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1 DOI:.38/n74 nti-cd63 P lls P CX AP BL lnxin Tsg Wnt5A Evi Apil suprntnt P Co-2 lls % Gol prtils Wg ntioy unspii ontrol <4 4- siz o lll struturs (um) 2 3 CD8 Ponu Bsoltrl K67 lls CX P Tsp42 Tsp96F Tsp42 Tsp96F HA Drosophil S2 lls TSP96F-HA Evi TSP42-HA Evi Figur S Chrtristion o xosoms rom humn n Drosophil lls. () Immunogol lling o P with nti-cd63 ntiois. Sl r nm. () Immunolot o ll xtrt (CX) n,g pllt (P) o polriz Co-2 lls grown on trnswll iltr support (AP) pil n (BL) soltrl suprntnt. () Rprsnttiv trnsmission ltron mirosop img o th P rtion rom suprntnt o Co-2 lls Sl rs is nm () Quntiition o Wg spii n unspii ontrol lling o th immuno-gol stining o Drosophil xosoms rom Fig.h. Immunostnings wr prorm in triplits rom thr inpnnt xosoml prprtions. Error rs rprsnt s.. Signiin lvl s init: P<.5, P<., P<. () Immunolot o ll xtrt (CX) n,g pllt (P) o Drosophil K67 lls trnst with Tsp42-HA or Tsp96F-HA () Rprsnttiv imgs rom immunoluorsn nlysis o Drosophil S2 lls otrnst with Tsp42-HA or Tsp96F-HA n Evi-GFP. Sl rs 5 μm Mmilln Pulishrs Limit. All rights rsrv.
2 Wnt rportr tivity knok own iiny qpcr sl DNA SN DNA SN GM Evi-C2 Lysotrkr L lls GM Evi-C2 Lysotrkr L lls sivps26 Wnt rportr tivity Wnt rportr tivity sl GL3 t Evi Tsg r5 r7 VPS26 VPS35 g r5a VPS35/26 /TSG VPS4B r27ab SN rl. Wnt rportr tivity rl. Wnt rportr tivity sl Bilomyin NH4Cl GW SN SN n.s. Bilomyin NH4Cl GW4869 Figur S2 Componnts rgulting Wnt tivity in th suprntnt. () lls wr trnsintly trnst with luirs-s TCF/Wnt rportr n stimult with suprntnt rom /Evi-GFP trnst lls or n tr ultrntriugtion. n=3, rror rs r s.., signiin lvl s init: P<.5, P<., P<. () Quntittiv PCR vlition o knokown iiny rom thr inpnnt knokown xprimnts, rror rs r s.. () Rprsnttiv imgs rom immunoluorsn nlysis o ontrol (uppr pnl) or sivps26 (lowr pnl) trnst L lls stin with Evi, Golgi mrkr GM n Lysotrkr, (n=3) Sl rs μm () Autorin Wnt Assy: lls s TCF/Wnt rportr wr trnst with init, (n=3) () Prrin Wnt Assy: Suprntnt rom lls trt with init wr us to inu TCF/Wnt rportr lls. Suprntnts rom thr inpnnt xprimnts wr us, rror rs r s.., signiin lvl s init: P<.5, P<., P<. ( n ) Control xprimnts or MVB mturtion: () TCF/Wnt rportr trnst lls wr trt with 2nM ilomyin A, mm NH 4 Cl, 5mM GW4869 or ontrol vhil lon. (g) Wnt tivity o ll ultur suprntnts rom inhiitor trt -L lls or n tr ultrntriugtion ws trmin in TCF/Wnt rportr trnst lls. ( n g) Suprntnts rom thr inpnnt xprimnts wr us, rror rs r s.., signiin lvl s init: P<.5, P<., P< Mmilln Pulishrs Limit. All rights rsrv.
3 Bilomyin MVB Fig. /3 Evi-C2 Tsg Clnxin ß-tin Ur CX RIPA CX P SN Ur CX RIPA CX P SN -L lls 5 5 Fig. Tsg CX CX LRP6 Fig. 2 Ponu CX Fig. Fig.2 /g ß-tin Evi Mit Fig. g CX P SN Wg CX P SN ApoII Wg 5 5 SN SN SN -L lls Supplmntry Fig. S3 CX shevi CX P shevi P -L lls Clnxin CX shevi CX P shevi P -L lls Figur S3 Control xprimnts or MVB mturtion. () Ex vivo trtmnt o wing iss with V-ATPs inhiitor ilomyin A (2 nm in ) or hour t room tmprtur n thn hour on i with Wg ntioy shows ru lvls o th xtrllulr Wg (right pnl). Control trtmnt with i not show signiint t on Wg srtion (lt pnl), sl rs 2 μm. () Eltron mirogrph o wing imginl iss shows ILVs /xosoms (init y rrow-hs) ontining MVB with mitohonrium. Sl r nm () Full immunolots with init r o sltion s shown in min igur n Mmilln Pulishrs Limit. All rights rsrv.
4 Chrry Wg (totl) GFP YFP Enhn Chrry wg::evi-chrry Chrry Wg (totl) Enhn EGFP wg::evi-egfp wg::evi-chrry wg::cd63-gfp wg::yfp-r4 g h 2 YFP YFP-R4/BF Mitotrkr 5/BF Dsh-YFP/BF Mitotrkr 5/BF Nr. o mitotrkr 5 positiv lls lso positiv or YFP YFP-R4 Dsh-YFP wg::yfp-r4 Figur S4 R4 is srt on xosoms n ololiss with Wg. () Evi-Chrry ws ovrxprss with wg-gal4 n shows Chrry-positiv punt srt outsi th xprssion omin, th mil pnl shows nhn imgs o th uppr pnl. Similrly Evi-EGFP xprss with wg-gal4 is srt (lowr pnl). () Totl Wg stining on iss ovrxprssing Evi-Chrry i not show signiint ololistion (inst). () Extrllulr Wg stining shows prtil ololistion o Wg with th xosoml mrkr CD63-GFP xprss with wg-gal4 (insts). () Wil typ YFP-R4 xprss using wg-gal4 shows prsn o YFP-positiv punt outsi th xprssing lls, whih ololiss with Wg (totl)- positiv punt (insts). () Extrllulr Wg lso ololis with YFP- R4 outsi th wg-gal4 xprssion omin (insts). () YFP-R4 xprssing lls mix with mitotrkr-5 ll lls or 5 hours show YFP-positiv punt in r lls (rrows). Morovr, th YFP-R4 xprssing lls i not show mitotrkr-5 stining (rrow-h). (g) Dsh-YFP xprssing lls mix with mitotrkr-5 ll lls or 5 hours i not show YFP-positiv punt in r lls (opn rrow-hs). (h) Quntiition o () n (g) showing numr o Mitotrkr-5 positiv lls tht ontin YFP-R4 positiv punt. Sl rs 2 μm ( g) Mmilln Pulishrs Limit. All rights rsrv.
5 singl pln WT Pt p:: singl pln WT Glu tivity sflu tivity Pt Fmi lls srt Glu-rportr TSG VPS26 VPS35 K67 lls srt Flu-rportr Sr TSG VPS26 p::rl WT p:: VPS35 srna knok own iiny g qpcr TSG VPS26A VPS35 GM Evi Tsg GM Evi Tsg GM Evi Tsg si si si Figur S5 pprs not to strongly t gnrl srtion. () ws knok own in th orsl omprtmnt o th wing iss using p- GAL4. Immuno-stining or Pt shows xprssion o Pt in th omin (ompr rrow-h to th rrow in th lt pnl). Singl onol pln shows prsn o Pt t th mmrn (rrow-hs, right pnl showing highr zoom o th mil pnl squr). () Knokown o gn Rlish ws us s ontrol or knokown. As n sn in mil pnl, Pt is xprss n lolis t th mmrn, similr to knokown. () ws knokown similrly s in, urthr shows norml mmrn lolistion o nothr trnsmmrn protin Flmingo (Fmi) in th omin (ompr insts rom th wil typ intrnl ontrol n omin). () Quntittiv PCR vlition o knokown iiny rom Fig. 7. rom thr inpnnt knokown xprimnts, rror rs r s.. () Srtion ssy in lls with srt Gussi luirs, lls wr tnst with init. (n=3), rror rs r s.. () Srtion ssy in K67 lls with srt Firly luirs. (n=3), rror rs r s.., signiin lvl s init: P<.5, P<., P<. or ( n ).(g) Cllulr lolistion o Evi-GFP n Tsg-Chrry in ontrol, si n si stin or Golgi mrkr GM. Sl rs μm or (g) n 2 μm ( ) Mmilln Pulishrs Limit. All rights rsrv.
6 Fig.3 Fig.3 Fig.4 Fig. 7 Ponu Tsg CX GFP CX GAPDH CX Evi CX P si CX si P Ponu Ponu Bilomyin NH4Cl GW4869 CD8 sigl3 si/tsg sivps35/26 si sigl3 si/tsg sivps35/26 si si CD63 si CD GL3 GFP 2 % o gol prtils 4 2 Lysosoms MVB ER Golgi + ssoit vsils plsm mmrn Mitohonri othr Supplmntry Fig. S6 Figur S6 () Full immunolots with init r o sltion s shown in min igur 3-7. () CD63-immunogol lling o ultrthin stions o ontrol n -plt lls n quntiition o lling or irnt orgnlls. n= gol prtils o thr gris wr ount, sl r 2 nm Mmilln Pulishrs Limit. All rights rsrv.
7 Supplmntry Tl S: Mss sptromtry rsults Exosoms rom K67 lls wr prpr s sri in th Min Mthos stions, sprt on n SDS-PAGE Gl n gl slis wr nlys y nnolc-esi-msms (Oritrp), or protins list t lst two pptis pr protins wr intii. Supplmntry Tl S2: In vivo srning rsults. Bs on th xosoml protin proil, th ov shown gns wr slt or n in vivo srn or Wg srtion. All lins (KK or GD) wr ross with wg-gal4 n rosss wr kpt t C. Ault wings wr sor or noth phnotyps. (shown in r) ws intii s strong nit or Wg srtion t Mmilln Pulishrs Limit. All rights rsrv.
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