Teeatoscnrbtdeulyt hswork this Neuherberg, to Science, equally Protein contributed Unit authors Research *These Health, Environmental for Center etr Tu Center, Tu University eiie ehin salIsiueo ehooy af,Israel. Haifa, Technology, of Institute Israel Technion, Medicine, d .Sabag Sema3B D. Adi and Sema3A in transduction plexin-A2 signal the of role The ARTICLE RESEARCH 5240 2014 October 13 Accepted 2014; May 2 Received ` these these of activity activity with the the 1 because modulate the interact the receptors, that such is that mechanisms of of cues of expression receptors and understanding and guidance cues the interpret the guidance cells of to which characterization by Essential guidance of mechanisms attractive factors. and variety repulsive a secreted by guidance of gradients destination cells include Migrating less correct which in 2012). their cues, as al., to much such et guided (Trepat diseases in are although of and forms animals various migrations, adult to the in place extensively Similar take migrate extensive, locations. cells development, distant embryonic During INTRODUCTION complex, Receptor Plexin, , guidance, Cell WORDS: KEY of gradients encountered of to responses cells . differential migrating of induced trigger populations plexins can different of cues expression the microenvironmental Our in level. by changes expression their that semaphorin on suggest functional depending in results plexin-A2-silenced flexible plexins be of in can identity signaling the receptors Sema3B that plexin-A4 follows restore It although to cells. plexin-A1-silenced failed contrast, in it By signaling cells, them, plexins. Sema3A restored between A functionally overexpression class differentiate can plexin-A2 other to overexpressed, replace ability when their that, suggesting nullified it semaphorins, both although to responses restored plexin-A1- cells in plexin-A4-silenced or plexin-A2 two of abolished the overexpression of on Interestingly, one completely and . plexin-A4 depended but required also which which plexin-A4, signaling, The signaling, Sema3B and cells. Sema3A plexin-A1 glioblastoma affect in neuropilin-1, not and did cells silenced endothelial silencing we in signaling, semaphorin expression the in study its receptor To plexin-A2 signals. associate the semaphorin of turn, transduce role to in anti-tumorigenic plexins which, A and neuropilins, class to with anti-angiogenic bind that are factors guidance semaphorins 3 Class ABSTRACT hita oansGloeckner Johannes Christian uhrfrcrepnec ([email protected]) correspondence for Author acrRsac n aclrBooyCne,TeBueRpaotFclyof Faculty Rappaport Bruce The Center, Biology Vascular and Research Cancer 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,54–22doi:10.1242/jcs.155960 5240–5252 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. ¨ ign Germany. bingen, ¨ ign nttt o ptamcRsac,MdclProteome Medical Research, Ophthalmic for Institute bingen, 1, ,TtaaSmolkin Tatyana *, 3 emot etu Mu Zentrum Helmholtz 2,3 1, n eaNeufeld Gera and ¨ ,Yln Mumblat Yelena *, ce,Gra Research German nchen, 2 bradKarls Eberhard 1, ` 1 aisUeffing Marius , eahrn.Hwvr tsol entdta nms studies most in that noted 3 class be the should of any it for specificity However, high be semaphorins. display class might not the might plexins that plexins A suggest A studies class to these the seems extension, By signaling turn, interchangeable. Sema3A in A for which, class that signals, four suggest Sema3A all Taken transduce that 2001). can suggest Strittmatter, to plexins seem and observations Takahashi cells these 2012; together, in al., signals levels Sema3A et plexin-A2 transduce expression also (Janssen which can higher plexin-A2 organization in that at suggested studies have cytoskeletal of expressed Additional ectopically affecting 2008). signals was that al., signals suggested et pro-apoptotic not (Ben-Zvi has but exclusively study Sema3A another transduces Interestingly, plexin-A3 2013). al., et oan htlmttersga rndcn blt,adthey and subfamily receptor ability, plexin A transducing class the signal of members their with associate limit intracellular the short that of contain one to domains Neuropilins bind receptors. 2005), al., neuropilin et (Gu two Sema3E six, subfamily of which exception a of as the are members, with seven semaphorins emerged containing 3 semaphorins Class secreted also and 2012). of al., responses et have Kikutani, Sakurai and immune 2013; but Kumanogoh 2012; , (Tamagnone, 1993) progression of tumor al., regulators et important (Kolodkin factors be will cascades transducing downstream. signal activated which determines receptors ta. 08 oet ta. 08 Bouvre 2008; Sema3A al., Schwarz et in 2005; Moretti al., role 2008; et a Suto al., 2005; evidence et play al., functional all et found (Yaron receptors transduction lacking receptors signal three these mice plexin-A4 that or suggesting knockout contrast, in plexin-A3 By plexins using extensively. plexin-A1, more additional investigated. conducted studied of been been Studies has roles not transduction possible signal have Sema3A the signaling (Nawabi but system Sema3B 2010), nervous the al., Sema3B of et of development ventral vertebrate the transduction the during at midline the projections well commissural for been guiding signals NRCAM, not the with have receptor along required, transduction be adhesion to signal found was Sema3B Plexin-A1 characterized. of class in inhibitor specific of plexins an roles as The A 2008). addition, al., in et binds (Varshavsky functions, that angiogenesis and 2001) a al., neuropilins (Kolodkin is et Sema3B both 2008). (Tomizawa (NP2) to al., suppressor et tumor neuropilin-2 Acevedo cancer 1997; al., to lung et Chen not 1997; al., but et (NP1) 2013). neuropilin-1 Giraudo, and (reviewed Gu subfamilies 2012; semaphorin al., signal 6 et class and Neufeld and in 5 binding of class direct 4, members class membrane-anchored as the some addition for receptors in These transducing signals. function semaphorin 3 also class plexins transduce to plexin-D1 with or h eahrn eeietfe srplieao guidance repulsive as identified were semaphorins The eaAi nat-nigncsmpoi htbnsto binds that semaphorin anti-angiogenic an is Sema3A 2,3 faKessler Ofra , 1 , ´ ta. 02 Katayama 2012; al., et e

Journal of Cell Science n hsmgtntb eeatpyilgcly(aaah and (Takahashi 2012). physiologically al., et relevant Janssen be 2001; not overexpressed, Strittmatter, derived were might the is plexins thus of specific experiments and which much culture in experiments Likewise, cell from direct from modulator. the a obtained secondary affects to targeting the information a due the whether of whether is or unclear expression Sema3A signaling, to Sema3A is on responsiveness it effect of models loss mouse observed -targeted using ARTICLE RESEARCH ieodhge splmnaymtra i.SA.W have We to two S1A). the is Fig. whereas NP1 material variations, encoding (supplementary cell some the per higher with types, the molecules fivefold similar and mRNA cell around A is of type both number NP2, plexins substrate encoding In D cell the per type arrows). molecules to mRNA 1C,E; of (Fig. number attached cell of remain appearance contracted the that with and focal contraction of ‘spikes’ loss cell of the by with disassembly followed associated localized contacts is which with cytoskeleton, Sema3A as their stimulation umbilical- such to human semaphorins respond by (HUVECs) and cells cells endothelial vein-derived multiforme glioblastoma U87MG cells in endothelial U87MG Sema6A in and and Sema3B cells of glioblastoma but that transduction for signal required Sema3A is for required not is Plexin-A2 RESULTS plexin-A4 that plexin-A2- given in signaling true, cells. Sema3B Sema3B. silenced be restore and to always failed Sema3A not overexpression between might distinguish this cells to such However, as ability are price, plexins their a further different at lost comes and the This of levels expressed interchangeable. threshold levels highly functionally for certain when expression requirement exceed that the plexins suggesting if the the abolished of that be some might suggesting Sema3A plexins both signaling, specific restored it Sema3B expression and plexin-A4 or silenced plexin-A1 cells in and for overexpressed was receptors, semaphorin. plexin-A2 appropriate Sema3B when the Interestingly, in by signaling plexin-A1 plexin-A2 abrogates of each and silencing presence receptors the expression by Sema3A determined in its for is specificity Sema3B of The and or signaling. Sema3A inhibition Sema3A Sema3B and both and Sema3A both of complexes nullifies component glioblastoma receptor required and a Sema3B provide cells is We endothelial plexin-A4 signaling. additional in cells, Sema3B as that, and well suggesting Sema3A as evidence to not plexin-A2 plexins of A in contribution class are studied clarify the we signaling, to detail Sema3A and plexins in more plexin-A2 transduction, of signal role the semaphorin possible the 3 class that in plexins and semaphorins complexes interchangeable. receptor specific of specificity 2011). to the plexins complex determine al., of combinations neuropilins a et specific and (Kigel that of suggest plexin-A4 observations consist and These physiologically plexin-A1 receptors NP1, that Sema3A containing suggesting cytoskeletal functional not Sema3A, the but inhibit relevant by plexins completely heterocomplexes. these induced to of sufficient collapse spontaneous one is any plexin-A3 of form of expression plexin-A1 the which U87MG of of cytoskeletal Inhibition in presence plexin-A4, affecting simultaneous and and the signaling cells requires Sema3A endothelial have organization we in cells, However, plexin. that, glioblastoma single observed a and recently NP1 requires signaling ogi etrudrtnigo h oe ftecasA class the of roles the of understanding better a gain To Sema3A successful that assumed is it studies these all In Fg CD upeetr aeilFg 1)a osplexin- contraction 2011). does al., the et as cell (Kigel S1E) cells, silencing Sema3A-induced Fig. plexin-A4 U87MG or material inhibit A1 supplementary and of not 1C,D; expression (Fig. HUVECs did S1B–D). the of in silenced expression silencing Fig. shRNA effectively the the material or plexin-A2 though plexins the supplementary even inhibit A However, not 1A; class (Fig. do other that neuropilins the species shRNA of of we specific expression U87MG possibility, transduction in two and this using HUVECs the examine in cells plexin-A2 To of in expression cells. the these inhibited participates in determine to signals also sought Sema3A and we plexin-A2 2012), (Takahashi al., receptors whether et in Sema3A Janssen participate 2001; functional to Strittmatter, co- of and signals by formation Sema3A also supported the transduce has is to These plexin-A2 reported that been 2011). Because assumption experiments. al., an immunoprecipitation receptors, et contains three inhibits complex (Kigel signaling these completely Sema3A Sema3A the plexins that suggest by cross- other observations not the transduction do of that signal in expression species these the (sh)RNA and of hairpin inhibit any HUVECs of short expression in specific the using collapse of Inhibition cytoskeletal cells. Sema3A-induced U87MG in of transduction resulting not the but signals for plexin-A4, required and are plexin-A1 plexin-A3, NP1, that observed previously swt lxnA splmnaymtra i.S3C). Fig. material (supplementary plexin-A4 plexin-A1 with with well complexes as of S3B), formed Fig. as (Serini plexin-A2 material form observed Sema3A truncated (supplementary plexin-A2 The of been truncated full-length (A2ExTm) 2003). inhibitor has al., tag dominant-negative similarly et a FLAG It a as a S3A). functions that with Fig. C-terminus which previously material its plexin-A2, of at (supplementary domain plexin-A2 tagged the intracellular of was the form truncated lacking a cells receptor U87MG the in expressed HUVECs the S2A–C). cells. Fig. in for material these plexin-A2 (supplementary cytoskeletal Sema6A-induced collapse in essential of and signals Sema3B- expression is inhibited Sema6A completely the plexin-A2 and silencing cells the re- Likewise, Sema3B that of silenced of specificity following indicate the the transduction rescued to and in attest was observations silencing plexin-A2 These U87MG Sema6A 1B–D). recombinant the (Fig. and of of response Sema3B expression contractile to Sema6A The enable own. to cells its sufficient of not on concentration probably (supplementary signaling the is low rather S1A), that al., seems Fig. et indicating which material cells, Okada 2011), these 2007; in al., plexin-A4 al., et signal- et a Kigel as (Suto 2007; function also receptor to cells also known Sema6A these is transducing that that silencing plexin considering a result plexin-A4, express surprising cells The a these 1C,D), of the 1C–E). (Fig. contraction (Fig. in Sema6A-induced cell inhibited inhibited completely strongly cells subsequent were the Sema3B plexin-A2-silenced and by signaling contacts induced disassembly 2008). contraction focal the al., cytoskeleton, Sema3F vinculin-containing et actin Schwarz the of of prior 2005; inhibited collapse al., with the et However, (Yaron agreement plexin-A3 of 2B) plexin-A2 in (Fig. contraction completely the silencing Silencing whereas, Sema3F-induced of 1C,D), semaphorins. on (Fig. reports, transduction effect 3 cells determined the no U87MG class we had for other expression signals, required of semaphorin is signals 3 plexin-A2 class whether of transduction nodrt eiyteerslsb ifrn ehd ealso we method, different a by results these verify to order In the in plexin-A2 of role the of understanding better a gain To ora fCl cec 21)17 2055 doi:10.1242/jcs.155960 5240–5252 127, (2014) Science Cell of Journal 5241

Journal of Cell Science EERHARTICLE RESEARCH P nypasascnayrl Vrhvk ta. 08.By 2008). al., 5242 et (Varshavsky role secondary and transduce a NP1, plays primarily to in only using However, NP2 signals plexin-D1 transduction. transduces signal Sema3B Sema3B or for HUVECs, that the neuropilins observed both plexins and previously are utilize can have own A semaphorins We their signals. class on 3 semaphorin The signals with 2005). class transduce al., associate et of cannot Gu which 2004; receptors additional neuropilins, al., 2011; to et binding al., Artigiani belonging et primary 2005; Kigel plexins al., 2007; et and al., Basile et plexins (Shraga-Heled A subclasses as plexin class well as four neuropilins two the the express transduction cells U87MG signal cells and for HUVECs neuropilins whereas both these NP1, utilizes using sema3B exclusively in signals cells transduces transduction Sema3A U87MG signal S3D,E). the Fig. Sema6A for material required (supplementary and indeed of is plexin-A2 Sema3B contraction that suggesting Sema3B- independently, Sema6A-induced inhibited cells U87MG and in A2ExTm of Overexpression 50 bars: Scale contraction. cell after behind ple stimula left were for ‘spikes’ restored silenced cytoplasm was Cells expression attached (C) plexin-A2 show vinculin. which Arrows or in photographed. plexin-A2 plexin-A2 and for against minutes silenced directed cells 30 qRT-PCR. or for using with shRNA semaphorins examined non-targeting probed indicated then a were was expressing lysates plexin-D1 cells cell and control cells. from plexins expression, U87MG prepared A in blots class signaling Western indicated Sema3A the expression. for of not expression mean but The the shRNAs. represent signaling (sh-PlexA2) Sema3B plexin-A2-targeting for or required Control) is Plexin-A2 1. Fig. eesiuae rntwt eaB fe 0mnts h el eesandwt loecn hlodnt iulz ci ieso iha an with or fibers actin 50 visualize bars: percentage to Scale the phalloidin antibodies. and fluorescent anti-vinculin C, with using stained in by were shown cells stained experiment the contacts the minutes, focal to 30 show similar After Arrows experiments Sema3B. vinculin. independent with not two or from mean stimulated the obtained were represent fields Data microscopic determined. was eight cells in contracted counted were Cells (D) 6 .. toidpneteprmns.()Rcmiatpei-2wsepesd(hPeA+lx2 rnti el iecdfrplexin-A2 for silenced cells in not or (Sh-PlexA2+PlexA2) expressed was plexin-A2 Recombinant (B) experiments). independent (two s.e.m 6 ...()Clsslne o lxnA xrsino oto el xrsiganntreigshRNA non-targeting a expressing cells control or expression plexin-A2 for silenced Cells (E) s.e.m. ohnuoiist rndc eaBsgasi 8M cells. U87MG with in associate signals to Sema3B to transduce seems to sufficient (supplementary plexin-A2 neuropilins Thus, both was contraction S4A,B). Fig. alone cell cell material NP2 Sema3F-induced Sema3A-induced silencing abrogate inhibited whereas completely contraction, NP1 alone NP2 silencing signaling contrast, Sema3B or By expression inhibit S4A,B). NP1 to Fig. material sufficient either (supplementary not of was of Sema3B inhibition alone expression expression abrogated whereas the efficiently transduction, of and signal inhibition completely only neuropilins cells, both U87MG in contrast, ls lxn nHVC n 8M el Fg A using 2A) (Fig. cells U87MG and HUVECs in additional any plexins of Sema3B- expression whether A for the out class of silenced plexin-A2 we find transduction besides contraction, To cell the required induced 2011). for are al., NP1, plexins et additional to (Kigel addition one signals in than Sema3A more required, that is demonstrated Sema3A plexin cells on U87MG work and previous HUVECs Our in the signals for Sema3B simultaneously of required transduction are plexin-A4 and Plexin-A2 ora fCl cec 21)17 2055 doi:10.1242/jcs.155960 5240–5252 127, (2014) Science Cell of Journal m m. A etvrsswr sdt xrs oto (Sh- control express to used were Lentiviruses (A) e ihthe with ted xin-A2 pcfcfor specific m m. Data of

Journal of Cell Science eahrn nadto oGPwr edda lnldniyo o fteHVC.Clswr htgahdatr2 or ypaecnrs and phase-contrast by mar hours are 24 cells 100 after semaphorin-expressing bars: photographed by Scale monolayer were cells. indi HUVEC Cells HEK293 the the HUVECs. shRNA GFP-expressing in expressing the or show created cells of clearings shRNA Arrows or top of control line. GFP on borders non-targeting yellow expressing density The a a cells pictures. clonal expressing by HEK293 merged at lentiviruses Control are seeded with Shown selection. were microscopy. infected following GFP fluorescence HUVECs semap subconfluency to (F) indicated to addition the 1D. grown in with Fig. were semaphorins stimulated for plexin-A4 were described or cells as plexin-A2 the B targeting selection, in Following 50 shown (sh-PlexA1–4). bars: conditions plexins Scale experimental A photographed. class and different minutes the 30 targeting shRNAs with or shRNA EERHARTICLE RESEARCH eut eeosre hnteepeso fpei-4was plexin-A4 of Identical These S2B,C). expression 2B,C). Fig. (Fig. material the (supplementary Sema3A HUVECs when in to silenced observed and were contract Sema3B to results plexin-A2 failed to plexin-A4 response of silencing in expression in cells had the However, silenced but before, contraction. we Sema6A cell which Sema3A-induced and As on Sema3B effect to no 2B–E). responses (Fig. plexin-A1 aborted completely and Sema3F silencing Sema3A, induced to contraction by because cell response inhibited not completely the plexin-A3 silencing was silencing inhibited effect insufficient an completely of expression lack to This Sema6A-induced 2B,C). due or (Fig. Sema3B- 2011). that in or transduction role al., suggesting essential signal Sema3B- an et cells, play inhibit not U87MG (Kigel do not they of plexins did contraction A plexin-A3 Sema6A-induced class or plexin-A1 or other neuropilins Silencing of of expression expression the affect the not do that species shRNA (sh-control shRNA mean control the a represent with Data or 100%. plexins as A taken class was the cells of control each the targeting cells. in U87MG shRNA plexin of encoding each contraction lentiviruses of Sema3B-induced with expression for infection mean plexin-A2 following to qRT-PCR addition in by required determined is Plexin-A4 2. Fig. m .(–)Tepretg fcnrce el namcocpcfedwsdtrie ne aho the of each under determined was field microscopic a in cells contracted of percentage The (C–E) m. m m. lxnA aiiae h soito fpei-2with plexin-A2 to of responses inhibits that plexin-A4 association hypothesize silencing We that the 2F). and (Fig. neuropilins, facilitates not Sema3B but Sema3A expressing plexin-A4 expressing cells cells by shRNA, by plexin-A2 repulsed which were in non-targeting silenced HUVECs was whereas were control Sema3E, silenced by was a plexin-A4 repulsed of only expression of the which in expression HUVECs Sema3A, lentiviruses the with low expressing infected inducing HUVECs a cells repulsed Sema3E Although of or of Sema3B HUVECs. seeding top on of the cells monolayers HEK293 by semaphorin-producing produced of concentration to semaphorins HUVECs cultured of were of observations response its gradients the These observing in by plexin-A2. confirmed that the and also despite plexin-A1 and inhibited of are complexes signaling presence receptor Sema3B and Sema3B Sema3A absence of and component shared Sema3A essential an the is plexin-A4 that suggest results ora fCl cec 21)17 2055 doi:10.1242/jcs.155960 5240–5252 127, (2014) Science Cell of Journal 6 ...()U7Gclswr netdwt non-targeting with infected were cells U87MG (B) s.e.m. A h xrsino ls lxnmNswas mRNAs plexin A class of expression The (A) oisfor horins cated .The ). 5243 s ked

Journal of Cell Science n ehv o enal opromasmlreprmn in experiment similar a perform receptors. endogenous to only plexin-A4, able express been of that be not cells independently co- might have neuropilins it we in with overexpressed and associate is NP1 to plexin-A2 able the with when Fig. inhibit However, material plexin-A2 S4C). to (supplementary seem experiments overexpressed with immunoprecipitation not associate of to did able association plexin-A4 longer Silencing no is neuropilins. plexin-A2 because Sema3B ARTICLE RESEARCH 5244 plex the with co-purified proteins for wi ratios incubated isotope were (forward (HL) acids panel) heavy:light amino (right show heavy Sema6A plots with or The labeled panel) experiment. Cells (left reverse protocols. Sema3B the (PLXNA2). SILAC recombinant for protein to expressing switched according cells were isotopes HEK293 Labels heavy heavy-isotope-labeled plexin-A2. experiment). or from and light derived NP1 containing medium between acids complexes conditioned amino of with formation labeled the were induces Sema3B 3. Fig. main plexin-A2 the is cells that these suggest in in Sema6A results of results these receptor contraction signal-transducing together, which cell Taken silencing, Sema6A-induced 2B,C). of (Fig. plexin-A2 inhibition by complete modest with almost produced compared effect a effect weaker much the a the produced is This contraction, Sema6A. only to response cell expression Sema3B-induced plexin-A4 , on of had silencing plexin-A4 of LGwr xrse nPEcls oimnpeiiainwspromduiga niFA nioyo oto nioy(ot) rcpttswer plexin-A4. Plexin- Precipitates (D) of (Cont.). column. antibody tag affinity control FLAG the a the to or bind antibody against anti-FLAG not antibodies an did or using that performed antibodies lysate was anti-plexin-A2 cell Co-immunoprecipitation the with cells. of PAE fraction in expressed the were i.e. FLAG through, flow F.T., immunoblotted; IB, immunoprecipitated; PXA)a ela h oprfe rtisSm3,Sm6,pei-4(LN4 n P NP)aeidctd BC 8M el xrsigStrep/ were expressing Cells cells Sema3B. U87MG recombinant (B,C) containing bai indicated. medium The are (red). conditioned (NRP1) quadrant or 37 fourth NP1 (control) at the and medium in minutes (PLXNA4) appear conditioned 5 ligand plexin-A4 control for the Sema6A, with stimulated of Sema3B, stimulated presence proteins in were decreased co-purified plexin-A2 proteins the FLAG-tagged (green), as quadrant well second the as in (PLXNA2) appear ligand the of presence 0 euto ntepretg fclsta otatdin contracted that cells of percentage the in reduction 20% ncnrs otevr togihbtr fetta h silencing the that effect inhibitory strong very the to contrast In x ai,HLrto o h ees xeiet(Lreverse); (HL experiment reverse the for ratios H:L -axis, ˚ n ye,adtelstswr uiido nat-LGclm.N1o P eete eetdb etr ltig IP, blotting. western by detected then were NP2 or NP1 column. anti-FLAG an on purified were lysates the and lysed, and C y ai,HLrto o h owr xeiet(Lfrad.Poen nihdi the in enriched Proteins forward). (HL experiment forward the for ratios H:L -axis, n htpei-4fntosa esefcieSm6 receptor Sema6A effective cells. less these a as in functions plexin-A4 that and salse tbeiooelbln yaioaisi cell following the in with labeled isotopes acids acids Pimienta amino lysine incorporated 2010; amino that nitrogen Mann, Cells and 2009). by and al., (Walther et light labeling arginine protocols (SILAC) isotope culture with or stable then metabolically We established heavy cells. cells U87MG in containing 2007) the Strep/FLAG al., C-terminal et labeled a expressed (Gloeckner we tag with Sema6A, epitope tagged and Sema3B plexin-A2 plexin- to the recombinant In response by formed in plexin-A4. associations receptor the and detail A2 plexin-A2 a more in of NP2, map consists to or order cells NP1 U87MG functional in containing the and complex HUVECs that in suggested receptor above Sema3B described plexin-A4 experiments with but plexin-A2 The plexin-A2 of with association NP1 the of not association the induces Sema3B A 8M el xrsigpei-2tge ihaSrpFA tag Strep/FLAG a with tagged plexin-A2 expressing cells U87MG (A) ora fCl cec 21)17 2055 doi:10.1242/jcs.155960 5240–5252 127, (2014) Science Cell of Journal 2adplexin-A4– and A2 nA bait in-A2 probed e protein t th

Journal of Cell Science rmtecnrladsiuae el eeidpnetyaffinity independently were cells lysates stimulated and, cell and second NP-40, debris, control a insoluble the using to from remove solubilized in order to then switched centrifugation In were following were controls. cells as labels The served experiment. hits, isotopes false-positive acids light secreting exclude amino the containing cells with Cells labeled Sema6A. HEK293 medium or Sema3B conditioned heavy-isotope-labeled recombinant with from stimulated then derived were isotopes heavy ARTICLE RESEARCH eimcnann auaigcnetain fete eaBo h oul xrclua oano eaA cl as 50 mean bars: the Scale show of Sema6A. Data presence of 1D. or domain Fig. absence extracellular for the soluble described in the as grown or determined were Sema3B wit was Cells infected either cells plexin-A4. were of contracted of repressor concentrations tetracycline expression saturating the directing of containing vector control medium aga lentiviral the directed under a additionall antibodies plexin-A2 with with that targeting probed or cells (1 shRNA blots (control) such an western vector as expressing are cells well expression Shown expression. U87MG doxycycline. as lentiviral plexin-A2 (B) of sh-plexA2) for vinculin. addition (Tet-on silenced or by plexin-A2 cells plexin-A4 not targeting in A2, or shRNA Sema3B stimulated were to (Dox)-induced sh-plexA2+plexA4) responsiveness doxycycline (Tet-on restore a plexin-A4 not carrying does lentivirus plexin-A4 with of infected overexpression Ectopic 4. Fig. co- was plexin-A4 prepared which lysates found in cell plexin-A2, from We experiments with plexin-A2 complexes 2011). with by form immunoprecipitated al., to revealed able et also as (Kigel is plexin-A4 plexin-A1 that with These complexes 3C). of association (Fig. association Sema3B-induced this the cells although NP1. than with stable plexin- control plexin-A2 less with be of induce NP2 to to seems of those able nevertheless association is in the Sema3B with that not suggest co-purified observations Sema3B-stimulated NP2 but from that purified extracts found cells eluants cell we examined in only were analysis, when plexin-A2 column blot because affinity western weaker anti-FLAG by receptors, a the to from due these be recovered might between This association 3A). (Fig. Sema3B-induced association in plexin-A2 similar detect with NP2 not a of could experiments we SILAC plexin-A2 Surprisingly, of the 3A). association (Fig. As the neuropilins 3A,B). promote with (Fig. not plexin-A2 mass did with Sema6A by NP1 expected, of strongly and from analyzed association Sema3B the control that and eluants enhances revealed from experiments mixed The These medium were spectroscopy. resin. conditioned cells affinity with Sema3B-stimulated anti-FLAG loaded an columns on purified m ehv hw rvosyta lxnA om spontaneous forms plexin-A4 that previously shown have We /l o 2husadte eddfrtecnrcinasy otato a nue yteadto fete oto odtoe eimo conditio or medium conditioned control either of addition the by induced was Contraction assay. contraction the for seeded then and hours 72 for g/ml) 6 rmprieari nohla PE el nwihwe which 3A). (Fig. in formation Sema6A the Sema3B by by cells However, enhanced or not 3D). was (PAE) (Fig. complexes plexin-A2–plexin-A4 receptors of endothelial two the aortic expressed porcine from coial vrxrse nteslne el Fg A it 4A) (Fig. cells was silenced plexin-A4 the recombinant in When overexpressed 4B,C). following (Fig. ectopically Sema3B inhibited to to Sema6A response exposure contractile was and their Following lost these cells 4A). plexin-A2 these (Fig. In doxycycline, of doxycycline doxycycline. with of expression stimulation control the a the cells cells, U87MG under in certain shRNA expressed we a plexin-A2 differentiate possibilities, exceeds To two plexins signals. these Sema3B the between transduce of so to important the concentration required not in total threshold is participate the complex that as signaling plexins long Sema3B the the of of identity it formation the Alternatively, contribute. that cannot possible plexin receptor is other Sema3B the the a of that provides function complex plexin the the each cells for essential that U87MG property is inhibited unique explanation of likely completely cytoskeleton A HUVECs. the and plexin-A4 of collapse or Sema3B-induced plexin-A2 Silencing not but versa Sema3B vice plexin-A4 to for responsiveness silenced restores cells expression in plexin-A2 of Overexpression odce h eircleprmn,i hc eectopically we also which We in experiment, transduction. reciprocal signal the Sema6A be can conducted restore but signaling to to Sema3B are to interchanged plexin-A4 This respect response and with 4A). plexin-A2 interchangeable (Fig. the of not that expression overexpression suggests not plexin-A2 that therefore restore noted experiment but not be Sema6A did should plexin-A4 It to 4B,C). (Fig. response Sema3B the restored s.e.m. ora fCl cec 21)17 2055 doi:10.1242/jcs.155960 5240–5252 127, (2014) Science Cell of Journal m .()Tepretg of percentage The (C) m. A 8M cells U87MG (A) overexpress y doxycycline nempty an h ntplexin- inst 5245 ned

Journal of Cell Science EERHARTICLE RESEARCH e erso Fg AB.We P a o overexpressed, not was 5246 NP1 in the When of cells control 6A,B). the used (Fig. under we repressor is of NP1 tet experiment, silencing of overexpression this the ectopic For which following expression. Sema3B plexin-A2 to responsiveness restore in plexin-A2 signaling. replace Sema3B cannot restore plexin-A4 to as cells plexin-A2-silenced true, not thus is opposite and cells, the activity plexin-A4-silenced Sema3B plexin-A4 the in 5A). missing transduction (Fig. signal for plexin- to Sema3B compensate restore levels to expression responsiveness able expression elevated is at A2 restored although plexin-A4 Thus, fully 5B,C). plexin- (Fig. the for cells in plexin-A2 A4-silenced of silenced overexpression ectopic Surprisingly, were cells in doxycycline of that control the under plexin-A2 overexpressed Follo cells. HEK293 from medium Sema3F-conditioned or 37 photograph Sema3A- at and control, medium minutes not with Sema3A-conditioned 5 or or stimulated for stimulated control were stimulation with and plexin-A2 stimulated Scal puromycin assay, Strep/FLAG-tagged minutes. using contraction recombinant 30 selected for expressing 50 after seeded plexin-A4, bars: photographed were targeting Scale and Cells an shRNA minutes. indicated hours. anti-vinculin with 30 as 24 or infected Sema6A for anti-plexin-A4 were or ng/ml) Sema3B anti-plexin-A2, PlexA2) (100 (Tet-on with with doxycycline plexin-A2 stimulated probed assay, inducible blots contraction expressing western a cells are (Sh-Cont.) for Shown seeded shRNA were plexin-A4. hours. non-targeting 50 A a for 24 bars: in with silenced for described or cells ng/ml) cells plexin-A4 in (100 The targeting Sema3B doxycycline (B) shRNA and with with Sema3A not infected to or were responsiveness (Tet-on) stimulated plexin-A2 restores (Dox)-inducible plexin-A2 doxycycline of expressing overexpression Ectopic 5. Fig. ltig P muorcpttd B muolte;FT,fo hog,ie h rcino h ellst htddntbn oteafnt colum affinity the to bind not did that lysate cell the of fraction the i.e. through, flow F.T., immunoblotted; IB, immunoprecipitated; IP, blotting. eas eemndwehrteoeepeso fN1could NP1 of overexpression the whether determined also We m .()Tema ( mean The (C) m. m 6 .()Tema ( mean The (E) m. ˚ ... ecnaeo otatdclsi aho h utrssoni a eemnda ecie o i.1.()U87MG (D) 1D. Fig. for described as determined was B in shown cultures the of each in cells contracted of percentage s.e.m.) ,clswr ye n uiido nat-LGclm,adtepeec fc-uiidN1wseaie sn western using examined was NP1 co-purified of presence the and column, anti-FLAG an on purified and lysed were cells C, 6 ... ecnaeo otatdclssoni a eemnda ecie o i.1.()U7Gcells U87MG (F) 1D. Fig. for described as determined was D in shown cells contracted of percentage s.e.m.) eaA(i.5,) niaigta o eaAsignal Sema6A for plexin-A4 to interchangeable. that response for fully are in indicating plexin-A4 silenced and contraction plexin-A2 5B,C), cells transduction (Fig. enhanced in in Sema6A resulted plexin-A2 expression thus of Doxycycline-induced 2C). NP1, (Fig. expression Sema6A with to response associate the affected to cell silenced transduction. signal the enabling remaining that the the in of suggesting percentage not 6C,D), higher plexin-A2 a but (Fig. by enables restored NP1 Sema3B, of overexpressed was to upregulation Sema6A, response ectopically and to signal the response was doxycycline, Sema3A-induced Sema3B- of NP1 not addition expected, When but as transduction. signaling inhibited, Sema6A-induced plexin-A2 silencing iecn lxnA nteU7Gclsol minimally only cells U87MG the in plexin-A4 Silencing ora fCl cec 21)17 2055 doi:10.1242/jcs.155960 5240–5252 127, (2014) Science Cell of Journal A 8M cells U87MG (A) n. el were Cells . tibodies. with dafter ed wing e

Journal of Cell Science EERHARTICLE RESEARCH gi htwe lxnA shgl xrse tcan it of expressed independently highly signals Sema3B suggest is Sema3B to results and plexin-A2 response These Sema3A when in S2D). transduce that in Fig. contracted NP2 again material to cells co-expressing only response cells (supplementary these in plexin-A2 whereas When strongly Sema3B, in contracted and and S2D). cells Sema3A expressed the both Fig. NP1, was to these to addition material plexin-A2 response in all recombinant (supplementary at PAE contracted contract whereas not the Sema3B, NP1 semaphorins did or In NP2 expressing Sema3A expressing NP2. either cells cells to or response PAE NP1 in either plexin-A2, weakly expressing of was cells absence plexin-A2 PAE cells completely However, recombinant in when 7A,B). expression expressed obtained in Fig. 2B). were 5D,E; (Fig. plexin-A1 results (Fig. plexin-A2 Sema3A Similar or to effect responsiveness of in their plexin-A4 restored no or for overexpression silenced cells has ectopic whereas U87MG conditional contraction, plexin-A2 in cell silencing plexin-A4 Sema3A-induced inhibits or HUVECs plexin-A1 Sema3A to Silencing or responsiveness plexin-A1 restores for mean expression silenced the plexin-A4 cells represent o in Data presence plexin-A2 A. The of under minutes. Overexpression shown 15 100 cultures bars: for cell Scale semaphorins the vinculin. indicated of for doxy the each without specific containing in or antibody medium with determined an conditioned p incubated was using of of then field determined expression were addition microscopic then Cells The the (Tet-On). per was (C) by repressor analysis. (arrows) contacts p tetracycline stimulated blot contacts of the then western focal expression of using were of the control expres determined cells which absence the plexin-A2 then in The under on was repressor NP1 hours. silencing NP1 Tet expressing 24 the the of cells of of Expression for U87MG effect control (Dox). in the the doxycycline silenced. silenced and under with was was expression NP1 not expression A2 plexin-A2 expressing or plexin-A2 for ectopically stimulated which not cells were or in U87MG silenced silenced (B) cells was were analysis. in repressor blot Sema3B Tet western to the using responsiveness of determined control restore the can under NP1 NP1 of expressing overexpression Ectopic 6. Fig. htatog lxnA a o bet etr Sema3B plexin- for restore to silenced able 7C,D). cells to (Fig. was in it expression able Sema3A expression, A1 to plexin-A2 cells not found responsiveness of we loss restore was in to surprise, owing plexin-A4 S3D,E). our plexin-A4 signaling To although Fig. expression. overexpressed that and plexin-A1 inhibited material ectopically for Sema3B silenced also of we (supplementary inhibition cells Indeed, Finally, to signaling addition activity. U87MG Sema6A in Sema3A in signaling inhibit These Sema3A overexpression also threshold. the certain in should A2ExTm A2ExTm a cells of exceeds overexpression U87MG that plexin- membrane predict of also cell concentration observations the the if in lost between 5F). be distinguish A2 (Fig. can to Sema3B association ability and an the of Sema3A such that association imply promote observations to the whereas These able induce plexin-A2, not overexpressing to was cells able Sema3F in levels Indeed, was NP1 2012). containing with Sema3A expression al., plexin-A2 that et receptors (Janssen found plexin-A2 through plexin-A2 we and high signals NP1 previous of transduce with at complexes agreement can that in Sema3A also showing are reports results These plexin-A4. ora fCl cec 21)17 2055 doi:10.1242/jcs.155960 5240–5252 127, (2014) Science Cell of Journal m .()Tepretg fclslcigfocal lacking cells of percentage The (D) m. 6 s.e.m. A 8M cells U87MG (A) inwas sion lexin-A2 cycline 5247 lexin- r

Journal of Cell Science EERHARTICLE RESEARCH hs el ssfiinl iht nbesga rndcin and 5248 transduction, signal enable to high sufficiently in plexin-A2 is endogenous cells of expression these of cells, level these the in that Sema3A signaling indicating Sema3B affect inhibited not completely it did receptors. signaling, expression three in these plexin-A2 containing receptor silencing These complex Sema3A Although a silenced. functional of consists is minimal or cells plexin-A2, the these plexin-A1 that or NP1, suggest of plexin-A3 results expression not the the but that if cells plexin-A4, indicate these inhibited 2011) in completely cytoskeleton al., actin is et the (Kigel actin of study collapse the can the Sema3A-induced previous and of a results and of Our collapse contraction. results binding the cell consequent of induces and U87MG cytoskeleton Sema3A specificity in and cells, cells the endothelial glioblastoma In affect a transduction. signal can contain modulate that that membrane-anchored additional complexes proteins as well frequently as receptor are ligand-binding receptors functional regulate that mechanisms the migration thus of cell is understanding semaphorins the correctly for by cells actin important delivered that ensure instructions that the guidance mechanisms of interpret molecular direction in the receptors the of semaphorin the in and changes of changes characterization The into movement. local of translated their activation are triggers the that migrations, cytoskeleton receptors their of of gradients course semaphorin transverse the cells in When in semaphorins semaphorins. changes as guidance sense for such to receptors factors include order receptors in These environment. receptors their surface cell employ Cells DISCUSSION eerhcnutdi h attodcdshsrvae that revealed has decades two last the in conducted Research nvivo in . ihpei-4peeiti UEsadU7Gcls We 2011), cells. U87MG al., and et plexin-A2 HUVECs (Kigel and in plexin-A1 pre-exist plexin-A1 of plexin-A4 with with with heterocomplexes complexes as that spontaneous well suggesting forms as have we plexin-A4 plexin-A2 results, these that with Sema3A agreement both observed suggesting In of receptors. component expression, shared Sema3B plexin-A4 essential and an of is plexin-A4 silencing that the by within inhibited Sema3B and complexes. the receptor Sema3A not Sema3B to and and these Sema3A plexin-A2 specificity together, and confer observations Taken plexin-A1 neuropilins plexin-A2. that by NP2 suggest and with supported NP1 experiments complexes both was Sema3B stable with form stimulation This upon Sema3A that receptors. functional showing of essential Sema3F not an but as or receptors, functions when it Sema3B in cells that of of expression U87MG component of in experiments silencing level and physiological cells these the its endothelial plexin-A2 at from following expressed following is conclude only plexin-A2 inhibited inhibited We not was plexin-A3. was and specific the Sema3F silencing their by ligand induced as contraction presence NP2 NP2 cell the However, require utilize plexin-A2. might receptor of that binding primary semaphorins or that exclusive by suggest NP1 observations both transduction These utilizes transduction. signal signal Sema3B signals for receptor, which NP2 NP1 and Sema3A, the unlike using that, exclusively revealed receptor of Sema3B receptors. component Sema3A essential of an not but is receptors plexin-A2 Sema3B that suggesting further neetnl,Sm3-nue inln a locompletely also was signaling Sema3B-induced Interestingly, functional the of composition the of examination Further ora fCl cec 21)17 2055 doi:10.1242/jcs.155960 5240–5252 127, (2014) Science Cell of Journal 0mnts cl as 50 bars: Scale minutes. 30 after photographed and medium Sema3A-conditioned or stimulated control were with Cells hours. 24 ng/ml) for (100 doxycycline or of absence presence the in grown and puromycin using selected targeting plexin-A1, shRNA with infected were PlexA2) (Tet-on plexin-A2 induced (Dox)- doxycycline expressing cells plexin-A1. for silenced cells in Sema3A to restore responsiveness can plexin-A4 or of plexin-A2 overexpression Ectopic 7. Fig. eimadpoorpe.Scale photographed. and medium Sema3A-conditioned with or stimulated control then were cells The indicated. as plexin-A4 lentivirus expressing with or vector expression lentiviral empty with infected and puromycin with selected Control), (Sh- shRNA non-targeting control or (Sh-plexA1) plexin-A1 shRNA targeting with U87MG infected (C) were 1D. cells Fig. for determined described was as cell A the in of shown each cultures in cells contracted of ( mean The (B) as 50 bars: eemnda ecie o i.1D. Fig. was for C described in as shown determined cultures cell the field in microscopic per cells contracted m .()Tepretg of percentage The (D) m. 6 ... percentage s.e.m.) A U87MG (A) m m.

Journal of Cell Science EERHARTICLE RESEARCH h orcasApeis nldn lxnA,mgtb beto able be might plexin-A2, including plexins, A class and four the plasticity cell Thus, provide receptors. component single each simple signals. in available complexes not enable is semaphorin that flexibility might receptor interpret mechanism differently multicomponent to this to destined factors population are in guidance that cases gradients cells intersecting of several In encounter migrating targets 8A). different of to (Fig. migrate populations migration the of two affect direction which a the cells as that and, semaphorins change render several to might result, factors or semaphorin changes specific a local Such to and ‘blind’ plexins. encounter Sema3A specific of of also expression gradients might intersecting two Sema3B encounters that migrating a cell example, For semaphorins. signals interpret of cells gradients migrating encountered the of which a in as way the and, change complexes of result, receptor expression semaphorin the modulate of microenvironments They constituents might essential the cells migrating receptors. in the encountered multicomponent by factors traversed local of that advantages suggest and the cells, migrating by highlight interpreted guidance are semaphorins to as responses such which factors by mechanisms the of understanding levels. expressed physiological are be plexin-A2 their might or this at plexin-A1 and when increase neuropilins, importance for to crucial plexins able of two is these in it of is plexin-A2 affinity plexin-A4 or the when plexin-A1 that either possible is with It complex pre-formed NP2. such or NP1 recruits to Sema3B complexes with stimulation suf not that is postulate plexin-A expression which plexin-A2 endo in for causes cells silenced plexin-A4 in cells Sema3 or plexin-A2 in of plexin-A2 recombinant plexin-A4 effects either of recombinant repulsive of Overexpression of the Sema3B. expression Sema3B. overexpression to to Reduced to profo whereas blind responsiveness up (B) a responsiveness Sema3B, rendered restore migrate have gradients. their to be can to semaphorin lose Sema3B responsiveness will continue encountered or completely restores cell will Sema3A the to the silenced and of interpret cells receptors as semaphorins cells multicomponent glioblastoma the right, the which and of or in in cells either contained left way component to veer a the respond to of Chan on to (No expression cell effect able the semaphorins the in the be cause by variation not direction Thus, would to will respectively. opposite plexin-A2 responses cell Sema3B, the or biological in the repelled affect plexin-A1 expression be complexes of plexin-A4 would receptor Downregulation downregulate cell the Sema3B would Normally and signal Sema3B. Sema3A local and a Sema3A of of composition gradients the intersecting in two encounters changes how of semaphorins. representation these schematic A 8. Fig. rvossuishv rvddeiec ugsigta l of all that suggesting evidence provided have studies Previous our for implications profound have observations These A hsdarmdpcsaU7Gcl htnral xrse l h erpln n ls lxn n hti irtn pad and upwards migrating is that and plexins A class and neuropilins the all expresses normally that cell U87MG a depicts diagram This (A) sbcuei osntpa elfntoa oei eaBsignal Sema3B with in role functional real complexes a play not expressed form does reason highly it when the because even is can that plexin-A2 replace hypothesize cannot it We plexin-A4 of plexin-A4. When overexpressed receptor overexpression without versa. neuropilins vice why signaling is not but explain functional plexin-A4 plexin-A2 for also substitute the can thus might plexin-A2 of and This neuropilins formation for complexes. plexin-A2 the of for affinity enables the silenced increases A2 cells overexpressed in strongly was signaling it 8B). when (Fig. Sema3B even expression restore was plexin-A2 plexin-A4 to to because general, be unable ability plexins not might A assumption class their revealed this experiments the that further all lost However, expression interchangeable. of be cells levels might observations higher These at the that Sema3B. suggest Sema3A and and Sema3A both between signaling, restore differentiate to Sema3B silenced, able was was and expression plexin-A2 U87MG plexin-A1 overexpressed or in the plexin-A4 plexin-A2 overexpressed which overexpressed in ectopically we cells using when of conducted Indeed, many that plexins. were given We levels, studies expression result plexins. these plexin the be high essential might abnormally our discrepancies specific of apparent contradict for these contain complexes that seemingly hypothesize Sema3B receptor results functional and that These Sema3A suggest 2005). which al., findings, et Yaron rndc eaAsgas(aaah n titatr 2001; Strittmatter, and (Takahashi Bouvre signals Sema3A transduce ehpteieta h soito fpei-4wt plexin- with plexin-A4 of association the that hypothesize We ´ ta. 02 ase ta. 02 aaaae l,2013; al., et Katayama 2012; al., et Janssen 2012; al., et e ora fCl cec 21)17 2055 doi:10.1242/jcs.155960 5240–5252 127, (2014) Science Cell of Journal was 4 iin to ficient Aor wards. thelial 5249 e.If ge). und

Journal of Cell Science eahrn nasmlrmne,adi inltasuto by expression the transduction 3 in signal changes class by if similarly and other gated is manner, by semaphorins similar these induced a transduction in semaphorins signal regulate plexins on neuropilins with of associate is cannot own. recruitment it plexin-A2 its which the of at facilitate concentration threshold a the to below when is neuropilins role to only plexin-A2 its and transduction ARTICLE RESEARCH 5250 (FBS), 50 serum bovine glutamine, the fetal 10% mM from with 2 previously purchased supplemented were medium MEM- cells Earl’s in described U87MG maintained Eagle were 2011). and (ATCC) al., Collection Culture as et Type American (Gluzman-Poltorak Kigel previously 2000; described cultured and al., as cells et cultured (PAE) were and endothelial aortic cells Porcine HEK293 1978). isolated al., et (Gospodarowicz were HUVECs lines Cell from purchased were The NC). pEF-1 plasmids Hill, pENTR1A-GFP-N2, pLenti4/Block-iT-DEST Chapel The at Invitrogen. the Carolina North and pENTR/H1/TO of pLenti6.3/TO/V5-DEST, pLenti6/V5-DEST, from (University pDonor221, obtained were Kafri production lentivirus Tal for vectors pMD2-VSV-G PcDNA3- Oded and The by The NY). provided Israel). Jerusalem, were Medicine, University, (Hebrew cDNAs of Behar Plexin-A2/myc School the Sinai and plasmid (Mount Sema6A Akiri to Gal given by was us vector expression lentiviral NSPI-CMV-MCS-myc-His The Plasmids H1 Invitrogen. Inducible from was Block-it was the Fugene-6 System and Scientific, RNAi (Switzerland) Thermo Lentiviral Ltd from Roche was (Gaithersburg, from kit PerfectPure purchased cDNA from Verso was 5-Prime The kit MD). PCR Cruz reverse RNA The (Santa anti-c-myc Kits 3574). (Chemicon; mouse anti-vinculin mouse and anti-HA 1583-816), sc-789) ab72369), Biotechnology (Abcam; Manheim; anti-Sema6A anti-plexin- rabbit (Boeringer rabbit R30914), ab39357), (Sigma; Biotechnology; (Abcam; A4 Signaling Cruz anti-plexin-A2 (Cell (Santa rabbit anti-plexin-A2 sc-25640), anti-plexin-A1 mouse rabbit rabbit 3813s), Laboratories), Technology; R960-25), anti- Immunoresearch (Invitrogen; sc- Cy2-conjugated (Jackson (Jackson anti-V5 Biotechnology; antibody antibody 705-165-147), Cruz rabbit-IgG anti-mouse-IgG Laboratories; Biotechnology; (Santa donkey Cruz Immunoresearch NP2 Cy3-conjugated (Santa anti-human beads NP1 13117), agarose mouse anti-human anti-FLAG goat sc-7239), mouse A2220), F3165), (Sigma; (Sigma; A5316), anti-FLAG (Sigma; AC-74 mouse clone Cruz anti-actin conjugate (Santa mouse conjugate sc-2350), peroxidase peroxidase Biotechnology; peroxidase anti-goat-IgG anti-rabbit-IgG bovine anti-mouse-IgG goat A6154), (Sigma; Goat A4416), used: (Sigma; were conjugate antibodies following The reagents and Antibodies METHODS AND MATERIALS tumors. the the of in of plexins use monitoring of tumor the future levels to require expression the might successful microenvironment therapeutics tumor that in as the semaphorins cells indicating plexins in tumor semaphorins, found that of of specific cells sensitivity levels suggest endothelial the and expression change observations might as the Our microenvironment such in diseases 2012). of changes treatment be (Tamagnone, the to for potential cancer agents the therapeutic have as Semaphorins used plexins. specific of levels rvddb yu aih(aut fBooy ehin sal.The Israel). Aldrich. Technion, Sigma kindly from Biology, purchased was were of vectors lentiviral plasmid (Faculty containing shRNAs Nabieh pLKO-Tet-On Ayoub The pLenti-CMV-Neo-DEST by Addgene. provided from and were plasmids pLenti-CMV-Puro-DEST GFP-DEST, trmist edtrie hte iia obntosof combinations similar whether determined be to remains It m /lgnaii,2.5 gentamicin, g/ml a pNR,pLenti-CMV- /pENTRA, m /lfnioe mM 1 fungizone, g/ml D R pM R8.74) dR (pCMV NRF TG-3 TCTATGTCACCAACTCGAGTTGGTGACATAGAAATACAGCTTTT- AAACTCATCTGGGCGTTTTTG-3 5 shRNA, nti eim hc a upeetdwt 0 e-prvdFBS addition Tet-approved by induced 10% 1 was of with shRNA the supplemented of Expression was Industries). (Biological which grown medium, were Industries, lines this (Biological cell stable in acids shRNA-expressing All amino Israel). Haemek, non-essential Beth and pyruvate sodium h tpn otae(ple isses sn h relative the using Biosystems) (Applied C Software Quantification-comparative with StepOne (Applied analyzed the manufacturer were Expression Data the RPLP0. the Gene using of was gene TaqMan instructions normalizing the The performed with Biosystems). to System according was PCR Mix, Time Master (qRT-PCR) Real Plus PCR StepOne real-time Quantitative PCR real-time Quantitative lentiviral 2011). PGK-eGFP al., the et from (Chrenek generated vector lentiviruses a were semaphorins with various infected expressing 2008). also cells al., HEK293 et assays, various (Kigel the repulsion previously containing by For described media of as provided containing conditioned performed production were of kindly The semaphorins plasmid generation Israel). was the Jerusalem, and expression cDNA University, lentiviruses Sema6A (Hebrew An Behar the Oded 2008). 2008; of al., domain al., et extracellular (Kigel et previously described Varshavsky as NSPI-CMV-myc-his vector the into expression subcloned lentiviral were cDNAs semaphorin 3 semaphorins Class of production and Expression s)Nswspromda ecie rvosy(asasye al., et (Varshavsky previously interfering described small as of 2008). transfection performed and using was NP1 cells of (si)RNAs U87MG as Inhibition in Israel) 2009). Technion, expression al., Biology, NP2 et and of (Wiederschain Faculty previously generated Nabieh; pLKO- described Ayoub vector were of expression (gift shRNA shRNAs inducible Tet-On double-stranded single-vector desired the plasmids, into the cloned shRNA-expressing encoding inducible oligonucleotides generate To of siRNA downregulation using and expression species neuropilin shRNA of expression Inducible 5 shRNA, AGTTGTCAATGATGAAATTGCCGTTTTTG-3 first A2 were DNA shRNAs 5 shRNA-encoding AGATCTCGAGATCTTGGACGTGAAGAAGTGCTTTTTG-3 plexin-A1, various following used: The of were Aldrich. sequences expression Sigma the from purchased directing vectors lentiviruses Lentiviral shRNA-expressing with expression gene of Inhibition in neuropilin and cells. plexin derive each U87MG to for and cell process per HUVECs the molecules in mRNA made of dilutions number moles/ mRNA the to the converted consideration each and into of curve taking amount standard well the The from above. calculated was was described PCR (ng/well) and as Real-time triplicate cDNA. HUVECs to in from cycle converted performed of and RNA curve extracted concentration. was standard cells a plasmid U87MG build versus to used (Ct) were cDNA threshold neuropilin the or containing plexin Plasmids quantified each assay. was of neuropilins expression cells gene and TaqMan U87MG plexins the and the using of HUVECs cell non-stimulated per in molecules expressed mRNA of number real- by The cell per PCR molecules reverse mRNA time of number the of Quantification lxnA,H0309;pei-3 s0518 plexin-A4, Hs00250178; plexin-A3, Hs00413698; Hs00300697; plexin-A1, Hs00187290; NP2, plexin-A2, Hs00826128; NP1, used: s0936 lxnD,H0821;RL0 Hs99999902. RPLP0, Hs00892410; plexin-D1, Hs00297356; E23clswr efre sdsrbdpeiul Vrhvk tal., et (Varshavsky previously described 2008). as performed were in cells cells expression HEK293 shRNA of selection the and cells of infection lentiviruses, GTTAATGCGGTCATTTATCTGCTTTTTG-3 m 9 ;plexin-A4,5 /ldxccie(im)dsovdi eoie ae o 2hours. 72 for water deionized in dissolved (Sigma) doxycycline g/ml ora fCl cec 21)17 2055 doi:10.1242/jcs.155960 5240–5252 127, (2014) Science Cell of Journal 9 -CCGGCGCCCAGATGAGTTTGGATTCTCGAGAATCC- 9 -CCGGGCAGATAAATGACCGCATTAACTCGA- 9 -CCGGCGGCAATTTCATCATTGACAACTCG- T ehd h olwn saswere assays following The method. 9 -CCGGGCACTTCTTCACGTCCA- 9 lxnA,5 plexin-A3, ; 9 h rdcino the of production The . 9 lxnA second plexin-A2 ; 9 -CCGGGCTGTATT- 9 plexin- ;

Journal of Cell Science h aepoeue.Pouto flniiue sn hs lsisand plasmids these to using according lentiviruses constructed of was Production of tag procedures. FLAG 1–1323) same C-terminal acids the a (amino to fused domains protein transmembrane the of containing and instructions plexin-A2 the extracellular of to mutant the according deletion tag A expression V5 (Invitrogen). manufacturer C-terminal lentiviral the a with pLenti6.3/TO/V5-Dest frame in into or vector recombination by pLenti6/V5-DEST the transferred into then the cloned and were plasmid plexin-A4 pDonor221 and gateway plexin-A2 of cDNAs full-length The plexins recombinant of Expression ARTICLE RESEARCH ersnainwspromduigi-os cit.W only We graphical scripts. and R analysis in-house Downstream Software using 2009). MaxQuant performed the al., to was using bind et representation by to (Cox induced performed were were mass v.1.3.0.2 was resulting labels that the data of light proteins quantification spectrometry and stimulation, identify the heavy to following mass plexin-A2 where order tandem In performed, Fisher) artifacts, switched. was labeling (Thermo by labeling caused Velos hits reverse mass false-positive Orbitrap exclude an To tandem spectrometer. to Thermo RSLC, and coupled 3000 chromatography liquid Ultimate Fisher) trypsin (Dionex high-performance system liquid nano-flow with (HPLC) a chromatography cleaved using by (LC-MS/MS) ratio proteolytically 1:1 spectrometry analyzed heavy-isotope- a content, in and mixed protein subsequently were light-isotope- columns on previously anti-FLAG Briefly, based described the from 2010). as eluants al., labeled performed et was analysis (Gloeckner plexin-A2 spectrometric with co-purified Mass proteins of analysis Proteomic or medium Sema3B conditioned recombinant 37 with at of minutes stimulated containing 5 concentrations acids for then amino Sema6A saturating with 2009). were labeled al., containing cells et isotopes the (Pimienta amino ligands, these protocols heavy with the SILAC proteins prepare standard cell To passages of to labeling ten according or complete for acids achieve nitrogen Laboratories) to light carbon, cells order a Isotope (K8/R10) containing in (Cambridge heavy HEK293 arginine or 2011) or isotopes and al., as cells) hydrogen 2008) lysine et control well (Kigel (Sigma; with al., Sema6A (K0/R0) labeled as et of metabolically domain 2007), (Varshavsky were extracellular al., soluble Sema3B secreted et recombinant Strep/FLAG a (Gloeckner with expressing in-frame tag fused plexin-A2 epitope expressing Strep/ cells a U87MG with tagged tag plexin-A2 epitope of FLAG purification affinity and SILAC previously described as performed PBS-gelatin. 2011). was with experiments, al., actin et coated (Kigel and immunofluorescence coverslips vinculin glass For of on 2007). Detection plated 2007). were al., cells al., et U87MG previously et Guttmann-Raviv described as 2008; (Shraga-Heled performed were al., described assays as et Co-immunoprecipitation performed were (Varshavsky cells as endothelial previously semaphorins of to assays response repulsion in as cells well U87MG and HUVECs of Contraction co-immunoprecipitation immunofluorescence assays, and repulsion transfection and 2007). contraction FuGENE6 Cell al., et using (Guttmann-Raviv expression cells previously Sema6A described target as The into (Roche) described reagent 2009). transfected as Neufeld, was essentially performed vector and were (Brekhman cells target previously of infection stable eeeue sn ufrcnann 0 Mgyie(H25 and 2.5) (pH proteins glycine bound mM and 100 TBS, nitrogen. containing liquid of buffer in ml a 1.5 snap-frozen using with eluted (150- were briefly were column treatment washed each affinity was (Roche) from anti-FLAG column protein cocktail an of inhibitor onto amounts lysis protease with loaded Equal with a NP-40]. briefly quickly 0.5% with washed lysed and supplemented then scraped, [TBS cells were (TBS), buffer the reverse saline Sema6A and For Tris-buffered or cells conditioned control ice-cold isotopes. Sema3B the with light with Both with incubated switched. stimulated labeled were were labels cells cells experiments, HEK293 Control from treatment). medium each for ˚ fv 0c imtrcnletplates confluent diameter 20-cm (five C m lbedvolume).The osdrdtoeht htwr dniidb tlatoeppiein one least at by experiments. identified reverse were and forward that both hits those considered lne h xeiet,itrrtdterslsadwoetemanuscript. the wrote and G.N. results writing. the the interpreted with and helped experiments, planning and the analysis, the planned their with with mass helped and experiments out O.K. of carried experiments. execution and these planned of their and and analyses in experiments M.U. spectroscopy involved SILAC experiments. were the SILAC and planned the experiments, C.J.G. conducted Y.M. the interpretation. of and most planning out carried T.S. and A.D.S. contributions Author interests. competing no declare authors manner: The following interests the Competing the in of 10%. error presented exceed standard is the not * Variations significance represent by did bars Statistical several triplicate. vary experiments error mean. stated, in single across not otherwise within Unless performed proliferation did data replicates were cell controls between the experiments In the normalized proliferation Student’s between experiments. paired we the differences most used which we in experiments, in used unless experiments was results Student’s similar unpaired with correction one-tailed twice The least stated. at otherwise repeated were experiments All analysis Statistical lza-otrk . oe,T,Hro,Y n efl,G. Neufeld, and Y. Herzog, T., Cohen, Z., Gluzman-Poltorak, Sarioglu, L., Wiesent, S., Helm, F., Zweydorf, von M. K., Ueffing, Boldt, J., and C. R. Gloeckner, Roepman, A., Schumacher, K., Boldt, J., C. Gloeckner, G. Neufeld, and V. Brekhman, and M. Tessier-Lavigne, Bouvre A., Yaron, M., Schachner, O., Manor, A., Ben-Zvi, S. J. Gutkind, and T. Afkhami, R., J. Giordano, Basile, D., Barberis, F., G. Gilestro, P., Fazzari, P., Conrotto, S., Artigiani, Go M., S. Weis, S., Barillas, M., L. Acevedo, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.155960/-/DC1 online available material Supplementary material M.U.). Supplementary (to Foundation (to Technion Kerstan of Charlotte the Sciences und (ISF), Medical Tistou Foundation the the Science in and Israel Research G.N.), the for from Institute grants Family by Rappaport funded was work This Funding o,J,Mtc . igr . aaa,N,Slah . le,J .adMn,M. Mann, and V. J. Olsen, M., Selbach, N., Nagaraj, M., Hilger, I., Matic, J., Cox, V. A. Makarevich, and M. Bauer, P., Chrenek, Che H., Chen, P nohla rwhfco VG)ado lcnagot atr2 u only but vascular factor-2, of VEGF. of growth form form acid placenta amino long 145 of the acid for Chem. receptor and 165-amino a as (VEGF) for functions neuropilin-2 factor receptors the growth are of endothelial Neuropilin-1 domain and Roc 2 the and LRRK2. N-terminus kinase the protein 1738-1745. in associated phosphorylation Parkinson-disease M. of Ueffing, clusters and H. and isolation complexes. efficient the protein for native strategy of purification characterisation affinity tandem novel A (2007). al. et V. formation. valve Fortuna, lymphatic for J., 111 required are Soueid, PlexinA1 Y., and Neuropilin-1, Xu, T., Mathivet, development. ganglia root dorsal during apoptosis O. the Behar, and Src, PYK2, of activation pathway. the 3-kinase-Akt through phosphatidylinositol migration cell endothelial induces L. Tamagnone, 5A. and semaphorin for M. receptor P. Comoglio, S., 20) rcia ud oteMxun opttoa ltomfrSILAC- for platform computational MaxQuant the proteomics. to quantitative guide based practical A (2009). constructs. gene EGFP different III. with Sema embryos not but receptor IV affinity Sema high and a E is 559. Sema family, neuropilin semaphorins the the of for member novel a Neuropilin-2, invasion. cell tumor of study the a as acts factor. yet permeability angiogenesis vascular VEGF-mediated suppresses 3A Semaphorin (2008). , .5 ** 0.05, 437-445. , e . rnt . e oo . odn . rht . rsoao B., Cristofaro, C., Prahst, E., Gordon, R., Toro, Del I., Brunet, K., ´e, ora fCl cec 21)17 2055 doi:10.1242/jcs.155960 5240–5252 127, (2014) Science Cell of Journal 275 dtl . e . oda,C .adTsirLvge M. Tessier-Lavigne, and S. C. Goodman, Z., He, A., ´dotal, 18040-18045. , 20) h eahrnrcpo lxn3mdae neuronal mediates PlexinA3 receptor Semaphorin The (2008). P , .1ad*** and 0.01 21) hshppieaayi eel w discrete two reveals analysis Phosphopeptide (2010). Blood MORep. EMBO 20) oe smerc3 nvtoasyfor assay in-vitro 3D asymmetric novel A (2009). P a.Protoc. Nat. , M Cancer BMC 0.001. 111 2674-2680. , 5 710-714. , o.Cl.Biol. Cell. Mol. 4 hr,J .adCeeh .A. D. Cheresh, and R. J. thert, ¨ 21) ult ftasei rabbit transgenic of Quality (2011). 20) lxnB safunctional a is Plexin-B3 (2004). 20) eahrn4D/plexin-B1 Semaphorin (2005). 698-705. , Proteomics 9 Zygote 415-427. , t ts,tkn aeta the that care taking -test, .Neurosci. J. 19 21) Semaphorin3A, (2012). .Poem Res. Proteome J. t 85-90. , 7 ts ihWelch’s with -test 25 4228-4234. , 20) Neuropilin- (2000). 6889-6898. , 28 Neuron 12427-12432. , . 0.Cell 50%. ic Res. Circ. 19 (1997). .Biol. J. 5251 547- , 9 ,

Journal of Cell Science aai . Brianc H., Nawabi, Y. Yoshida, and F. Suto, F., Imai, K., Katayama, utanRvv . haaHld . asasy . Guimaraes-Sternberg, A., Varshavsky, N., Shraga-Heled, N., Guttmann-Raviv, Henderson, J., Merte, F., Mann, V., D. Reimert, J., Livet, Y., Yoshida, C., Gu, E. Giraudo, and C. R. Gu, B. Zetter, and R. C. Birdwell, D., K. Brown, D., Gospodarowicz, ARTICLE RESEARCH 5252 O. Kessler, and N. Rabinovicz, D., A. Sabag, G., Neufeld, M., Marra, R., Testa, R., M. Rippo, R., Lazzarini, A., Procopio, S., Moretti, Ginty, and J. H. Kikutani, R. and Giger, A. T., Kumanogoh, Y. Tai, G., E. Rowe, V., D. Levengood, L., A. S. Kolodkin, C. Goodman, and J. D. Matthes, L., A. Kolodkin, G. Neufeld, and O. Kessler, A., Varshavsky, N., Rabinowicz, B., Kigel, G. Neufeld, and O. Kessler, A., Varshavsky, B., Kigel, and C. Siebold, Z., M. Cader, A., G. Weir, T., Malinauskas, J., B. Janssen, kd,A,Tmng,M,Hruh,M n ook,Y. Tomooka, and M. 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