretic mobility shift assaySee srrl-.plentcntrLl lvlcthorls.

ResultsMEISI putatively regulated genes are important determinantsof the PLT lineageTher exprcssiot\ ol' MEISI is restr.icted to rhe mesakaryocyticlineagc, anrong dillerentiared blood cclls (supplenrental Iri-surc1).3 e To urrp N,IEIS i binding cvenrs. we pertbnried Ctrlp-seq in thehuman urcgakar\ocvtic cell line CHRF. a close rnodel fbr NiKsbased on its transcriptional profilc (supplcrnental Figure 2). NIEISIpeaks r.r'el'e citlled usiug magnetic-activatecl cell sortingri anJPICSTe frcnr \\,hich a high-confidence daraset of l3 842 bindingevents detcoecl bi both algorithnrs \ as serrerated (Figure lA).Gcnes nearbr, NiEIS 1 binding events \\,ere analvzed with GREATTt(Genomic Ilegions Enrichntent of Annotations Tool). revealing thatputativeiv reculated genes are significantll, over-represented innlegakaryopoiesis and PLT biology,' categor-ies (?rble L; supplemen-tal Fi_eure -j). To increase stringency in assigning putativel),regulated scnes, ii list of 1285 potentialiy N{IJISl-regulared senesr.r'as ser.ieratecl hv filtcring firr pelks \\'ith a rninirrunl height oi'30 reuris (S()tir ircrrcntile oi peak hei-ithil ancl r.vithin - -i kb o1,

p[otein .o(ilnL {e]tes (sLri)pleinental X13thods). Anall,sis of their.c.\pressir)n Pxttilits slt0ri eil e sigltiticiUtt o\'cr-rcprescntl,rion ol -i7q*nes ol il sct ol'l-jl tltat $cre sh()\\'it to he sfrnn-ulr,()\.1-r.L-.{Dr.c,ssdJin \lK. r'iiilti\'3 tr) '.itJ i)thar - tntrttu-i bloocl e eil l_t lc:,(P < -5 x l0-rrt Figure lBl in the HaernAtlas colnpcndium ofbiood ceil trtnscripts.s Thcse obser.r,ations are coltsistent with ourll,pethcsis that t'1El.91, hc:cause of'its character-istic finclge resn.i.-tion, r'esulates rnirnv gcnes critical to the identitv ol N{Ks and pLTs.Incleed. thesc -57 gcnes contain ntan\, \\,ell-known resulators oftheir firnction. such rs the coilrrgen siqnaling rcceptor Glycr)protein(GP) \rl ((i['rtl. 1 of the .tr suhLurits oi'tlre PLT recepror for vou\\iillchrancl liactor. GPV iG'P-i1. and rhe r qranule rnctnbreneprotei n P-sclectin (.lfLP).