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1 DOI:./n67 GATA E-rin 8 Control Gt 5 α-gata % o mx 6 α-nulolin GATA-APC T-Control T-Gt Spontnous lun mtstsis GATA # o pospo-iston H nuli pr il 8 6 sorn T-Control T-Gt Dys in ultur VEGF in srum (p/ml) p-histon H Fiur S GATA supprsss lun mtstsis in mous T rst nr lls, witout tin prolirtion. () Immunoluorsn stinin or GATA (rn) n E-Crin (r) in t norml mous mmmry ln monstrts tt GATA is xprss only in t luminl pitlil lls. Sl r = 5 mm. ( ) Rltiv GATA protin xprssion in T lls ± Gt usin wstrn lot () n intrllulr low ytomtry (). ( p<. y proility innin tst). () Immunoistomistry (IHC) stinin or GATA in spontnous lun mtstss rom mi injt wit T ± Gt primry ortotopi tumors. Sl r = mm. () Numr o IHC stin pospo-iston H positiv nuli in T ± Gt tumors, wit rprsnttiv ims sown low. Vlus r riv rom n=8 inpnnt mi pr roup n 8 rnom ils pr tumor. Sl r = mm. () MTT ll viility ssy o T lls ± Gt rown in D ultur (n= inpnnt xprimnts prorm in triplit). () Srum VEGF lvls in mi rin primry T ± Gt tumors, msur y -s multiplxin ssy (n= inpnnt mi pr roup, p<.5). Dt r rport s mn ± SEM. Mmilln Pulisrs Limit. All rits rsrv.
2 pospo-iston H # o P-iston H nuli/il 6 i CD mn intnsity 8 6 Rltiv GATA xprssion CD 8 lv sps- MDA-GATA # o lv sp- lls/il MDA-Control j k 6 F/8 F/8 mn intnsity 6 8 Srt VEGFA (μ/ml) 8 6 α-nulolin 5 α-gata Tumor siz (mm ) 5 5 MDA-GATA MDA-Control l Dys in ultur Rltiv sorn...6 MDA-Control MDA-GATA.8 MDA-GATA MDA-Control GATA Supplmntry Fi. S Fiur S GATA supprsss primry tumor rowt n xprimntl lun mtstsis in umn MDA rst nr lls. ( ) Rltiv GATA mrna () n GATA protin () xprssion in MDA lls ± GATA, y qpcr n wstrn lot, rsptivly. (n=6 inpnntly otin ioloil smpls, p<.). () Tumor siz msurmnts o primry ortotopi MDA ± GATA tumors. (n=6 inpnnt mi pr roup, p<.5). () IHC stinin or GATA in MDA ± GATA primry ortotopi tumors. ( ) Mn intnsity ± SEM o CD IHC () n F/8 IHC () in MDA ± GATA primry tumors. Rprsnttiv ims r sown low. (n=6 inpnnt tumors pr roup, ils nlyz pr tumor, p<.5.) () Srt VEGFA rom MDA lls ± GATA rown in D ultur nlyz y nzym-link immunosornt ssy (ELISA). (n= inpnnt xprimnts prorm in triplit, p<.5). () MTT ssy to msur viility o MDA lls ± GATA rown in D ultur. (n= inpnnt xprimnts prorm in triplit.) (i j) IHC stinin or pospo-iston H (i) n lv sps- (j) in MDA ± GATA tumors. T numrs o lls pr il r quntii n rprsnttiv ims r sown low. (n=6 inpnnt mi pr roup n 5 inpnnt ils nlyz pr tumor, p<.5.) (k) Rprsnttiv H&E ims o primry ortotopi MDA ± GATA tumors, wit t nroti rs outlin y t s lins n mrk wit t strisk. (l) Bioluminsn imin o nu mi injt i.v. wit MDA lls ± GATA. Mi wr im t tr wks post injtion. Rprsnttiv mi r sown. (n=5 inpnnt mi pr roup). Dt r rport s mn ± SEM. Sl rs = mm (,, k) n mm (, i j). Mmilln Pulisrs Limit. All rits rsrv.
3 MDA-Control MDA-GATA Hs578T-Control Hs578T-GATA Rltiv GATA xprssion Control GATA 8 6 GATA-APC Hs578T-Control Hs578T-GATA KRT8 CDH ZEB ZEB T-Control T-Gt T-Control T-Gt. T-Control T-Gt i Rltiv VEGFA-Lu tivity Control GATA Fiur S GATA ts ll mirtion in D n D ultur n promots luminl irntition prorm. () Ps-ontrst ims o MDA ± GATA lls sujt to woun srt ssy in srum-r onitions. Ims wr tkn t ours tr wounin. () Ps-ontrst ims o umn sl rst nr Hs578T lls ± GATA m into D Mtril n rown in srum-r onitions wit.5 nm FGF or tr ys. ( ) Rltiv GATA xprssion in Hs578T lls ± GATA msur y qpcr () or rltiv GATA protin y intrllulr low ytomtry stinin () (n=5 inpnntly otin ioloil smpls, p<., p<.). () Rltiv xprssion o pitlil n msnyml mrkrs in Hs578T lls ± GATA msur y qpcr. (n=5 inpnntly otin ioloil smpls, p<.5 or ll ns). () Rltiv xprssion o pitlil n msnyml mrkrs in T lls ± Gt msur y qpcr. (n=6 inpnntly otin ioloil smpls, p<.5). () Ps-ontrst ims o T lls ± Gt in D ultur. () Psontrst ims o sinl T lls ± Gt m into D Mtril n rown or ys. (i) Rltiv VEGFA-Lu promotr luirs tivity in MDA lls ± GATA. Vlus r riv rom n= inpnnt xprimnts prorm in triplit. Dt r rport s mn ± SEM. All sl rs = mm. Mmilln Pulisrs Limit. All rits rsrv.
4 MDA-Control - TGF MDA-Control + TGF MDA-GATA - TGF MDA-GATA + TGF SUPPLEMENTARY INFORMATION Rltiv mir-9 xprssion.5.5 mir-9 mir-9 mir-9 GATA Sit # 5 -UAGCACCAUCUGAAAUCGGUUA- 5 -UAGCACCAUUUGAAAUCAGUGUU- 5 -UAGCACCAUUUGAAAUCGGUUA- GATA Sit # mir-9/ GATA Sit # Supplmntry Fi. S mir-9 MDA-Control MDA-GATA Hs578T-Control Hs578T-GATA mir-9 + PBS + TGFβ NMuMG-Control NMuMG-Gt 5 NMuMG-Control NMuMG-Gt NMuMG-Control + TGF NMuMG-Gt + TGF C Krt C Snil Slu Twist. MDA-Control MDA-GATA Rltiv mirna xprssion.5 PBS TGF srankl TNF mir-9 mir9 mir9 mir-9 i Rltiv mir-9 xprssion MDA-Control - TGF MDA-Control + TGF MDA-GATA - TGF MDA-GATA + TGF ANOVA p<. NS Fiur S GATA inus xprssion o mir-9 mily mmrs, iniits TGF-inu EMT, n trnsriptionlly mpns TGF sinlin. () Rltiv xprssion o mir-9 in Hs578T lls ± GATA msur y qpcr. (n=5 inpnntly otin ioloil smpls, p<.5). () Squns o t mtur orms o mir-9, mir-9, n mir-9. T onsrv s squn is ilit in r n t squn irns r ilit in lu. () Rltiv xprssion o mir-9 n mir-9 in MDA n Hs578T lls ± GATA msur y qpcr. (n=5 inpnntly otin ioloil smpls, p<.5). () A smti o t mir-9/ promotr, wi ontins tr irt GATA inin sits or GATA. () Ps-ontrst ims o NMuMG mmmry lls ± mous Gt stimult wit n/ml TGF or ours to inu EMT. Sl r = mm. () Rltiv xprssion o pitlil n msnyml mrkrs in NMuMG lls ± mous Gt msur y qpcr, wit n witout TGF stimultion. (n= inpnntly otin ioloil smpls pr roup.). () Rltiv xprssion o TGF n BMP ns in MDA lls ± GATA. (n=5 inpnntly otin ioloil smpls, p<.5 or ll ns.), () Rltiv xprssion o mir-9 n mir- 9 in MDA lls stimult wit 5 n/ml TGF, n/ml srankl or n/mltnf or 8 ours. (n=5 inpnnt xprimnts, p<.5). (i) Rltiv xprssion o mir-9 in MDA lls ± GATA stimult wit 5 n/ml TGF or 8 ours. (n=5 inpnnt xprimnts, p<.5, NS = not siniint). Dt r rport s mn ± SEM. Mmilln Pulisrs Limit. All rits rsrv.
5 CD-5 CD9-APC.. Luminl Bsl Gt ER CK8 CK8 CK p6 Rltiv mirna xprssion.5 Norml MECs Bsl Luminl mir-9 mir9 mir9 mir-9 mir9 mir-9 Rltiv mirna xprssion.5 68FARN TO7 T mir-9 mir-9 mir-9 Mtstti Cpility Fiur S5 mir-9 mily mmrs r nri in luminl pitlil lls n is orrlt wit ru mtstti pility. () Norml mous luminl n sl mmmry pitlil lls (MECs) wr rtiont y FACS usin t prviously sri mrkrs CD9, CD n lin mrkrs (CD, CD5 n Tr9). A rprsnttiv FACS plot, t rom t Lin-ntiv popultion, is sown. () Rltiv xprssion o luminl n sl mrkrs wr msur in rtiont popultions y qpcr to vlit sussul rtiontion o t two popultions. (n=6 inpnntly otin ioloil smpls, p<. or ll ns.) Dt r rport s mn ± SEM. () Rltiv mir-9, mir-9 n mir-9 xprssion in norml mous luminl n sl MECs msur y qpcr (n=6 inpnntly otin ioloil smpls, p=.7 or mir-9, p=.6 or mir-9 n p=.5 or mir-9). Dt r rport s mn ± SEM. () Rltiv mir-9, mir-9 n mir-9 xprssion lvls in mtsttiomptnt 68FARN, TO7, n T lls. Mirorry tst pt rom Dykxoorn t l. (9). 5 Mmilln Pulisrs Limit. All rits rsrv.
6 Rltiv mirna xprssion.5.5 MDA-Control MDA-Zip9 mir-9 mir9 mir9 mir-9 mir9 mir-9 Rltiv GATA xprssion.5.5 TO7-Control TO7-Zip C TO7-Control TO7-Zip9 Vimntin Rltiv mirna xprssion.5.5 HMLE-Control HMLE-Zip9 mir-9 mir-9 mir i HMLE-Control HMLE-Zip9 BMI MYC LRP6 NANOG TET j PyMT-Control PyMT-Zip9 PyMT-Control PyMT-Zip9 T-Control T-Zip9 Bmi My Nno Krt Krt8 C p6 Sni Sni Vim T-Control T-Zip9 Bmi Kl Lr5 Lrp6 Msi Sni Sni Z C Fiur S6 mir-9 loss uss -irntition n n EMT/pronitorlik pnotyp. () Rltiv xprssion o mir-9, mir-9 n mir-9 in MDA lls ± Zip9 msur y qpcr to msur knokown. (n=5 inpnntly otin ioloil smpls, p<.5). () Rltiv xprssion o GATA in MDA lls ± Zip9 msur y qpcr (n=5 inpnntly otin ioloil smpls, p<.5). () Ps-ontrst ims o mous TO7 lls ± Zip9 knokown. Elont n spinl-lik lls r mrk y rrows. () Rltiv xprssion o msnyml mrkrs C n vimntin in TO7 lls ± Zip9 msur y qpcr. (n= inpnntly otin ioloil smpls, p<.5). () Rltiv xprssion o mir-9, mir-9 n mir-9 in HMLE lls ± Zip9 msur y qpcr. T Zip9 knokown onstrut iniits multipl mmrs o t mir-9 mily. (n= inpnntly otin ioloil smpls, p<.5). () Rltiv xprssion o stm/pronitor-lik mrkrs in HMLE lls ± Zip9 lls msur y qpcr. (n=5 inpnntly otin ioloil smpls, p<.5). () Ps-ontrst ims o mous PyMT lls ± Zip9 knokown. () Rltiv xprssion o stm/ pronitor-lik n msnyml mrkrs in PyMT lls ± Zip9 msur y qpcr. (n=5 inpnntly otin ioloil smpls, p<.5). (i) Ps-ontrst ims o mous T lls ± Zip9 knokown. (j) Rltiv xprssion o stm/pronitor-lik, pitlil n msnyml mrkrs in T lls ± Zip9 msur y qpcr. (n=5 inpnntly otin ioloil smpls, p<.5). Dt rport s mn ± SEM. All sl rs = mm. 6 Mmilln Pulisrs Limit. All rits rsrv.
7 Rltiv mir-9 xprssion Numr o lun mtstss/mous Rltiv mir-9 xprssion PyMT- Control PyMTmiR MDA-miR-9 MDA-Control Anptl.5.5 It6 It Lox Loxl Loxl Mmp9 P P Pr T T VA PyMT-Control PyMT-miR-9 MDA-miR-9 MDA-Control MDA-Control MDA-miR-9 α-vegf α-lox α-tin Fiur S7 mir-9 supprsss pro-mtstti rultors o t tumor mironvironmnt n iniits lun mtstsis. () Rltiv mir- 9 xprssion in stly trnsu MDA lls ± mir-9 (n=5 inpnntly otin ioloil smpls, p<.). () Rltiv xprssion o init mir-9 trt ns in MDA lls ± mir-9 msur y qpcr. (n=5 inpnntly otin ioloil smpls, p<.5 or ll ns). ( ) Nu mi wr injt i.v. wit GFP-ll MDA lls ± mir-9 n srii svn wks post-injtion. T numr o lun mtstss pr mous wr ount n rp (). Rprsnttiv luorsn ims o lun mtstss (lt) n H&E stin (rit) r sown (). (n=8 inpnnt mi pr roup, p<.5, sl r = mm (lt) n mm (rit)). () Nu mi wr injt wit MDA lls ± mir-9 to orm primry ortotopi tumors in t mmmry t p n srii it wks post-injtion. Rprsnttiv H&E ims o lun mtstss r sown. Tumor sizs wr not siniintly irnt (t not sown). (n=6 inpnnt mi pr roup, sl r = mm). () Rltiv mir-9 xprssion in stly trnsu PyMT lls ± mir-9 msur y qpcr (n=6 inpnntly otin ioloil smpls, p<.). () Rltiv xprssion o init mir-9 trt ns in PyMT lls ± mir-9 msur y qpcr. (n=6 inpnntly otin ioloil smpls, p<.5 or ll ns). () Rltiv protin xprssion o VEGFA n LOX in PyMT lls ± mir-9 msur y Wstrn lot. Atin ws us s t loin ontrol. Dt rport s mn ± SEM. 7 Mmilln Pulisrs Limit. All rits rsrv.
8 MDA-Control MDA-Zip9 T-Control T-Zip9 E- Vim T-Control T-Zip9 Tumor siz (in mm ) 8 6 T7D-Zip9 T7D-Control T-Control T-Zip9 wks 5 T-Control T7D-Control T7D-Zip9 T-Zip9 VIM TWIST GATA i T-Control T-Zip9 T7D-Control T7D-Zip9 j T-Control T-Gt T-Gt-Zip9 Fiur S8 GATA promots irntition n supprsss mtstsis y rultin mir-9 xprssion. () Bioluminsnt imin o nu mi injt i.v. wit MDA lls ± Zip9 knokown. Mi wr im six wks post injtion. (n=8 inpnnt mi pr roup). ( ) Bioluminsnt imin o Bl/ mi injt i.v. wit T lls ± Zip9 knokown (n=8 inpnnt mi pr roup). Mi wr im two wks post injtion () n t lun mtstss wr stin or E-rin (rn) n vimntin (r) (). ( ) Bl/ mi wr injt wit T lls ± Zip9 into t mmmry t p to orm primry tumors. Tumors wr msur wkly or tr wks () n mi wr srii 5 ys post-injtion (n= inpnnt mi pr roup). Rprsnttiv ross luns () n H&E ims () o t lun mtstss (rrows) r sown. () Ps-ontrst ims o umn luminl T7D lls ± Zip9 knokown. () Rltiv xprssion o msnyml mrkrs n GATA in T7D lls ± Zip9 msur y qpcr. (n=5 inpnntly otin ioloil smpls, p<.5). (i) Rltiv xprssion o mir-9 trt ns in T7D lls ± Zip9 msur y qpcr. (n=5 inpnntly otin ioloil smpls, p<.5). (j) Bl/ mi wr injt i.v. wit T lls ± Gt ± Zip9 knokown n srii two wks post injtion. Rprsnttiv H&E ims o lun r sown (n=8 inpnnt mi pr roup). Dt r rport s mn ± SEM. Sl rs = 5 mm (), mm (), mm (, j), n 5 mm (). 8 Mmilln Pulisrs Limit. All rits rsrv.
9 Fi. i Supplmntry Fi. S α-pospo-sm (5 kd) α-vimntin (57 kd) Fi. 5 α-tin ( kd) α-angptl (7 kd, ull lnt) α-lox (58 kd) α-vegfa ( kd) α-tin ( kd) Fi. S Fi. S α-gata (5 kd) α-nulolin ( kd) α-gata (5 kd) α-nulolin ( kd) Fiur S9 Wstrn lot iitl ims. Full-lnt ims o wstrn lots wit t molulr wit lrs r sown. Boxs ilit t lns us in t iurs. 9 Mmilln Pulisrs Limit. All rits rsrv.
10 Fi. S α-vegf ( kd) α-lox (58 kd) α-tin ( kd) Fiur S9 ontinu Mmilln Pulisrs Limit. All rits rsrv.
11 Supplmntry Vios Supplmntry Vio S Tim-lps imin o MDA-Control rst nr lls ultur in D Mtril. MDA-Control lls wr m into rowt tor ru Mtril tr r-rtion ovrnit on low sion plts. D ulturs wr rown in srum-r mi wit.5 nm FGF. Clls wr im usin Ziss Axiovrt invrt ritil mirosop vry minuts or 8 ours t 7 o C n 5% CO. Ims wr ssml n ply t 8 rms/s. Supplmntry Vio S Tim-lps imin o MDA-GATA rst nr lls ultur in D Mtril. MDA-GATA lls wr m into rowt tor ru Mtril tr r-rtion ovrnit on low sion plts. D ulturs wr rown in srum-r mi wit.5 nm FGF. Clls wr im usin Ziss Axiovrt invrt ritil mirosop vry minuts or 8 ours t 7 o C n 5% CO. Ims wr ssml n ply t 8 rms/s. Mmilln Pulisrs Limit. All rits rsrv.
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DOI:.38/n74 nti-cd63 P lls P CX AP BL lnxin Tsg Wnt5A Evi Apil suprntnt P Co-2 lls % Gol prtils 6 4 2 Wg ntioy unspii ontrol
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