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![DOI:./n h Woune fsh n ~ µl soton gr - Imgng + ~ ml of superntnt [hypoton(h)/ soton() + ompouns] Aton of soton (upper pnel) or hypoton (lower pnel) meum - mn mn mn mn mn mn Leukoyte # (t= ) ** () () h](/docs-images/96/129224064/images/1-0.jpg "Trns/Sytox-Ornge h Nerot re [.u.] () h (9) Leukoyte # (t= ) Ton Comp () () () () () () h - - AA.")
1 DOI:./n h Woune fsh n ~ µl soton gr - Imgng + ~ ml of superntnt [hypoton(h)/ soton() + ompouns] Aton of soton (upper pnel) or hypoton (lower pnel) meum - mn mn mn mn mn mn Leukoyte # (t= ) ** () () h Trns/Sytox-Ornge h Nerot re [.u.] () h (9) Leukoyte # (t= ) Ton Comp () () () () () () h - - AA. mm ATP mm ATP ytosol Fgure S Isoton nhton of leukoyte rerutment s reversle n s not result of neross or ytoplsm lekge. () Sheme of Expermentl esgn. Lrve re woune wthn smll volume (- µl) of soton low meltng grose, woune n mge for mn. ml of soton (, mm NCl) or hypoton meum ( h, mm NCl) re then e on top of the soton gr p, n woun rerutment of leukoytes wthn t = mn fter meum ton s quntfe y lght trnsmsson mrosopy (.e. llowng mn for equlrton of slt onentrton throughout the gr p s ompre to our stnr t = mn ssys). Rply mgrtng ells (whh orrespon to leukoytes) re hghlghte wth oloure trks. () Left pnel, representtve tme-lpse mges of woun rerutment fter shftng the meum tht the lrve were woune n from soton to ether hypoton meum ( h shft) or soton meum ( shft). Rght pnel, quntfton of leukoyte rerutment wthn mn fter tonty shft. Numer of lrve (n) use for the nlyses s gven n prentheses on the grphs. Error rs, SEM. **, t-test p<.. Sle r, µm. () Stnng of nerot ells wth Sytox ornge. Lrve were woune ether n hypoton (h) or soton meum supplemente wth µm Sytox Ornge. Neross ws quntfe s re of Sytox-fluoresent ells t the woun ste t t = mn fter njury. Sle r, µm. () Leukoyte rerutment (wthn mn) n response to tl fn nsons n hypoton ( h ) or soton ( ) meum n the presene or sene of µm AA,. n mm ATP or ytoplsm extrt from Co- ells (see Methos seton for etls). Numer of lrve (n) use for the nlyses s gven n prentheses on the grphs. Error rs, SEM., t-test p<.. Mmlln Pulshers Lmte. All rghts reserve
2 pl tn NL L α-pla woune pl MO PLA-mKte Leukoyte # (t= ) () () () Leukoyte # (t= ) 9 () () pl MO PLA-mKte ACA Fgure S () pl mrna expresson n leukoyte vs. non-leukoyte tssue. Leukoyte/non-leukoyte totl mrna ws generte y FACS sortng of ssote, trnsgen zerfsh lrve expressng re fluoresent proten (mkte) uner the ontrol of the lysc promoter. pl mrna expresson etween these smples ws ompre y sem-quntttve RT-PCR. () Immunofluoresene stnng of PLA n ntt n woune wt, pl morphnt n PLA-mKte overexpressng lrve (expresson of the ltter shown n re). Sle r, µm. () Averge leukoyte rerutment fter hypoton tl fn wounng of wt, pl morphnt, or pl morphnt lrve tht hve een onjete wth mrna enong PLA-mKte for resue. Dt for the wt n pl morphnt lrve erve from the sme t set s shown n Fg.. () Averge leukoyte rerutment fter hypoton tl fn wounng n the presene of µm non-seletve PLA nhtor N-(p- Amylnnmoyl) nthrnl A (ACA). Numer of lrve (n) use for the nlyses s gven n prentheses on the grphs. Error rs, SEM., t-test p<.. Mmlln Pulshers Lmte. All rghts reserve
3 Leukoyte # (t= ) Leukoyte # (t= ) e Leukoyte # (t= ) 9 Ton AA () (h) Comp () ** () MK (h) h h () EDBC (h) ASA - - ASA () wt () () () lth MO * () () f Leukoyte # (t= ) Leukoyte # (t= ) Ton AA Comp lox mpx tn () () () (h) Besttn (h) Zleuton (h) (9) ** () () Besttn NL L Fgure S Lpoxygenses, ut not prostglnns or nonl leukotrenes re nvolve n woun rerutment of leukoytes. Averge rerutment of leukoytes fter hypoton tl fn wounng n the presene of () µm MK- (ALOX nhtor, more seletve for ALOX), µm EDBC (ALOX nhtor, more seletve for ALOX/), () µm Besttn (LTA H nhtor), or µm Zleuton (ALOX nhtor more seletve for ALOX). () Averge rerutment of leukoytes fter tl fn wounng t nte tonty n the presene/sene of µm AA n µm etylslyl (ASA, ylooxygense nhtor). () Averge rerutment of leukoytes fter soton tl fn wounng n the presene/sene of µm AA n µm Besttn. (e) Averge rerutment of leukoytes fter hypoton tl fn wounng of wt or lth morphnt lrve, usng prevously pulshe trnslton trgetng morpholno. (f) lox mrna expresson n leukoyte vs. non-leukoyte tssue. Leukoyte/non-leukoyte totl mrna ws generte y FACS sortng of ssote, trnsgen zerfsh lrve expressng re fluoresent proten (mkte) uner the ontrol of the lysc promoter. lox mrna expresson etween these smples ws ompre y semquntttve RT-PCR. Numer of lrve (n) use for the nlyses s gven n prentheses on the grphs. Error rs, SEM. *, t-test p<.. **, t-test p<.., t-test p<.. Mmlln Pulshers Lmte. All rghts reserve
![Tme post wounng [mn] () Fgure S () Averge rerutment n mgrtory prmeters of leukoytes fter hypoton wounng of lrve tht h, or h not een pretrete wth -KETE.](/docs-images/96/129224064/images/4-1.jpg "For OXE-R esenstzton, lrve were soke for 9 mn n µm -KETE pror to wounng. -KETE ws wshe out, n leukoyte rerutment to hypoton wouns ws mesure n trete n non-trete smples.")
4 () * -KETE prenuton Leukoyte # (t= ) () (9) v l Dw Dp. +/-. µm/mn (9) +/- µm (9). +/-. (9). +/-. (9) -KETE prenuton. +/-. µm/mn () +/- µm (). +/-. (). +/-. () * (9) () ** NS Leukoyte # (t= ) () Leukoyte # (t= ) oxer mpx tn + AA NL L e oxer MO + AA oxer MO pl MO wt.. HyPer rto HyPerwoun/HyPeroy pmo... p MO+oxer MO. wt. pl MO oxer MO. Tme post wounng [mn] () Fgure S () Averge rerutment n mgrtory prmeters of leukoytes fter hypoton wounng of lrve tht h, or h not een pretrete wth -KETE. For OXE-R esenstzton, lrve were soke for 9 mn n µm -KETE pror to wounng. -KETE ws wshe out, n leukoyte rerutment to hypoton wouns ws mesure n trete n non-trete smples. () Averge rerutment of leukoytes wthn mn fter soton tl fn wounng of wt or oxer morphnt lrve n the presene of µm AA n the meum. () Averge rerutment of leukoytes wthn mn fter hypoton tl fn wounng of p morphnt n p+oxer morphnt emryos. () oxer mrna expresson n leukoyte vs. non-leukoyte tssue. Leukoyte/ non-leukoyte totl mrna ws generte y FACS sortng of ssote, trnsgen zerfsh lrve expressng re fluoresent proten (mkte) uner the ontrol of the lysc promoter. oxer mrna expresson etween these smples ws ompre y sem-quntttve RT-PCR. (e) HyPer mgng of woun mrgn H O prouton n response to wounng wt, pl morphnt n oxer morphnt lrve. Upper pnel, representtve HyPer-rto mges. Re, hgh [H O ]. Blue, low [H O ]. Lower pnel, normlze HyPer-rto s funton of tme fter wounng. Numer of lrve (n) use for the nlyses s gven n prentheses on the grphs. Error rs, SEM. NS, t-test p >.. *, t-test p<.. **, t-test p<.., t-test p<.. Sle r, µm. Mmlln Pulshers Lmte. All rghts reserve
5 Tssue Integrty Survellne n Zerfsh WOUND Streth? Cell njury? Juntons? Reue osmot pressure n the tssue Inrese ytosol C + ref 9 Duox tvton Cell swellng AND PLA tvton NADPH epleton?, ref H O lyn/pten oxton ref, OXE-R OXE-R lgns (e.g. -KETE) LEUKOCYTE CHEMOTAXIS Fgure S () Two prgms of tssue mge eteton. Left pnel, lss ell-ntegrty prgm : Pssve lekge of ytoplsm DAMPs (mge ssote moleulr ptterns) from roken ells proues leukoyte nerotxs. Rght pnel, tssue ntegrty prgm : Epthell rrer rekge nues ell swellng n e novo prouton of hemottrtnts tht ttrt leukoytes. () Sheme of propose regultory ruts. Blk rrows, mehnsms propose y ths stuy. Grey rrows, mehnsms propose y prevous stues (see referenes, 9,, ). Dshe grey rrows, speultve mehnsms. Mmlln Pulshers Lmte. All rghts reserve
6 Supplementry Veos Veo S - Hypotonty s requre for rp leukoyte rerutment to lrvl zerfsh tl fn wouns Ths veo shows leukoyte rerutment fter tl fn wounng of wt lrve n hypoton (ontrol) or soton (mm NCl) meum. Imgng strts ~ mn pw ( se/frme). Sle r: µm. Veo S Isotonty reversly nhts rp leukoyte rerutment to lrvl zerfsh tl fn wouns Ths veo shows leukoyte rerutment fter tl fn wounng of wt lrve n soton meum tht ws shfte to hypoton meum ( h shft) or soton meum ( shft). Imgng strts ~ mn pw ( se/frme). Sle r: µm. Veo S - Hypotonty lolly tvtes PLA t the woun ste Ths veo montge shows PLA-mKte trnsloton to the nuler memrne nue y UV-lser wounng n hypoton (hypo), soton (so) or hypoton meum supplemente wth µm G + (hypo+g + ). Lser wounng t ~ mn ( se/frme). Sle r: µm. Veo S - G + exposure enhnes woun mrgn swellng Ths veo shows leukoyte rerutment fter hypoton tl fn wounng of wt lrve n the presene or sene of µm G +. Imgng strts ~ mn pw, G + e t mn ( se/frme). Sle r: µm. Veo S - Extrellulr C + s requre for PLA tvton Ths veo montge shows PLA-mKte trnsloton to the nuler memrne nue y lum-swth n hypoton meum. Hypoton C + -free meum supplemente wth mm EGTA ws e t mn to the lrve tht were prewoune n soton meum wth EGTA. Lrve were mge for mn wth (A, C) or wthout (B) reton of C + t mn. Veos from fferent experments. Low mgnfton sle r: µm, hgh mgnfton µm, ( se/frme). Veo S - Tl fn njury rply nreses ytoplsm [C + ] t the woun ste rrespetve of meum tonty Ths veo shows the ytosol C + sgnl nue y UV-lser wounng of lrvl zerfsh tl fns mntne n hypoton (h) or soton () meum. Lser wounng t ~9 se ( se/frme). Sle r: µm. Veo S - PLA s requre for rp leukoyte rerutment to lrvl zerfsh tl fn wouns Ths veo shows leukoyte rerutment n pl morphnt lrv (pl MO) vs. wt lrv pf. Imgng strts ~ mn pw ( se/frme). Sle r: µm. Veo S -KETE s hemottrtnt for leukoytes n zerfsh Ths veo shows leukoyte rerutment fter soton tl fn wounng of wt lrve n the presene or sene of µm -KETE (-oxo-ete). Imgng strts ~ mn pw, -KETE e t mn ( se/frme). Sle r: µm. Veo S9 - OXE-R s requre for rp leukoyte rerutment to lrvl zerfsh tl fn wouns Ths veo shows leukoyte rerutment n oxer morphnt lrv (oxer MO) vs. wt lrv pf. Imgng strts ~ mn pw ( se/frme). Sle r: µm. Mmlln Pulshers Lmte. All rghts reserve
Supplementary figure 1
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