Preventive and therapeutic effects of Smad7 on radiation-induced oral mucositis
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1 Prvntiv n thrputi ffts of on rition-inu orl muositis Gngwn Hn, Li Bin, Fulun Li, An Cotrim, Donn Wng, Jino Lu, Yu Dng, Grgory Bir, Anstsi Sowrs, Jms B. Mithll, J. Silvio Gutkin, Rui Zho, Dvi Rn, Ptr tn Dijk, Yosf Rfli, Qinghong Zhng, n Xio-Jing Wng Th uthors ontriut qully to this work. Supplmntry Informtion
2 psm1/5/8 /K14 Moriun rt (%) CD4 BM8 Ly-6G 8 Gy x 3, y 9 Nxt to mg 8 Gy x 3, y 9 Dmg Nonirrit Hn, t l. K5. M 46 kd 43 kd Atin Non-irrit 8 Gy x 3, y 9 K /8 K5. 4 2/7 2 22Gy K5. K5. Non-irrit 8 Gy x 3, y 9 Supplmntry Figur 1. trnsgn xprssion in orl muos us rsistn to ritioninu orl muositis ut i not fft psm1/5/8. () wstrn lots: unttl in nonirrit wil-typ () tongu n rly ttl ftr rition. K5. tongus hv omprl protin lvls for n ftr rition. M: molulr mrkr. () immunostining. Not tht nuli in som irrit pithlil lls r hyprtrophi. Dott lins lint pithlil-stroml ounry. () Ru inin of orl muositis-inu moriity in K5. mi. : P =.7 trmin y Fishr s xt tst. () Immunostining of nutrophils (Ly-6G), mrophgs (BM8) n CD4 T-lls in n K5. tongus on y-9 ftr initition of 8 Gy x 3 irrition. Frozn stions wr tkn outsi of th ulr zon. Dott lins lint th pithlil-stroml ounry. () No signifint iffrn in psm1/5/8- nulr positiv lls (grn) twn n K5. orl muos for or ftr rition. Krtin (K14) immunostining (r) highlights th pithlil omprtmnt. Not tht nuli of irrit pithlil lls r hyprtrophi. Th sl r: 5 µm for ll pnls.
3 Prntg of woun losur TGF-β1 xprssion (fol) sitgf-β1 Srml si-2 si-1 Srml Hn, t l. h 24h 48 h 72 h 96 h h 24 h 48 h 72 h Srml sitgf-β h 48 h 72 h Srml sitgf-β1 Supplmntry Figur 2. Migrtion in spontnously immortliz humn orl pithlil lls (NOK-SI) ws ly y knoking own ut lrt y knoking own TGF-1. (, ) Rprsnttiv imgs of ll migrtion. Pirs of ott lins lint th srth woun. Quntifition of ll migrtion n ffiiny of knokown r prsnt in Fig. 2 n Fig. 2. Srml: srml sirna. () Quntifition of ll migrtion ftr TGF-1 knokown from 3 sprt xprimnts. () qrt-pcr showing TGF-1 knokown ffiiny. Dt r prsnt s mn ± s.. : P <.5, : P <.1, : no signifin trmin y two-til Stunt t-tst.
4 48 h 24 h h BrU/K14 Prntg of BrU ll lls Hn, t l Srml sir1-1 sir kd R kd Tuulin 2 1, srml, sir1-1, srml, sir1-1 srml sir1-1 sir1-2 srml sir1-1 sir1-2 Supplmntry Figur 3. Knoking own R1 ru prolifrtion n migrtion of wil-typ () n trnsgni krtinoyts. () Wstrn lot for R1 48 hours ftr R1 sirna (sir1-1, sir1-2) trnsftion. Control: Srml sirna. () Prntg of BrU ll lls in n trnsgni krtinoyts with or without R1 knokown. Dt from 3 sprt xprimnts wr prsnt s mn ± s.. : P <.1 trmin y two-til Stunt t-tst. () Rprsnttiv immunofluorsn of BrU positiv lls prsnt in (). An ntioy ginst krtin 14 (K14, r) ws us for ountrstin. () In vitro ll migrtion ssy for n trnsgni krtinoyts ftr R1 knokown. Pirs of ott lins lint th srth woun. Quntifition of ll migrtion is prsnt in Fig. 2g.
5 72 h 48 h 24 h h Protin lvls (fol) Hn, t l. 22 kd M GTP-R kd Totl-R kd 55 kd 46 kd Tuulin Tuulin GTP-R1 Totl R1 f Srml sictbp1-1 sictbp1-2 6 kd M Srml sism2 Sm2 36 kd GAPDH 54 kd M Srml sism3 Sm3 36 kd GAPDH 65 kd M Srml sism4 Sm4 36 kd GAPDH Supplmntry Figur 4. Inrs R1 xprssion y, knokown ffiinis of iniviul Sms, n lrt krtinoyt migrtion y knoking CtBP1. () Wstrn lot for GTP-R1 n totl R1 in wil-typ () n trnsgni krtinoyts. Aitionl smpls r shown in Fig. 3. M: molulr mrkr. () Quntifition of GTP-R1, totl R1 n in n trnsgni krtinoyts shown in () n in Fig. 3. Th protin lvl in krtinoyts of h lot ws normliz s "1". Dt is prsnt s mn ± s.. : P <.1, : P <.1 trmin y two-til Stunt t-tst. (-) Wstrn lots for Sm2, Sm3 n Sm4 knokown in humn krtinoyts. Thir ffts on R1 xprssion r shown in Fig. 3. M: molulr mrkr. GAPDH: intrnl protin ontrol y rproing th sm lot. (f) NOK-SI ll migrtion. Pirs of ott lins lint th srth woun. Quntifition of ll migrtion n ffiiny of CtBP1 knokown r shown in Fig. 4 n Fig. 4.
6 Ulr siz (mm) Tt- V5/K14 Elut Column Mrkr Hn, t l. RKKRRQRRR GLPIPNPLLGLDS GST Tt V5 Prission Enzym 5 kd Tt--V5 Prission Enzym GST Tt V5 5 min 12 h 12 h 5% Glyrol / Tt- Non-irrit ul tissu V5/K14 kd Lr Tt-Cr p Lr DNA f psm2/v5 DNA+ Tt-Cr g Prvntion, 2 Gy, y Supplmntry Figur 5. Purifition n hrtriztion of Tt- n Tt-Cr protins. () Shmti of Tt- protin. () Coomssi stin SDS-PAGE gl for purifi Tt-. () Tt- protin trnsution to krtinoyts. Lft n mil pnls: Tt- stining (grn) using V5 ntioy, ountrstin with K14 ntioy (r). Clls show Tt- in th nulus 5 min ftr trnsution n in oth nulus n ytoplsm 12 hours ftr trnsution. Right pnls: Tt- rogt Sm2 phosphoryltion (psm2, grn). V5 (r) ountrstin visulizs Tt- trnsu lls. () V5 ntioy stining tts Tt- trnsution in ul muos 12 hours ftr Tt- (1 µg) topil pplition. A K14 ntioy ws us for ountrstin. Sl r: 5 µm for oth pnls. () Slivr stin SDS-PAGE gl for purifi Tt-Cr with th sm Tt n V5 tgs in (). (f) Agros gl showing tivity of Tt-Cr: Tt-Cr uts out 1,46 p flox frgmnt from th 7,65 p vtor pll3.7 (Agn). (g) Tt- protin prvntiv trtmnt ru 2 Gy rition-inu orl ulrs (mn ± s..m.). : P <.5, : P <.1 trmin y two-til Stunt t-tst.
7 psm2 NF-kB p5 CD45/K14 psm2 + lls (%) R1-Lu tivity (fol) BrU/K14 TUNEL/PI CD45 + lls pr mm Nulr NF-kB p5 + lls (%) Ulr siz (mm) Apoptoti lls pr mm BrU + lls pr mm Hn, t l. Trtmnt, y Glyrol Tt- (.8 µg) Gy 2 Gy Glyrol Tt- 12 Glyrol Tt f 25 Glyrol Tt Glyrol Tt- 1 Glyrol Tt g Glyrol Tt- h Tt- R1-SBE mut R1-SBE i CtBP1 on R1 SBE 18 Gy, y 1, trt y 6 to y 9 Supplmntry Figur 6. Effts of Tt- trtmnt on orl muositis. () Ru ulr siz (mn ± s..m.) in Tt- (.8 µg ily, y 6 to y 9) trt orl muos. Smpls wr hrvst on y 1. N = 8 pr group, xpt N= 7 for 2 Gy Glyrol (5% glyrol/) group. () Immunostining of molulr mrkrs for smpls from (). Sl r: 5 µm for th top two pnls n 25 µm for othr pnls. Propiium ioi (PI) n K14 wr us s ountrstin. (-g) Quntifitions (mn ± s..) of immunostining shown in (). 3-4 smpls wr us. (h) Luifrs ssy. Tt- trtmnt inrs tivity of th R1 promotr with SBE ut not th mutnt SBE in mous krtinoyts (mn ± s..). (i) ChIP ssy for CtBP1 ining to th SBE of mous R1 promotr in Tt- trt mous krtinoyts. : P <.5, : P <.1, : P <.1, : no signifin trmin y two-til Stunt t-tst.
8 Prntg of woun losur Cl27 Tt- Prntg of woun losur 6 h 48 h 36 h 24 h 12 h 72 h Tt-, 4 h, 4 h Non-irrit h 48 h 24 h h Tt- Hn, t l. NOK-SI Tt- NOK-SI, 3 Gy psm2/k14 NF-kB p5/k14 V5/K14 f MSK921 Tt- 21 kd 55 kd g M 12 1 R1 Tuulin Tt Tt- 12 h 24 h 36 h 48 h 6 h h h 24 h 48 h 72 h h 48 h 72 h Supplmntry Figur 7. Effts of Tt- trtmnt on migrtion of humn krtinoyts n tumor ll lins. () Tt- lrts NOK-SI ll migrtion. Quntifition from 4 sprt xprimnts is shown in Fig. 6f. Pirs of ott lins lint initil wouns () Tt- trtmnt in NOK-SI lls ttnut rition-inu psm2 n NF-kB p5 nulr loliztion. () V5 stining of MSK921 lls 2 hours ftr Tt- trtmnt. K14 ws us s ountrstin. () R1 wstrn nlysis in MSK921 6 hours ftr th initil ily Tt- trtmnt. M: molulr mrkr. () Quntifition of MSK921 ll migrtion from 3 sprt xprimnts. (f) A rprsnttiv MSK921 ll migrtion ssy trt with Tt- n. Pirs of soli lins lint initil wouns. Dott lins highlight th forfront of migrt lls. (g) Quntifition of Cl27 ll migrtion from 3 sprt xprimnts. (h) Rprsnttiv imgs for (g). Pirs of soli lins lint initil wouns. Dott lins highlight th forfront of migrt lls. Dt r xprss s mn ± s.. : no signifin trmin y two-til Stunt t-tst.
9 Hn, t l. Rition NF-kB TGF-β1 CtBP1 P Sm4 CtBP1 P Inflmmtion Apoptosis Growth Arrst -1.5K R1 Orl muositis R-pithliliztion/rpir Rition NF-kB TGF-β1 CtBP1 Sm4 CtBP1 Inflmmtion Apoptosis Prolifrtion -1.5K R1 R-pithliliztion/rpir Orl muositis prvntion/hling Supplmntry Figur 8. Potntil mhnisms of -mit prottion n hling of orl muositis. () Rition tivts NF-kB, inrss TGF-β1 n CtBP1. NF-kB n TGF-β1 inu inflmmtion. TGF-β1 inus poptosis, growth rrst n tivts Sm-2, -3 n -4 whih rruit CtBP1 to th R1 promotr to rprss R1 trnsription, ling to lunt r-pithliliztion. Th lk of pithlil rgnrtion n xssiv inflmmtion us orl muositis. () loks NFkB n TGF-β1-inu inflmmtion n loks TGF-β1-inu poptosis n growth rrst. rlivs R1 trnsriptionl rprssion y ithr prvnting TGF-β1-mit Sm tivtion (phosphoryltion) or ompting with signling Sms/CtBP1 trnsriptionl rprssion omplx in ining to th R1 promotr. Inrs R1 inu y ontriuts to krtinoyt migrtion uring r-pithliliztion. -mit lrtion of pithlil rgnrtion n rution in inflmmtion l to prvntion or hling of orl muositis.
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