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Contents
1. EMT HPMC FN EMT HPMC OF 40 6 DTGF 8 1 IRRIZ KS HPMC mRNA EMT 10ng ml TGF B 1 HPMC Snail m2. 2 DNA DNA DNA 75 2 DNA HS DNA
3. GDP glucose degradation products Nean Ne 4 Fig 1 5 1 1 3
4. xi lt gt TOYOBO FAS II Gel Pro Analyzer Version 3 1 for Windows Media Cybernetics amp 50N31000 11851 Windows IW UT PCR gt Q UV UV 11253 7 jpeg 4 Positive Control
5. EMT HPMC FN FN HPMC EMT HPMC TGE 8 1 Snail mRNA E cadherin o SMA a 5 integrin FN EMT HPMC hfi
6. EMT EMT 3 TOR Hig 1 8 STEP 1 tight junction occludins ZO 1 adherence junction E cadherin apical STEP 2 a SMA e SMA STEP3 MMP IS collagen Fig 1 8 EMT
7. Table 1 3 EMT 8 15 18 19 o TGF 8 1 trans forming growth factor er o ores EGF epidermal growth factor FGF 2 fibroblast growth factor IL 1 interleukin AGEs advancedglycation end products PDGF MEPA KIER E platelet derived growth factor 1 3 1 3 1 EMT 724 EMT
8. O 180C s Over Night gt Wide Mini Sub Cell amp GT System BIO RAD 170 4485 POWER PAC 300 BIO RAD 282 BR 11443 LIBROR AEL 200 76 100ml 100ml PIV IIT PARAFILM Pechiney Plastic Packaging Inc PM 996 v IAI NuSieve GTG Agarose BMA 50080 Ultra Pure Agarose Invitrogen 15510 019 50x TAE buffer INTERGEN S4300 10x TAE buffer Invitrogen 15558 042 Ethidium Bromide SIGMA E 1510 Blue Juice Invitrogen 10816 015 123 bp DNA Ladder Invitrogen 15613 011 DEPC CALBIOCHEM 298711 lt Agarose Gel 3 wt vol gt D Wide Mini Sub Cell amp GT System Gel caster 2 NuSieve amp GTG Agarose Ultra Pure Agarose
9. DNA GTC RNA RNA RNA Bee ISOLATION OF RNA RNA lt RNA DNA gt DNA mRNA CHS mRNA RNA 2 E RNase RNA RNase RNA
10. Gel Pro Analyzer Version 3 1 for WindowsrM lt gt Gel Pro amp Analyzer Version 3 1 for Windows HTD FO 2 CAT Os i PCR Size bp ii max OD control 1 81 A 6 BCA 2 1 1 2 BCA Bicinchoninic Acid SDS PAGE SDS 1g 1 4g SDS
11. Table B 1 1 Table B 1 1 180 200 C 0 5 1 250 C 0 5 X X lt WARE gt 2 UV gt UV 105 lt gt O x1 50 ml 100 ml X amp 1
12. 30 93C 94C i annealing Tm lt Primer gt 1 2 2 ii extension 72 1000bp 1 1000bp 1 lt Primer gt
13. O 75 EtOH DEPC 500 1 Vortex 10000G4C D 2 5cm PARAFILM 10 X PARAFILM DNA mini X 1 3 S D DEPC 10u Vortex LARL MINI CENTRIFUGE 2 X RNA 55 5 4C RNA RTLPCR A 4 RNA lt gt
14. D 7cm 180 C Over Night 106 B 2 Primer List Primer TableB 2 1 Primer List Size Primer Sequence Cycle 5 GAT GTC ATC ATA TIT GGC AGG TT 5 CCT GCA CCA CCA ACT GCT TAG CCC 3 5 TCA GAC GAG GAC AGT GGG AAA G 3 5 GCT TGT GGA GCA GGG ACA TTC 3 5 TCC CAT CAG CTG CCC AGA AA 3 5 TGA CTC CTG TGT TCC TGT TA 3 5 GGC ATC AGA GGT GGC TGG AGG CTT 3 5 GGC AGC TAT GGC GTC CCA CTG TGG 3 5 ACA GCC TGG CAT GGG GCA AGT 3 5 TTC TCC TCC ATC CAG TGG AG 3 Primer Invitrogen GAPDH antisense Snail l antisense E cadherin antisense c 5 integrinP 1 i antisense MMP 2 P7 antisense 107 B 3 RT PCR program RT PCR program RT program gt Table B 2 3 RTprogram Temp Time RT n as 95 0C 5 min roc lt 200C 20 RT UM ie 42 0 40 a3 95 0 EL min PCR program Table B 2 4 PCRprogram 94 0C 3 min 94 00C 730 sec 94 0 40 sec
15. PCR DNA DNA PCR DNA GAPDH 3 house keeping gene GAPDH a actin B actin 79 2 A B X N A B i X ArB a b A B X ab
16. 50G 10cm 750rpm cell pellet S0 EFBS M 199 2ml Vortex 50G 10cm 750rpm 5 10 FBS M 199 Imi 10 25 cm 37C 5 COz C lt 4 A 1 3 gt CO gt 10 0212 KUBOTA 2010 RD3132 Vortex BR 21B 73cm MS 21230 23Scm MS 21050 6 MS 80060 635mm MMS 80240 53 15ml MS 56130 50ml MS 56500 XU ET 10ml
17. Fig 1 7 ve 99 d Ms a s as a Vus f x w OB Fig 1 7 1 HPMC 9 GDPs glucose degradation products HPMC HPMC EMT 2 3
18. D Cuvette W lt gt 2 i i DEPC ug p 1 ODzeo x x RNA ug uguu x ul RNA ng ug DEPC uD ul RNA 0 04 gzD 60 fi AGPC 10 1 RNA 11 91 A 5 T PCR lt
19. 34 EI bres sail lt s a 5 integrin 1 n E MMP 2 1 E cadherin l Fig 5 2 TGE B1 Snail mRNA E Ss RE Snail mRNA E cadherin mRNA 35 5 3 EMT HPMC a 5 integrin MMP 2 mRNA OQ a SMA o integrin MMP 2 o SMA TGF B 1 HPMC A Table 1 Table 5 1 pre FN Collagen TGF 8 1 a S integrin mRNA Fig 4 5 MMP 2 mRNA Fig 4 6 o SMA Fig 4 11 3 EMT HPMC HPMC
20. 15000rpm 30 20 A B 50 1 100 1 BSA 1041 37 7 30 ODz 3 5 2 SDS PAGE running gel 10 stacking gel 4 5 30 21 1 1 TRAL 100C 3 40 1 5g1 15mA 2
21. 1x TAE buffer from 0x Ethidium Bromide 6 01 10 10x TAE buffer DEPC 10 1x TAE buffer from 10x y 10 Gel Electrophoresis Wide Mini Sub Cell amp GT System 1lx TAE buffer from 10x 600ml Ethidium Bromide 20 1 X PARAFILM 20cm Blue Juicerw 3 1 X Blue Juicer 1
22. O bu or O lt gt CR13B DNA mini HETO LAB EQUIPMENTID 872146 MINI CENTRIFUGE COSTER MVSS 06618 Vortex 1 5ml 72 690S Crash ice PARAFILM Pechiney Plastic Packaging Inc PM 996 TERUMOG NN 2432R RNA Bee ISOLATION OF RNA Tel Test Inc CS 104B 61 chloroform isoamylalcohol 24 1 SIGMA C 0549 isopropanol SIGMA I 9516 PBS SIGMA D 8537 75 EtOH DEPC EtOH DEPC EtOH Wako 321 00025 DEPC Diethylpyrocarbonate CALBIOCHEM 298711 Crash ice lt AGPC gt CD 6 PBS Iml well X 3 4 PBS RNA Bee MZ 1ml well 2 well AD MDS MAST ED CH
23. Arp2 3 Actin related protein a SMA a 5 f 1 integrin binding site Heparin I amp II binding site Heparin binding site ERK 130 Cas ossa E E m X _ Stress fiver 5 Arp2 3 y Calpain Rac formation Lamellipodia Focal adheslon o formation turn over _ MMP 2 mRNA 4a C Cell kivan Dut se 1 Fig 5 4 integrin FN a SMA TGF 8 1 a S integrin FN Talin MMP 2 mRNA cell invasion o gen Pai ap a im g 3 integrin FN ES a 38 5 4 FN RGDS EMT HPMC FN RGDSP21 Arginine Glycine Aspartic acid Serine RGDS FN integrin Fig 1 11
24. Fig 1 6 1 7 m 2 2 nt C 50 150um Py microvilli MV cilium C junctional complex JC larnellar body LB elastic lamina EL mast cell M fibroblast F Pericyte P endothelium E capillary CAP basement membrane BM Fig 1 6 1 1 4 Epithelial to mesenchymal transition EMT KROES
25. SDS ETA CHS 2 S S SDS C HRP
26. GIPSO MERRI CHAR Z AT A I IRE RF 10 trypsin PBS ZZL 10 15ml 750rpm 5 70 1 M199 Big Go E II D D 5 D CAMEDIA 200 10 IV gel D lower collagen gel 2 Collagenase S 1 401
27. FN Fnlll4 S YRLTVGLIR Full 12 14 HBS 1 Heparin binding site 1 HBS 2 4 syndecan FN FEn 4 S YRLTVGLTR FnIl 12 14 HBS 1 Fn13 L lt IX HBS2 FnIll 14 ABE CDGF Fig 1 9 8 syndecan FN eSP1 integrim Syndecan EMT FN 13 2 HPMC EMT o rc
28. 97 lt gt A 3 0mg ml pH3 0 Cellmatrix Type 1 A BB 10 C 0 05N NaOH 100ml NaHCO 2 2g HEPES4 77g Sat 022um NaHCO CO Sml gt Table A 6 2 D A B C 8 1 1 A B pH7 4 IN HCl NaOH 1 pH7 3 7 4 7 5 10 8 FN BSA M199 HHA NZ PRR TS
29. TGF B1 a 5 integrin FN Fig 4 6 TGF 8 1 a S integrin FN Snail fig 2 5 4 a 5 integrin FN FAK Focal adhesion kinase p FAK Src Paxillin 2 DOR Src ERK Extracellular 97 signal Regulated Kinase p 130 Cas Crk associated substrate Rac Small GTPase JNK Jun amino terminal kinase MMP 2 Paxillin Rac
30. 4 123 bp DNA Ladder 341 iX Blue Juice 341 1x TAE buffer from 50x Bul 5 PCR product 10 1 Blue Juice 34 D Positive Control 101 Blue Juice 341 Er X ECKO I340 EKF KEE CEU E M ie CD POWER PAC 300 80V 80mA 70 A 5 4 PCR lt gt 280nm UV 2 DNA Ethidium Bromide PECR
31. FN integrin M Syndecan 1 4 4 Syndecan FN Syndecan Heparan sulphate proteoglycan HSPG Chondroitin surface proteoglycan CSPG PY Fig 1 11 Heparan Sulphate chain G Chondroitin Surface chain PnlII4 TVPSPRDLQFVEVTDV KVTIMWTPPE SAVTGYRVDVIPVNLP GEHGQRLPISRNTFAEVIGLSPGVTYYFKVFAVSH GRESKPLTAQQTT PnIIIS KLDAPTNLQFVNETDS TVLVRWIPPR AQITGYSLTVGLTRR GQPRQOYNVGPSVSKYPLRNLQPASEYTVSLVAIKG NQESPKATGVFTT FnIII12 AIPAPTDLKFTQVIPT SLSAQWTPP NVQL GPMKEINLAPDSS EVSVYALKD TLTSRPAQGVVTTLE PnIII13 NVSPPR ARVTDATET TITISWRTEK TETITGFQVDAVPANG OTPTORTTKPDVRSYTTTGLOPGTDYKIYLYTLND NARSSPVVTDAST Fnl1114 AIDAPSNLRFLATTPN SLLVSWQPP RARITGYIIXYEXPGSP PREVVPSPSPGVTEATITGLEPGTEYTIYVIALEXN NQKSEPLIGRKXT Fig 1 12 Syndecan FN Syndecan
32. CAURE 42 7 FAK ERK FAK ERK L 8 EMT HPMC 48 10 11 12 13 14 8 ERE Dee DOSE at 1999 Gary A T etal
33. ODzz ODoso RNA PCR b RT PCR mRNA DNA DNA mRNA PCR 63 gt RNA ODzz ODoso OD o OD2sp RNA 1 5 RNA 0 2 0 5u gul OD 60 0 085 lt gt Smart Spec 3000 Spect
34. i 18 28 2 20 24 DNA Oo i CG G C A C CG 50 3 AT ii Tm Tm Melting Temperature PCR DNA 2 denature 1 55 a iv v 80C CHS 2 Tm Tm A T 2C C G 4 Tm 2 3 2
35. 1 2 EMT 1 2 1 EMT 1 2 1 1 EMT EMT MEATUS Mk 4 1 2 1 2 EMT mRNA mRNA EMT
36. 16h 1 2 24h 1 4 n 5 vs control o co without BSA diffusion 16h diffusion 24h Fig 4 8 vs control 29 4 4 3 EN RGDS Fig 4 9 EMT integrin RGDS FN EMT FN 1 EMT EMT RGDS EMT n 3 a a y P 0 0098 Fig 4 9 vs EMT HPMC 30 4 4 4 Fig 4 10
37. ECL HRP im A 6 1 lt gt 6 NS80060 HIMAC CRISB BIO RAD PBS SIGMA D8537 HEPES Wako 340 01371 protease inhibitor SIGMA P8340 1ML phosphatase inhibitor SIGMA P5726 1ML lt gt D confluent 75cm flask PBS10ml 2 0 1 TritonX 100 20mM HEPES NaOH pH7 2 1 0ml 3 protease inhibitor phosphatase inhibitor 1 25 3T C 6 5 20 D 1 5ml tube 15 000rpm 30 D 20
38. 10 50110012001 11 Image Analysis Image Quant 12 SCAN SCAN 94 Save As 13 X Image Quant 1 1 Grey Color Adjust 2 High 3 Low 4 OK Grey Color Adjust 2 1
39. 10ml 15ml 50G 750rpm5 10 FBS 37C 5 CO 75cm 15ml 6 2ml OCH DAHLE LBA ENY bCRIITS 10 FBS 1ml 3ml 10 D 37C CO 75Scm 1ml 6X 200 1 55 A 2 2 Image J Image J File bbe hat OU pw SOAR SE 400x320 pkeis 9 bi TE Plugins Window Help Ke primm mjurz7 8 ES ND IEE M ONE ie n gu JL 56 Threshold
40. table 1 1 1 2 Table 1 1 EMT up regulate up regulate IV Table 1 2 EMT down regulate down regulate E cadherin occludins ZO 1 desmoplakin EMT 43 2 9 Snail F CHS a SMA 4 EN collagen MMP 2 9 matrix metallopro
41. 12000G 4 10 75 EtOH DEPC Xt 500 1 10000G 4 5 17 DEPC 10 1 55C 5 4C Smart Spec 3000 Spectrophotometer mRNA Kk 3 3 2 mRNA cDNA Polymerase Chain Reaction PCR Snail E cadherin o 5 integrin cDNA mRNA mRNA mRNA Super ScriptTM RNase H Reverse Transcriptase Random Primers cDNA RT product RT product 2 4 1 Primer Table 3 1 AmpliTaq Gold DNA polymerase PCR Master Mix 48 wl cDNA PCR PCR product cDNA PCR product house keeping gene GAPDH Table 3 1 Primer List Size Primer Sequence Cycles bp 5 GAT GTC ATC ATA TIT GGC AGG TT 3 cappy Se 5 5 CCT GCA CCA CCA ACT GCT TAG C
42. 2 ASH DNA DNA DNA HOR 50 gt AGPC RT PCR better RNase Ethidium Bromide Ethidium Bromide Ethidium Bromide 2 AG DNA DNA Ethidium Bromide buffer
43. OK p na
44. R 4 n 3 10 FBS M199 0 1 FBS M199 0 1 FBS M199 10ng ml TGF 1 0 1 FBS M199 10ng ml TGF B 1 10 u g ml FN 10 FBS M199 growth arrest EMT Fig 4 10 31 4 4 TGF 8 1 a SMA Fig 4 11 C FN a 5 integrin a SMA 4 o SMA control 1 ol control n 3 TGF B1 meee FN o SMA RGDS TGF B 1 0 1 FBS M199 0 1 6FBS M199 10ng ml TGF B 1 24h BREE 0 1 FBS M199 10ng ml TGF 8 1 24h URE 10 u g ml FN 24h BREE 0 1 FBS M199 10ng ml TGF 8 1 24h BREE RGDS 30
45. 48 control 0 7 n 8 E cadherin TGF B1 Oh 12h 24h 48h P lt 0 001 E cadherin GAPDH vs control Oh 12h 24h 48h Fig 4 4 TGF B1 E cadherin mRNA 26 4 3 3 TGF 8 1 a 5 integrin mRNA Fig 4 5 TGF 8 1 10ng ml a 5 integrin mRNA TGF 8 1 a 5 integrin mRNA 12h 48h n 12 TGF B1 Oh 12h 24h 48h rms 4 Q 5 integrin GAPDH vs control control 12h 24h 48h Fig 4 5 TGF B1 aS integrin mRNA 27 4 3 4 TGF 8 1 MMP 2 mRNA Fig 4 6 TGF 81 10ng ml MMP 2 mRNA TGF 8 1 MMP 2 mRNA 12h 48h n 6 TGF p1 Oh 12h 24h 48h MMP 2 GAPDH 2 5 B 2 P tmt o gt 15 T o 1 Ss T 0 5 control 12h 24h
46. ae ptu E EISE 1 TGF 8 1 HMC mRNA EMT 2 EMT HPMC in vitro 3 EMT HPMC 4 14 3 I EMT D TGF B 1 HPMC TGF B 1 HPMC Snail E cadherin mRNA II FN a S integrin mRNA collagen MMP 2 mRNA EMT EMT FN gradient EMT
47. 0 5mg ml collagen gel 2001 37 C 30 FN BSA FN BSA 16h 24h EMT 3 5 1 19 3 4 3 RGDS EN FN 3 1 RGDS 35 subconfluent 10ng ml TGF 8 1 48h 5 g ml RGDS ZIRE L 30min 7 5X10 24 72h 3 4 4
48. RGDS Arginine Glycine Aspartic acid Serine integrin 1 4 2 integrin integrin 140 200kDa O a HL 90 110kDa B 123 1 1 o 18 BHA 8 24 o RGDS 5 Fig 1 10 integrin a 83 aA ej e Ge REN B1 kj zn collagen receptor BE Laminin receptor RGD receptor receptor w98 1 C Fig 1 10 integrin ECM 11 integrin ECM 1 4 3 a 5 f 1 integrin FN
49. ii N A B m n A N B N m n A N B N mn i i i N X a m bn Table A 5 5 Table A 5 5 _ AlB amp X Hor a N OFF N X CHE Ethidium Bromide PCR O E O O jpeg
50. gt ERORE QUEM mA ads Aon a Ke s D Process Binary Make Binary zf d aut 400x 120 ele 8 bit Im Fie Ean 6 DOA Smooth Threshold Apply 57 D Analyze Set measurement inm ME Imace t File Edt image P nolo 4 Fie Eat Image Process Anayze Plugins Window Help paalo 4 N AlQ iom s en 214 32132 ye 106 values 255 58 lAnalyze Analyze particle rw o TEE berandi ine 08082 1200x1024 pixels amp brt inverting LUT 1 LAGE 5 Size 1000 10000 X Y TG het me T 128071024 preis 6 bt nverting LUT 1 3MB 4 AQ e p mess us P 7 amp 780 157031053 1259 115875912 722 906759124 Se 1063481426004 804 652186938 BSTBUSB 54596919294 2227 425 535506511 904 955321060 4224 1143048295455 95
51. ECL 1 2 X 1 X Typhoon Image Quant Typhoon 1 Typhoon ON 2 Ctrl Alt Delete 3 Welcome OK 4 5 Typhoon 3 0 6 7 chemiluminescence 8 setup sensitivity high PMT 950 9 Orientation
52. 3 PCR Master Mix AmpliTaq GoldTM Vortex MINI CENTRIFUGE D PCR Master Mix 48 413 O72 Vortex MINI CENTRIFUGE 1 GeneAmp PCR system 2400 PCR MINERAL OIL SIGMA MS904 50 1 PROGRAM TEMP CONTROL SYSTEM PCR program B 5 RT PCR program 2H PCR product 20C A 5 3 Gel Electrophoresis lt gt
53. 5mm 88 II running gel stacking gel Tabale A 5 1 96 REAGENT Runnning gel 3096 2 0ml 3 0ml 4 0ml POUT SEB sm m 1 5M Tris HCI pH8 8 3 0ml 0 4 SDS HCI pH6 8 Cs 0 4 SDS TEMED Total Volume X AX 1 X D running gel stacking gel TEMED 2 running gel TEMED 12 1 D 2 running gel WIRE Adv X 5 10
54. E20 3 4 3 it ER COD DA aS an ee Gt ae a ae Se ee He P 20 3 4 4 Gh cw po gue Tarta dw o ToS ee Sr tae emt igs DAP UD ee She Se P 20 3 5 TGE B1 FN RGDS o SMA lt P21 3 5 1 BCA P21 3 5 2 SDS PAGE P21 3 5 3 P 22 4 P 23 4 1 TGF B 1 P 23 4 2 TGF B 1 P 24 4 3 TGF B 1 Snail E cadherin o 5 integrin MMP 2 mRNA P 25 4 3 1 Snail mRNA ee P 25 4 3 2 E cadherin mRNA gt ss ts ttt ttt t t t t t n n P 26 4 3 3 o S integrin mRNA gt scc tot t t t t t t t n sn P 27 4 3 4 MMP 2 mRNA e s ee eae P28 4 4 EMT i P 29 4 4 1 FN
55. P 29 4 4 2 BSA P 29 4 4 3 FN RGDS E30 4 4 4 P31 4 5 TGF B 1 FN RGDS a SMA lt P 32 5 2 TGF B 1 1 Snail mRNA E cadherin mRNA P 34 5 3 EMT HPMC a 5 integrin MMP 2 mRNA 33 X Ua SMA P36 5 4 FN RGDS p 39 5 5 E 40 6 e o o n o o ee PATI 7 ee ee o o 3 P43 C ES GUIDE P 44 ORE ri oo ccrte etch a 10 APPEND IX ee ee ee ee o oo P 50 A 1 Faz se eee n ol n on t ng n n P 50 A 2 2 Image J PS6 A 3 AGPC JET OU RaW e e e e
56. FN FN 40 50kDa 3 Fn I 2 52 53 54 55 Fig 1 9 FniIIl FniIi2 FnIII3 FnIII4 FnIIIS FnIII6 FnII17 FnIIIEDB FnIII8 FnIII9 FnIII10 FnIII11 FnIIIEDA FnIII12 FnIII13 FnIII14 FnlIII15 T FnIII3 RGDS RGD a5fi integrin 000000 mmm coo SS 1 amp 9 SGPVEVFITETPSQPNSHPIQWNAPQPSHISKYILRWRPKNS VGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQ YGHOEVTRFDFTTT SPLVATSESVTEITAS APDAPPDPTVDQVDDT TVPSPRDLQFVEVTDV KLDAPTNLQFVNETDS LQPGSSIPPYNTEVTET PLSPPTNLHLEANPDTG EVPQLTDLSFVDITDS AVPPPTDLRFTNIGPD GLDSPTGIDFSDITAN VSDVPRDLEVVAATPT EIDKPSQMQVTDVQDN NIDRPKGLAPTDVDVD AIPAPTDLKFTQVTPT NVS PPRRARVTDATET AIDAPSNLRFLATTPN PGLNPNASTGQEALS RLDAPSQIEVKDVTDT SFVVSWVSAS DTVSGFRVEYELSEEG DEPQYLDLPSTATSVNIP
57. 1 8ml TableA 6 2 FN BSA in Type I collagen gel mg ml FN BSA ul AE ACRE ge XFN BSA FN 10zgml gt Sg g ml BSA RE 75 ug ml gt 1 54 g ml A 7 3 HPMC TGF 1 EMT dodi gt 6 NS80060 TGF 81 SIGMA T7039 2 UG M199 SIGMA F9423 FBS SIGMA F9423 lt gt D 6 HPMC subconfluent 2 0 1 FBS M199 24 growth arrest Q 6 3 I10ng ml TGF 8 1 0 1 FBS M199 2ml well 48h A 7 4 HPMC lt gt PCF 12xm MILLIPORE PIXP 012 50 24 MS 80240 APPENDIXA 5 1 APPENDIXA 5 2 M199 SIGMA M2154 FBS SIGMA F9423 PBS SIGMA D8537 TRYPSIN EDTA SOLUTION 10x SIGMA T4174
58. 24 10m g ml FN 1 5mg ml collagen gel 2001 3770 30 control 1 5mg ml collagen gel 0 5mg ml collagen gel 200 gl 37C 30 FN FN 48h 72h I2um EMT 0 1 FBS M199 EMT 10ngml TGF B 1 0 1 FBS M199 30041 Fig 3 3a 35 subconfluent EMT HPMC 10ng ml TGF 8 1 48h 5 X10 24 72h Fig 3 3b Fig 3 3c 3 4 2 FN RU BSA 3 4 1 24 1 5 gml BSA 1 5mg ml collagen gel 2001 37C 30
59. DEPC X DEPC DEPC D REO IL 109 121 40 B 4 lt gt Stat View for Windows ver 5 0 SAS Institute Inc lt gt D Stat View COLANT I 342 E 222 9S35XtNwN M DESC LI Ed Ar oh 100mM glucose Fig B 1 Stat View 1 2 2
60. pH S0 10 Fig 1 1 Fig 1 2 ARO Figl 3 Fig 1 4 ELI MAS OO GE 1 1 2 Peritoneal Dialysis PD D glucose pH Fig 1 5 172
61. 2 B 1 PVDF C 3 D C SS 1cm 2mA 40 Do A 5 6 gt PBS SIGMA D8537 PBS HA 05913 TritonX 100 SIGMA T8787 50ML Monoclonal Mouse Anti Human Smooth Muscle Actin Dako M0851 Rabbit polyclonal to beta Actin abcam ab 16039 500 tum APE KR EBE CE AE i HRP Goat anti Rabbit IgG H L Zymed 62 6120 HRP Goat anti Mouse IgG H L Zymed 62 6520 lt gt PBS PBS 9 6g 1000ml 0 1 PBST PBS 19 Triton1 0ml 1 0 19 PBST PBSTS0ml 0 5g 1 0ml 2 0 1 300
62. FN integrin RGD ocSJ1 integrim integrin co 60 61 62163 Fig l4 1 sons cavam FNS o Cu GS je pO MO e A I i Fig 1 11 integrin FN a 5 8 1 integrin integrin o BIC Ca Ca eo FN Fnm10 RGD RGD c MOAI EF hand type domain 8 DDL a S51 integrin FN RGDS
63. HPMC informed consent I D PBS D i 3 II D 25 ci 10 FBS M 199 5ml 2 50m 5 0 2 0 EDTA 2m1 PBS18ml 0 5 2 0 EDTA PBS 50m 20ml 37 C 20 O
64. MS 66100 Lay es pu yes MS 93170 Counting chamber Erma 03 202 1 Medium 199 SIGMA M2154 PBS SIGMA M8537 TRYPSIN EDTA SOLUTION 10x SIGMA T4174 FBS SIGMA F9423 D PBS Sm1 25 cW 10ml 75 cW I meso M 199 fibro F 12 1ml PBS9ml 10 QE RYU PY UR KYA VV BR 5ml 25 ch7 F AAR 10mMV75 ci 7 FAALIMNZA D FBS1ml 25 ci 7 7 4 3 XX 2ml 75 c 54
65. 2 3 5X 10 ml 6 35 24 15 confluent subconfluent 3 1 3 1 2 HPMC TGF f 1 6 35 subconfluent HPMC OH 0 1 FBS M199 24h growth arrest 10ng ml TGF B 1 M199 12 24 48h 3 2 TGF 8 1 635 HPMC TGF 8 1 IXSO OLYMPUS Tokyo Japan Fig 1 1 MOS X cap 1 10 72h Virtual Dub Ver 1 617 Avery Lee Fig 3 1 TGF 8 1 0 24 48h HPMC
66. CK2 CAMEDIA C 3040 ZOOM Digital camera Sample CAMEDIA C 3040 ZOOM USB SmartMedia Reader Writer MAUSB 2 Phase contrast microscope CK2 Personal computer Fig 3 2 3 3 TGF B1 Snail RU E cadherin a 5 integrin MMP 2 mRNA HPMC TGF B 1 mRNA Snail E cadherin o 5 integrin MMP 2 mRNA AGPC ik RNA RT PCR APPENDIX A 3 AGPC A 3 RNA A 4 RT PCR 3 3 1 Acid Guanidium Phenol Chloroform AGPC mRNA HPMC TGF B 1 RNA Bee ISOLATION OF RNA 1ml well chloroform isoamylalcohol 24 1 200 1 15 12000G 4 C 15 350u1 isopropanol 500 u1 80C
67. PBS PBS APPENDIX M199 501 collagenase S 1 301 37 C 30 M199 50 1 3 4 5 APPENDIX A 7 635 HPMC X10 10 FBS M199 24h 0 1 FBS M199 24hgrowh arrest 10 FBS M199 0 1 FBS M199 0 1 FBS M199 10ng ml TGF 1 0 1 FBS M199 10ng ml TGF 1
68. RNase 60 121C RNase RNA DNA O O RNase free O O TENG m O AGPC
69. Snail mRNA TGF 8 1 12 Fig 4 3 E cadherin mRNA 12 24h Fig 4 4 mRNA TGF 8 1 HPMC TGF 8 1 CEU VIA TGF 8 1 SH2 SH3 PBK HAT 3 PI3K PIP2 4 5 PIP3 HAT 3 4 5 SARA Smad Anchor for Receptor Activation PIP3 RKU TGF Smad2 3 SARA TGF PP 2 Smad2 3 Co Smad Smad4 Snail
70. 1 3 2 EMT table 1 4 8 Table 1 4 Reference 19 26 27 28 FN fibronectin PDGFRI PDGF PDGFR2 0 1 2 GPCR 3 3 TGF
71. PCR PCR IH BT ALBZbSLNSEDPH CHS gt RNase A 4 AGPC PCR AE PROGRAM TEMP CONTROL SYSTEM PCR program O 4 2 O RT product cDNA PCR RT Reaction Tube with Cap gt PROGRAM TEMP CONTROL SYSTEM ASTEC PC 708 MINI CENTRIFUGE COSTER MVSS 06618 Vortex Reaction Tube with Cap
72. lt gt D 300410 M199 APPENDIXA 5 1 APPENDIXA 5 2 1 5mg ml 200 1 24 lower QA 3 37 m 30 99 O APPENDIXA 5 1 77 0 5mg ml 7 200 1 0 1 FBS M199 300 u1 37C FN 48h 72h BSA 16h 24h 6 0 1 FBS M199 10ng ml TGF B 1 0 1 FBS M199 300 1 upper lower KM lower M199 100 1 APPENDIXA 5 3 6 PBS
73. 0 02 101 1ml 10041 37C 30 lower 15ml 750rpm 5 M199 Im vortex D 750rpm 5 7138 23 BE 7c 1T 2 2 3 M199 70 1 pde co Meis rs 2s A 8 lt gt 6 NS80060 M199 SIGMA M2154 FBS SIGMA F9423 TGF 81 SIGMA T7039 2 UG FN 341635 CAMEDIA C 3040 ZOOM CK2 OLYMPUS CK2 lt
74. i PCR 25 30 i v PCR 30 30 35 71 gt PCR PCR i denature 94 C X30 b 1
75. 10 FN Fn BY A FR N C A B G FN A B E C D G F B FN Fn Fn 4 5 FnHI12 14 PG Fn7 i0 En I0 EN
76. 4 49 10 APPENDIX A 1 Fa p 50 A 2 2 Image J PS6 A 3 AGPC hub 4er rf f I t t l n n P 60 A 4 RNA has er rr f o gt t nr P 63 A 5 RT PCR 5525525 c5 5 5 5 5 5 c t t n aso P65 A 6 Um ABRwZuwbklkh 22 2 c P 82 A 7 o ttt trt trt nn P 96 A 8 tmage J P103 B 1 rrr rr t nt nt nt nS A Kee P 105 B 2 Primer List eee P 107 B 3 RI PCR program gt gt sss Se ee eee ee igs P 108 B 4 Sms ie jee a BS Se es re a ea eo ee P 110 A 1 A 1 1 lt gt pH D52 55 HORIBA 10ml TERUMO 50m TERUMO TERUMO NN 1838R Millex GV MILLIPORE Cat No SLGV025LS 4L 0 22 um Strivex GS MILLIPORE Cat No SVGSB1010 4L amp 0 22 um STERICUP MILLIPORE SCGVUOSRE 30ml 543 001S 0 5 m 72 699 S Medium 199
77. Q BRKT IZ AWS D DORREAN A D X 5 1 1 15 20mA 2 30mA X BPB Smm A 6 4 CBB lt gt Wako 134 01833 Wako 012 00245 CBB R250 09408 52 CREK TENE PSE i lt gt 20ml 7 3ml 72 5ml Ki CRS EH CBB R250 2 5g 500ml RERE 0ml 450ml ETR HE 30ml
78. 200ml 0 4 SDS gt SDS0 2g 50m 10 APS APS 0 1g 1ml Tris3 0g 144g 1000ml SDS1 0g gt 4 0 5M Tris HCI pH6 8 2ml 0 4 SDS 4ml 2 1 2ml 2ml 0 8ml 0 1 BPB rumning gel 1 5M Tris HCI pH8 8 0 5ml 0 4 SDS 20 1 1 48ml lt gt I fein amare D e s 87 comb
79. 3 5 3 running gel PVDF 20 BB 304i SUI running gel A 2 B 1 C 3 rnning gel B A 2 B 1 PVDF running gel C 3 72mA 40 PVDF over night 3 5 4 a SMA PVDF 1 PBST 10 3 PVDF 1 PBST 10 3 ECLI 2 750 gu 1 PVDF
80. Spectrophotometer SmartSpec Plus BIO RAD 1 2 3 enter 1 4 562nm 5 enter No 6 select enter YES 7 enter 3 8 Blank PBS Read Blank 9 10 11 Read Sample 3 12 13 10 11 85 A 6 3 SDS PAGE lt gt ATTO MAB 10 MB 00 ATTO RMS 01 ATTO RM10 12 AE 6410 ATTO S500 ATTO Tris Wako 207 06275 SDS Wako 199 07141 HC1 Wa
81. o 5 integrin RGDS EMT HPMC EMT HPMC EN RGDS a SMA 3 1 APPENDIX A 1 3 1 1 HPMC HPMC informed consent 10 trypsin PBS HPMC supplement 10 FBS M199
82. 1096trypsin PBS 1ml d 3 15m AD 750rpm 5 D 1ml 0 1 FBS M199 1 upper D 72h A 7 5 gt HIIRE LE LABINCO 2790001 OLYMPUS CK2 Collagenase S 1 Wako 038 10531 M199 SIGMA M2154 PBS SIGMA D8537 TRYPSIN EDTA SOLUTION 10x SIGMA T4174 16920 ne lt gt I upper D APPENDIXA 5 4 upper 2 5 Ez Hh zc DX ORY ml 750rpm 5 4139 73 BE 2711 2 70g1 M199 ZU AMAR ze TE
83. 10 1 5ml X 5 X well chloroform isoamylalcohol 24 1 200n1 15 RT 13 12000G 4 C 15 3 KERB ORR RNA 235 350 1 1 5ml D isopropanol 500 1 Vortex 80C Over Night bs 62 XAGPCik gt 12000G 4C 10 X
84. 1 Typhoon Image Quant Sm ACE RP Be Le Wh RB 22 4 4 1 TGF 8 1 Subconfluent HPMC 10ng ml TGF B 1 Fig 4 1 TGF B135 f Oh TGF 8 1 48h Fig 4 1 TGF B1 HPMC scale 250um 23 4 2 TGF B 1 Subconfluent HPMC Z 0 1 FBS M199 24h growth arrest 10ngml TGF 8 1 IR L 10 APPENDIX Image J 1h 6h TGF 8 1 OWRSEI do C 4 1 Tu WRB 24 36h q 20 Fig 4 2 P e Co eo e e x m N e
85. PECR E PCR n Fig A 5 3 Eq A 5 3 E Fig A 5 3 PCR 5 i ii ii iv v Taq DNA polymerase OB DNA
86. x2 D 121 20 O yox X1 X3 4 FHL X 1 HF XI RAR 20cm X20 121C 20 O Reaction Tube with Cap PCR 121 20 O 10 ml D 20 24 24 2 10ml 3 6 180C 4
87. gt D 6 10 FBS M199 2ml 2 75cm confluent HPMC 10 1 6 24h 0 1 FBS M199 2ml 24h Growth arrest 4 2ml 10 FBS M199 0 1 FBS M199 0 1 FBS M199 10ng ml TGF f 1 0 1 FBS M199 10ng ml TGF B 1 10 u g ml FN D 3 0 24 48 72 96h A 8 1 Image J 103 Plugins cell_counter cell counter 8 cell counter Initiahze Type 1
88. BSA control 1 2 FN control 2 1 EMT EMT HPMC 41 EMT HPMC TGF B 1 HPMC a 5 integrin mRNA MMP 2 mRNA BSA FN FN FN RGDS EMT HPMC EN
89. Rectangle 2 Moe Ce 3 Select 4 v 5 6 3 1 Select 2 1 3 Volume Review 95 A 7 A 7 1 Type I collagen gel gt Millex GV MILLIPORE Cat No SLGV023LS 0 22 um Strivex GS MILLIPORE Cat No SVGSB1010 1L amp 0 22 um 15ml MS 56150 50ml iE MS 56500 Cellmatrix
90. 69 Reverse Transcription RT Master Mix RNA AGPC Reaction Tube with Cap NCAR UL 941 91 RT Master Mix 91 Vortex ic k OHH L MINI CENTRIFUGE PROGRAM TEMP CONTROL SYSTEM RT program 01 B 5 RT PCR program RNase inhibitor Super ScriptTM 11 X 2 21 4 0 PROGRAM TEMP CONTROL SYSTEM RT program 02 B RT PCR program D RT product 20 A 5 2 Polymerase Chain Reaction lt gt PCR 3 DNA 2 DNA 1 denature
91. DNA 2 DNA 2 annealing DNA 3 DNA DNA extension 2 2 RF DNA 3 2 DNA EK DNA DNA 20 30 DNA DNA fig A 3 2 69 cDNA Primer antisense TITTTT cu 5 gree 3 AAAAAA Primer antisense a TTTTTT T TTTTTT Primer sense 3 5 Pnmer 2
92. DNA RNA 2 PCR denature 1 DNA mRNA AX RNA RNase cDNA Fig A 5 1 ya XI lt A RNase A 3AGPC RT GEC e PROGRAM TEMP CONTROL SYSTEM RT program 01 02 O Reaction Tube with Cap Reaction Tube with Cap Reaction Tube with Cap gt PROGRAM TEMP CONTROL SYSTEM ASTEC PC 708 MINI CENTRIFUGE COSTER MVSS 06618 Vortex Reaction Tube with Cap
93. FN 30 FN integrin RGDS EMT HPMC FN RGDS RGDS Fig 4 9 X RGDS a SMA RGDS Fig 4 11 RGDS EMT HPMC integrin FN integrin integrin FN RGD RGDS FN RET TO integrin Syndecan HSPG Heparan sulphate proteoglycan CSPG Condroitin surface pro
94. 6 12 18 24 30 36 42 48 54 60 66 72 TGF 18 fal hr Fig 4 2 TGF 8 1 HPMC 24 4 3 TGF 81 Snail RU E cadherin o 5 integrin MMP 2 mRNA Snail E cadherin o 5 integrin MMP 2 mRNA RT PCR GAPDH mRNA control 1 4 3 1 TGF 8 1 Snail mRNA Fig 4 3 TGF 81 10ng ml Snail mRNA TGF 8 1 Snail mRNA 24 control OM 1 8 48h control mn 8 TGF B1 Oh 12h 24h 48h Snail GAPDH vs control control 12h 24h 48h Fig 4 3 TGF B1 Snail mRNA 25 4 3 2 TGF B 1 E cadherin mRNA Fig 4 4 TGF 81 10ngml E cadherin mRNA E cadherin mRNA
95. DNA annealing extension 5 3 3 5 TTTTTT AAAAAA 3 5 5 y 3 ee denature 5 3 5 3 IAB ADR Y E TTTTTT 2 Fig A 5 2 PCR lt PCR gt PCR PCR PCR 1 DNA 2 n Eg A 5 1 I IX2 ee Eq A 5 1 DNA f 100 2 n E ddn Eq A 5 2 TH d dn E XI ceses Eq A 5 2 BE E Eq A 2 Eq A 5 3 I XE GXE e cree Eq A 5 3
96. gt PCR DNA mRNA 65 PCR DNA mRNA DNA mRNA cDNA cDNA PCR mRNA PCR RT PCR Fig A 5 1 mRNA RNase sense Primer 1 TTTTTT 2 3 2 DNA PCR 1st cycle sense Primer ee 3 AAAAAA TTTTTT 3 antisense Primer PCR 5 3 2 DNA 3 5 Fig A 4 1 RT PCR A 5 1 Reverse Transcription lt gt mRNA Primer Random Hexamer mRNA 1 DNA cDNA CUKA 66
97. EMT EMT TGE 8 1 85 MMP 2 collagen o SMA MMP 2 a 5 integrin TGF 1 Snail mRNA fig 2 a SMA TGF B 1 TGF 8 1 G Rho RhoA ES RhoA mpDia mammalian diaphanous DIP diaphanous interacting protein Arp2 3 mDia ec SMA RhoA Rho ROCK Rho associated coiled coil containing protein kinase ROCK MLC myosin light chain o SMA Fig 3 RhoA ROCK 14 39 o a 5 integrin 36 D receptor RhoA E mDia RO
98. MYW 1998 BR A KE EBM 2000 epithelial to mesenchymal transition EMT 2007 Gotloib L et al Functional structure of the peritoneum as a dialyzing membrane In R Gokal et al eds Textbook of Peritoneal Dialysis Second Edition Dordrecht Kluwer 2000 37 106 Selgas R et al Epithelial to mesenchymal transition of the mesothelial cell its role in the response of the peritoneum to dialysis Nephrol Dial Transplant 2006 21 112 ii7 Jimenez Hefferman J A et al Immunohistochemical characterization of fibroblast subpopulations in normal peritoneal tissue and in peritoneal dialysis induced fibrosis Virchows Arch 2004 444 247 256 Yanez Mo M et al Peritoneal Dialysis and Epithelial to Mesenchymal Transition of Mesothelial Cells N Engl J Med 2003 348 403 413 Bucala R et al Circulating fibrocytes difine a new leukocyte subpopulation that mediates tissue repair Mol Med 1994 1 71 81 Luiz S Aroeira et al Epithelial to Mesenchymal Transition and Peritoneal Membrane Failure in Peritoneal Dialysis Patients Pathologic Significance and Potential Therapeutic Interventions J Am Soc Nephrol 2007 18 2004 2013 LaGamba D et al Microarray Analysis of Gene Expression During Epithelial Mesenchymal transformation Dev Dyn 2005 234 132 1
99. X 20 X 89 C stacking gel TEMED 61 stacking gel 30 X II D 100 200z g BCA Q 1 1 CHATS D 100 3 IV OD
100. gt 10 u g ml FN 24h BRB a SMA P00039 pe 10 00 Q SMA vs control a 0 00 TGF B 1 FN RGDS Fig 4 11 co SMA control 92 5 B 5 1 TGF 8 1 glucose GDP HPMC Cla PKC protein kinase C 1 PKC c Fos c Jun AP 1 activated protein 1 TGF B 1 AP 1 TGF B 1 mRNA TGF 8 1 Fig 5 1 9 HPMC TGF B1 TGF 81 HPMC EMT High glucose GDPs se LIN m 3 t Y AP 1 TGF 811 Inu pes 7 aw Fig 5 1 TGF B1 33 5 2 TGF B 1 Snail mRNA E cadherin mRNA TGF 81 HMC
101. E l 60 0C 10 72 00C 7 1 5 min Snail annealing n 58 0C 1 min dg 72 0 id 10 EER mn E cadherin 95 0C 3 min 94 00C 7 1 min eme 58 0 1 min 108 Ld PC rive 95 0 30s 950C 30s 580C 30s n 72 0 7 min se 940C lmin 60 00C 1 min 72 00C 7 1 min ee 94 0 C 5 min 5 integrin MMP 2 Primer 72 0 30s 72 0 7 min DEPC D Milli Q FAO 50m 500ml Milli Q DEPC 50041 0 1 X DEPC 37C 2 DEPC RNase D 121C 40 1 500m DEPC
102. Strizzi L et al Epithelial mesenchymal transition is a characteristic of hyperplasias and tumors in mammary gland from MMTV Cripto 1 transgenic mice J Cell Physiol 2004 201 266 276 Grinnell F et al Nested collagen matrices a new model to study migration of human fibroblast populations in three dimensions Exp Cell Res 2006 312 86 94 Schnyder B et al Role of interleukin 8 phosphorylated kinases in stimulating neutrophil migration through fibrin gels Lab Invest 1999 79 1403 1413 Takahashi M et al Effects of endogenous endothelial interleukin 8 on neutrophil migration across an endothelial monolayer Cardiovasc Res 1995 29 670 675 Zhuang S et al Requirement of the epidermal growth factor receptor in renal epithelial cell proliferation and migration Am J Physiol Renal Physiol 2004 287 F365 F372 Becker P M et al Differential regulation of diverse physiological responses to VEGF in pulmonary endothelial cells Am J Physiol Lung Cell Mol Physiol 2001 281 L1500 L1511 HAS2 mRNA 17 2006 Leung J C et al Glucose degradation products downregulate ZO 1 expression in human peritoneal mesothelial cells the role of VEGF Nephrol Dial
103. 2 DNA 280nm UV Ethidium Bromide 590nm Ethidium Bromide 300nm 300nm 280nm UV Ethidium Bromide Ethidium Bromide DNA 2 DNA Ethidium Bromide 2 DNA x DNA 2 DNA Ethidium Bromide lt gt
104. DEPC 2 ml 0 5 mnm 200 pl POMEL RT D L G P S 5 ml ITS HC 50 10 ml Millex GV Medium 199 pH 3 pH PH 1N NAOH aq IN HCL aq 51 pH 7 2 pH 7 4 pH 0 2 A 1 2 gt CO pondo d 10 0212 FORCED CONVECTION OVEN ADVANTEC DRS620DA KUBOTA 2010 RD3132 HPE DHE LABINCO 2790001 Vortex 25Scm MS 21050 15ml MS 56150 50m EOE MS 56300 10ml MS 66100 Ec PAR Medium 199 SIGMA M2154 PBS SIGMA D8537 TRYPSIN EDTA SOLUTION 10x SIGMA T4174 FBS SIGMA F9423 gt
105. 30731 Takagi J et al Structure of integrin a5b1 in complex with fibronectin EMBO 2003 22 4607 4615 Sambra D et al Defining Fibronectin s Cell Adhesion Synergy Site by Site directed Mutagenesis JBC 2000 149 521 527 Jean Louis B et al The Cation binding Domain from the a Subunit of Integrin aSb1 Is aMinimal Domain for Fibronectin Recognition JBC 1998 273 24744 24753 Shiokawa S Functional Role of Arg Gly Asp RGD Binding sites on b1 Integrin in EmBryo Implantation Using Mouse Blastocysts and Human Decidua Biology of Reproduction 1999 60 1468 1474 Sharma A et al Crystal structure of a heparin and integrin binding segment of human fibronectin EMBO 1999 18 1468 1479 i 9 SEXUS OK MI
106. 48h Fig 4 6 TGF B1 MMP 2 mRNA 4 4 EMT 4 4 1 FN Fig 4 7 FN FN 1 FN FN FN 48h Control 1 6 72h 2 1 75 vs control without FN diffusion 48h diffusion 72h Fig 4 7 vscontrol 4 4 2 BSA Fig 4 8 BSA OH BSA 1 BSA T BSA
107. 70ml 880ml lt gt 3 30 30 0 D 2 3 XO A 6 5 gt PVDF ATTO 2392390 ATTO 2392393 Tris Wako 207 06275 Wako 134 01833 6 Wako 018 09642 Elm gt A 0 3 M Tris 5S 4C Tris 3 63
108. 8 Analyze Plugins Window Help Beaoiaiel Alae roms ure S a Imace t File Edt image Process Anayze Plugins Window Help daoago laiiAls f GO mw si lt cell counter 104 B 1 gt ti JB U7 vas Re SERI O3 f 121 20 50 121C 1L 20 lt REAREA gt
109. 83 A 6 2 BCA lt gt Spectrophotometer SmartSpec Plus BIO RAD 04841 4 Wako 197 13975 NazCO Wako 196 01595 NaOH Wako 196 05635 NaHCO Wako 191 01305 HH S Wako 033 04415 lt gt D A B A Ev E LINE lg 0 16g NazCO3 2g 100ml NaOH NaOH 0 4g pH11 25 NaHCO 0 95g DW BK CuSO4 5H 0 4g DW 100ml Q 1mg mA BSA 1 1mg ml BSA ABSA ul ul 84 A B 50 1 CHATS Oro TVA CAH 10 1 2 100 37C 307 CHET D C ODse
110. EART D lt gt D over night 4 PBST 10 3 X O PVDF 1 lt gt 1 0ml HRP HRP Goat anti Rabbit IgG 1 01 1000 FARTS lt gt D PBST 10 3 PVDF 1 XO D 93 A 6 7 lt gt Variable Model Imager Typhoon 8600 Amersham ECL Enhanced ChemiLuminescence Amersham RPN2109 14kL2i4Xx500u 3 25 1 2 0 12Smlcm ECL Ais BRE D
111. FFE L EAIA Table A 5 4 S 50x TAE buffer DEPC 50 1x TAE buffer from 50x Table A 5 4 Table A 5 4 Agarose Gel 3 wt vol DEPC Ultra Pure Agarose g NuSieve amp GTG Agarose pdf 50x TAE buffer 1 2 ml 77 1x TAE buffer from 50x buffer X 1 X buffer
112. P11 1 4 3 o5Bl integrin P12 1 4 4 Syndecan FN P 13 2 eee ee ew ew n n n n P14 3 ee ee ee ee ew ww Pp 15 3 1 e e e e e e e o o e e e e e e e e e oo P 15 3 1 1 HPMC P 15 3 1 2 HPMC TGF B1 P 16 Growth arrest TGF p1Hm amp 3 2 TGF B REZ X 25 REL OO HBE 0O XE E COS RR IE EU 3 3 TGE B C Snail E cadherin gwS integrin MMP 2 mRNA P 17 3 3 1 AGPC mRNA P 17 3 3 2 mRNA cDNA PCR Snail E cadherin a 5 integrin MMP 2 DNA P 17 3 3 3 P 18 3 4 EMT Me 2 e e P19 3 4 1 EN BSA P 19 3 4 2 RGDS FN
113. SIGMA M2154 Penicillin streptomycin SIGMA P 0781 SR AR AE eR EB 50 Insulin Transferin Selenate SIGMA I 1844 L Glutamine SIGMA G 7513 Hydrocortisone SIGMA H 0888 IN NaOH aq Wako 196 05635 IN HCl aq Wako 080 08065 FBS SIGMA F9423 e Te OH gt Fetal bovine serum FBS 37C 5 56C 30 D 50 ml Strivex GS Stericup 7 30 ml 20 ml 20C O ITS Insulin Transferin Selenate ITS 15m 500 1 POMEL A 4C L G L Glutamine 15 ml 5 ml 4C P S Penicillin Streptomycin Solution Sm 5 ml 20 HC Hydrocortisone HC 4 mg 2 ml ethanol CHHL
114. Type I A 637 00653 HEPES 2 4 2 Hydroxyethyl 1 piperazinyl ethanesulfonic acid Wako 340 01371 NaHCO Wako 038 10531 INNaOH 196 05635 Wako 1NHC 1 080 08065 Wako 10 M199 02 069 010 lt YES gt A 3 0mg ml pH3 0 Cellmatrix Type I A B 10 C 0 05N NaOH 100ml NaHCO 2 2g HEPES4 77g St 022um NaHCO CO S5ml lt gt Table A 6 1 D A B 8 1 1 50m A B C pH7 4 IN HCl NaOH 1 LEIER HE 5bkv pH7 3 7
115. 10 u g ml FN 24h 96h 20 23 5 o SMA HPMC 0 1 FBS M199 0 1 FBS M199 10ng ml TGF 8 1 24h BREE 0 1 FBS M199 10ng ml TGF 8 1 24h BREE 10 u g ml FN 24h BR BE 0 1 FBS M199 10ng ml TGF B 1 10 u g ml FN 5 u g ml RGDS 30min 10u g ml FN 24h BRE c SMA BCA SDS PAGE APPENDIX A 6 3 5 1 BCA 300 1 protease inhibitor phosphatase inhibitor 21 15
116. 3644649621 1781 309926726556 974 226242560 3461 945016951608 968 952042886 A 3 AGPC 6 lt gt RNA DNA 2 1 DNA DNA RNA AGPC RNA Guanidium thyocyanate GTC RNase
117. 4 7 5 10 FET HS HU TUS Wo Sil M199 1 8m Table A 6 1 Type I collagen gel MEE C 3s is 1 9 I A ul 199 u I 1 M199 1 A 7 2 Fibronectin in Type I collagen gel X BSA in typeI collagen gel lt gt Millex GV MILLIPORE Cat No SLGV025LS 1L amp 0 22 um Strivex GS MILLIPORE Cat No SVGSB1010 0 22 um 15ml Cat No MS 56130 S0ml Cat No MS 56300 Cellmatrix Type I A 637 00633 HEPES 2 4 2 Hydroxyethyl 1 piperazinyl ethanesulfonic acid Wako 340 01371 NaHCO Wako 191 01305 INNaOH 196 05635 Wako 1NHC 1 080 08065 Wako 10 M199 02 069 010 BSA Wako 019 07494 Fibronectin 341633 1g ml gt M199 50 u g ml 20C
118. 42 Dokic D et al VCAM 1 inhibits TGFbeta stimulated epithelial mesenchymal transformation by modulating Rho activity and stabilizing intercellular adhesion in epicardial mesothelial cells Dev Biol 2006 299 489 504 Zeisberg M et al BMP 7 counteracts TGF betal induced epithelial to mesenchymal transition and reverses chronic renal injury Nat Med 2003 9 964 968 Maszi A et al Central role for Rho in TGF betal induced alpha smooth muscle actin expression during epithelial mesenchymal transition Am J Physiol Renal 44 16 17 18 19 20 21 22 23 24 25 26 27 Physiol 2003 284 F911 F924 Tian Y C et al Epidermal growth factor and transforming growth factor betal enhance HK 2 cell migration through a synergistic increase of matrix metalloproteinase and sustained activation of ERK signaling pathway Exp Cell Res 2007 313 2367 2377 Ha H et al High glucose induced PKC activation mediates TGF beta 1 and fibronectin synthesis by peritoneal mesothelial cells Kidney Int 2001 59 463 470 Offner F A et al Transforming growth factor beta synthesis by human peritoneal mesothelial cells Induction by interleukin 1 Am J Pathol 1996 148 1679 1688 Okada H et al Early role of Fspl in epithelial mesenchymal transformation Am J Physio 1997 273 F563 F574 White L R et al The characterization of alphaS integrin expression on tubular epithelium during renal injury A
119. 42g 50ml Sml 100ml 91 B 2SsmM Tris 5969 7 7 IRI 4 Tris 0 3029g 50ml RAL SmI OA F 7 v JI AC 100ml C 25SmM Tris 40mM6 59923 Z 7 Visi 4 Tris 0 3029g 40mM 6 0 5247g 50ml Sml 100ml lt gt D PVDF runnin gel 20 BEI U3041 P uS 6 running gel A 2 HK BRIT 1 C 3 4 stacking gel running gel LC B A
120. A MAL JSE sananoav ro Master s Thesis HHHHHHHHHHHHHHHHHHL D BLDLELEL ELE E EMT ET E 30 EI U UU 0UU 2008 HHHHHUHHHHHHHHHHHHHHHHHHHHH http hdl handle net 10076 10886 20 EMT 3 1 a a 1 1 rr ee ee B ew we we ww P 1 1 1 1 BARE ee dee ea ae ee ee Oe DL OL RC P 1 1 1 2 CAPD P 2 1 1 3 9 cw SS ee te ee ee er ee ee ie es a ee E P 3 1 1 4 P 3 1 2 EMIT P 5 1 2 1 EMT ee P 5 1 2 2 EMT P 5 1 3 RHONE P 7 1 3 1 EMT P7 1 3 2 ee ee ee ee a a PS 1 4 FN coS8p 1 integrin P 10 1 4 1 FN ee P 10 1 4 2 integrin
121. B TGFR 1518 om me m EGF HER1 ErB1 1 VEGF 0 5 8 32 VEGFR1 3 33 4 Integrin o 5 B 1 Integrina v B 1 Do vascular endothelial growth factor HA CD44 hyaluronan RHAMM 45 SUS 3 1 4 FN o5 8B 1 integrin 1 4 1 EN FN
122. CC 3 38 sense 5 TCAGAC GAG GAC AGT GGG AAA G 3 5 GCT TGT GGA GCA GGG ACA TTC 3 5 TCC CAT CAG CTG CCC AGA AA 3 E cadherin 36 500 5 TGA CTC CTG TGT TCC TGT TA 3 sense 5 GGC ATC AGA GGT GGC TGG AGG CTT 3 a 5 integrin 5 GGC AGC TAT GGC GTC CCA CTG TGG 3 sene 5 ACA GCC TGG CAT GGG GCA AGT 3 29 292 5 TTC TCC TCC ATC CAG TGG AG 3 MMP 2 3 3 3 PCR 3 wt vol Agarose gel 0 3 u g ml ethidium bromide 600ml 1x Tris acetate EDTA buffer 80V 80mA 70 PCR product 1041 jpeg Gel Pro amp Analyzer Version 3 1 for Windowsr f GAPDH Snail KU E cadherin a 5 integrin 18 TIR LE 3 4 EMT APPENDIX A 6 3 4 1 EMT EMT
123. CK wae qan dui TW w SMA 1 Fig 5 3 TGF B1 a SMA TGF B 1 HPMC MMP 2 a SMA oe SMA TGF B 1 HPMC FN TGF 1 Fig 4 11 Z TGF B 1 HPMC FN collagen 7 FN FN fig 4 7 BSA EMT HPMC control Fig 4 8 FN RGDS FN EMT HPMC EMT HPMC fig 4 9 FN
124. DLLPGRKYIVNVYQISE DGEOSLILSTSOTT SIVVRWSRPQ APITGYRIVYSPSVE GSSTELNLPETANSVTLSDLOPGVOYNTTTYAVEE NONESTPVVTOOET KVTIMWTPPE SAVTGYRVDVIPVNLP GEHGQRLPISRNTFAEVTGLSPGVTYYFKVFAVSH GRESKPLTAQQTT TVLVRWTPPR AQITGYRLTVGLTRR GQPRQYNVGPSVSKYPLRNLQPASEYTVSLVAIKG NOESPKATGVPTT TIVITWTP APRIGFKLGVRPSQG GEAPREVTSDSGSIVVSGLTPGVEYVYTIQVLRD GOERDAPIVNKVVT VLTVSWERSTTPDITGYRTTTTPTNGOOGNSLEEVVHADOSSCTEDNLSPGLEYNVSVYTVKD DKESVPIDSTIIP SIGLRNTPLNSSTIIGYRITVYAAGE GIPIFEDFVDSSVGYYTVTGLEPGIDYDISVITLIN GGESAPTTLTQQT TMRVTWAAPPSIDLTNFLVRYSPVKNE EDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYE OHESTPLRGROKT SFTVHWIAP RATITGYRIRHHPEHFS GRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNG REESPLLIGQQST SLLISWDAP AVTVRYYRITYGETGGN SPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT SISVKWLPS SSPVTGYRVITTPENGP GPTKTKTAGPDQTEMTIEGLQPTVEYVVSVYAQNP SGESQPLVQTAVT SIKIAWESP QGQVSRYRVTYSSPEDG IHELFPAPDGEEDTAELQGLRPGSEYTVSVVALHD DMESQPLIGTQST SLSAQWTPP NVQLTGYRVRVTPKEKT GPHKEINLAPDSSSVVVSGLMVATKYEVSVYALKD TLTSRPAQGVVTTLE TITISWRTK TETITGFQVDAVPANG QTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTILND NARSSPVVIDAST SLLVSWQPP RARITGYIIKYEKPGSP PREVVPRPRPGVTEATITGLEPGTEYTIYVIALKN NQKSEPLIGRKKT QTTISWAP FQDTSEYIISCHPVGTD EEPLQFRVPGTSTSATLTGLTRGATYNIIVEALKD OORHKV TALITWFKP LAEIDGI ELTYGIKDVP GDRTTIDLTEDENQYSIGNLKPDTEYEVSLISRRGD MSSNPAKETFTT 1V V EBA GFCD Fig 1 9 FN 1 y gt NAS
125. MicroAmp N801 0540 Crash ice Random Primers Invitrogen 48190 011 dATP Amersham Biosciences 27 2050 01 dTTP Amersham Biosciences 272080 dCTP Amersham Biosciences 272060 dGTP Amersham Biosciences 272070 10x PCR buffer PERKIN ELMER E12387 Recombinant RNasin Ribonuclease Inhibitor Promega N2511 67 Super Script RNase H Reverse Transcriptase Invitrogen 18053 017 DTT Invitrogen Y00147 DEPC CALBIOCHEM 298711 Crash ice RT Master Mix Table A 5 1 RT Master Mix RT Master Mix 1 2 Table A 2 RNase inhibitor Super ScriptTM Table A 5 1 RT Master Mix MR Sampl 1 9 22 ee fooled ca bed feo Random Primers 100mM bebe Table A 5 2RNase inhibitor Super ScriptrM ul EE CENERETUFAEAET Sample 6 1 2 3 4 RNase RNase inhibitor p cp MH C EHE 2 nm jJ z 28 se DR APACE BE
126. MicroAmp N801 0540 Fa wc Crash ice 73 dATP Amersham Biosciences 27 2050 01 dTTP Amersham Biosciences 272080 dCTP Amersham Biosciences 272060 dGTP Amersham Biosciences 272070 Primer sense antisense AmpliTaq GoldTM Applied Biosystems N808 0242 10x PCR buffer PERKIN ELMER E12387 DEPC CALBIOCHEM 298711 Crash ice PCR Master Mix Table A 5 3 PCR Master Mix B 4 Primer List RT Master Mix 1 2 PCR Master Mix AmpiiTaq GoldrW MTA es Table A 5 3 PCR Master Mix asin Primer antisense oe 11 25 TE 26 25 35 20uM 10x PCR buffer aan Se NT NE AmpirqGoam 2s t5 25 ss 525 7 PI Talem Fam KFK EBE COHR n lt Polymerase Chain Reaction gt D PCR Master Mix AmpliTaq GoldTM RT RT product 241 Reaction Tube with Cap
127. RNA 24h Ly E cadherin mRNA EMT 3 EMT mRNA FN a 5 integrin mRNA TGF 8 1 12 48 MMP 2 mRNA a 5 integrin 12 48 EMT HPMC a 5S integrin FN MMP 2 QEMT in vitro
128. Transplant 2005 20 1336 1349 Medici D et al Cooperation between snail and LEF 1 transcription factors is essential for TGF betal induced epithelial mesenchymal transition Mol Biol Cell 2006 17 1871 1879 Itoh F et al The FYVE domain in Smad anchor for receptor activation SARA is sufficient for localization of SARA in early endosomes and regulates TGF beta Smad signaling Genes Cells 2002 7 321 331 18 2007 AGER 2002 Yang J et al Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis Am J Pathol 2001 159 1465 1475 Yang L et al Gene targeting of Cdc42 and Cdc42GAP affirms the critical A 42 43 44 45 46 47 48 49 50 51 52 53 involvement of Cdc42 in filopodia induction directed migration and proliferation in primary mouse embryonic fibroblasts Mol Biol Cell 2006 17 4675 4685 Garcia Vicuna R et al CC and CXC chemokine receptors mediate migration proliferation and matrix met
129. alloproteinase production by fibroblast like synoviocytes from rheumatoid arthritis patients Arthritis Rheum 2004 50 3866 3877 Zhao Z et al Rho associated kinases play a role in endocardial cell differentiation and migration Dev Biol 2004 275 183 191 Kalluri R et al Epithelial mesenchymal transition and its implications for fibrosis J Clin Invest 2003 112 1776 1784 Flessner M F et al The transport barrier in intraperitoneal therapy Am J Physiol Renal Physiol 2005 288 F433 F442 Ogata S et al Glucose and prednisolone alter basic fibroblast growth factor expression in peritoneal mesothelial cells and fibroblasts J Am Soc Nephrol 2001 12 2787 2796 Chen S et al High glucose induced endothelin dependent fibronectin synthesis is mediated via NF kappa B and AP 1 Am J Physiol Cell Physiol 2003 284 C263 C272 Isaji T et al N glycosylation of the beta propeller domain of the integrin alphaS subunit is essential for alphaSbetal heterodimerization expression on the cell surface and its biological function J Biol Chem 2006 281 33258 33267 Inagi R et al Glucose degradation product methylglyoxal enhances the production of vascular endothelial growth factor in peritoneal cells role in the functional and morphological alterations of peritoneal membranes in peritoneal dialysis FEBS Lett 1999 463 260 264 Ding J X et al The reinforcement of invasion in epithelial ovarian cancer cells by 17 beta Estradiol is associated with
130. een n n n n P 60 A 4 RNA P 63 A 5 RT PCR P 65 GGRKCEOKCEWD A 6 ey Li ee a te te se gt ae Sap cit rm i ae fears Gas De a ae Be P 82 A 7 ee te tt te ee et ee P 96 A 8 woe UTOR he Se C DO OR OL UNO PROS ey ee m CO See ist OR we a P 102 B 1 Nr CG EC P 105 B 2 Primer List c ot ot ttt tet P 107 B 3 RT PCRprogram s ss sc tt ctt ttt tr ttt t n g P 108 B 4 WW w C Fer eh a We OR UO CMS ROO 9 09 189 09 0S9 SRL ON LCD CCS NE P 110 dE Dil 1 1 1 1 1 FR EE X DRE SIKRE FA Fig 1 1 1 2 Fig 1 3 1 4 pH
131. ko 080 08065 Wako 016 00765 Bis NN Wako 138 06032 APS 02602 15 Wako 077 00735 BPB Wako 021 02911 2 Wako 135 07522 Wako 079 00614 TEMED NNN N SIGMA T9281 25ML BIO RAD 161 0318 lt gt 1 1 SM Tris HCl pH8 8 0 4 SDS gt 4C Tris54 5g SDS1 2g 5N HCI 17ml pH8 8 300ml 0 5M Tris HCl pH6 8 0 4 SDS gt 4C Tris6 0g SDS0 4g 5N HCI 9ml pH6 8 100ml 0 SM Tris HCl pH6 8 gt 4C Tris3 0g 5N HCI Sm pH6 8 0ml 30 gt 4C 38g Bis2 0g
132. m J Physiol Renal Physiol 2007 292 F567 F576 Strutz F et al Role of basic fibroblast growth factor 2 in epithelial mesenchymal transformation Kidney Int 2002 61 1714 1728 Fan J M et al Interleukin 1 induces tubular epithelial myofibroblast transdifferentiation through a transforming growth factor beta1 dependent mechanism in vitro Am J Kidney Dis 2001 37 820 831 Oldfield M D et al Advanced glycation end products cause epithelial myofibroblast transdifferentiation via the receptor for advanced glycation end products RAGE J Clin Invest 2001 108 1853 1863 Yang L et al P68 RNA helicase mediates PDGF induced epithelial mesenchymal transition by displacing Axin from beta catenin Cell 2006 127 139 155 Liu Y et al Epithelial to mesenchymal transition in renal fibrogenesis pathologic significance molecular mechanism and therapeutic intervention J Am Soc Nephrol 2004 15 1 12 3 2001 Greiling D et al Fibronectin provides a conduit for fibroblast transmigration from collagenous stroma into fibrin clot provisional matrix J Cell Sci 1997 110 861 870 Bates R C et al Transcriptional activation of integrin beta6 during the epithelial mesenchymal transition defines a novel prognostic indicator of aggressive colon carcinoma J Clin Invest 2005 115 339 347 45 28 29 30 3l 32 33 34 35 36 37 38 39 40 41
133. mRNA Snail mRNA 136 MAPK Ras Raf MEK ERK Snail mRNA 9 Snail mRNA E cadherin mRNA E cadherin mRNA adherence junction E cadherin TGF 8 1 E cadherin EMT tight junction ZO 1 Occludins 72 4 ZO 1 Occludins glucose down regulate 78 Padherence junction tight junction EMT
134. rophotometer BIO RAD 170 2501 Cuvette BIO RAD 170 2505 MINI CENTRIFUGE COSTER MVSS 06618 Vortex 0 5ml 72 699S 7 S 200 62011 0 1N HCl aq Wako 080 08065 DEPC CALBIOCHEM 298711 lt RNA gt D AGPC 10 1 11 0 5ml DEPC WEEK 59u1 Vortex 7 CHHE MINI CENTRIFUGE 3 HCI aq 0 1N Cuvette D Cuvette DEPC 601 An Smart Spec 3000 Spectrophotometer Zt y FL RAHE Read cup FK E RU AE Blank DEPC 1 601 Smart Spec 3000 3 Read Sample
135. teinase 1 EMT 1 2 2 EMT EMT growth factor EMT table 1 3 TGF B 1 transforming growth factor EMT EMT J TGF 8 1 EMT
136. teoglycans P Fig 1 9 Fig 1 12 integrin FAK Fig 4 RhoA Ras homolog gene family member A eo SMA Fig 5 5 Heparin binding site 1 59 1 Integrin binding site Heparin I amp II binding site PG PG HSPG or CSPG HSPG or CSPG Stress flver l formatio Fig 5 5 Syndecan integrin 99 FN Syndecan integrin RGDS integrin Syndecan FN Heparin RGDS 5 5
137. up regulation of Snail Gynecol Oncol 2006 103 623 630 Han S et al Control of COX 2 gene expression through peroxisome proliferator activated receptor gamma in human cervical cancer cells Clin Cancer Res 2003 9 4627 4635 Gao M et al Structure and functional significance of mechanically unfolded fibronectin type III1 intermediates PNAS 2003 100 14784 14789 Jose V et al A synthetic peptide from the heparin binding domain III repeats III4 5 of fibronectin promotes stress fibre and focal adhesion formation in 47 54 55 56 57 58 59 60 6l 62 63 64 melanoma cells Biochem 2003 371 565 571 Jose V et al Cooperative Role for Activated a4b1 Integrin and Chondroitin Sulfate Proteoglycans in Cell Adhesion to the Heparin III Domain of Fibronectin BioChem 1999 274 135 142 David C et al Tuning the Mechanical Stability of Fibronectin Type III Modules through Sequence Variations Structure 2004 12 21 30 DeannaLee M et al Syndecans in tumor cell adhesion and signaling BioMed 2004 doi 10 1186 1477 7827 2 3 Hirahara I et al Methylglyoxal induces peritoneal thickening by mesenchymal like mesothelial cells in rats NDT 2008 doi 10 1093 ndt gfn495 Martin P et al The interplay between Src and integrins in normal and tumor biology Nature 2004 23 7928 7946 Zhenyu L et al Two Waves of Platelet Secretion Induced by Thromboxane A2 Receptor and a Critical Role for Phosphoinositide 3 Kinases JBC 2003 33 30725
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