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TGFβig-h3 IGF-I - + - IGF-II - - + Supplementary Figure 1. Stimulated release of TGFβig-h3 by IGF-I and IGF-II.

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Presentation on theme: "TGFβig-h3 IGF-I - + - IGF-II - - + Supplementary Figure 1. Stimulated release of TGFβig-h3 by IGF-I and IGF-II."— Presentation transcript:

1 TGFβig-h3 IGF-I IGF-II Supplementary Figure 1. Stimulated release of TGFβig-h3 by IGF-I and IGF-II. Western blots shows both IGF-I (50ng/ml) and IGF-II (100ng/ml) stimulated TGFβig-h3 release from gastric myofibroblasts. Cells GAPDH Cells TGFβig-h3 Media TGFβig-h3 Ionomycin Supplementary Figure 2. Calcium-dependent stimulated release of TGFβig-h3 from gastric myofibroblasts. A) Western blot showing that IGF-II (100 ng/ml) stimulated release of TGFβig-h3 was decreased in Ca2+ free media. B) The calcium ionophore, ionomycin (1µM), stimulated TGFβig-h3 release from gastric myofibroblasts. 37kDa 68kDa 74kDa A B IGF-II Ca2+ free

2 MMP-7 MMP-7 + AG1024 F/Fo 1.20 0.95 Supplementary Figure 3. Intracellular Ca2+ transients induced by MMP-7 are inhibited by AG1024. MMP-7 (2 g/ml) increased intracellular calcium in myofibroblasts (top) and this was inhibited in the presence of AG1024 (3.2µM) (bottom); records from a region of interest placed in the centre of the cell.

3 A. Decorin B. Galectin-3 Sequence: SAIQLGNY Sequence: IALDFQR Sequence
IGF-II Mean ratio IGF-II vs control Quantified in (out of 3 replicates) DLPPDTTLLDLQNNK 1.26 3 SAIQLGNYK 0.99 VPGGLAEHK 1.51 2 VVQCSDLGLDK 1.18 VVQCSDLGLDKVPK 0.43 Sequence: SAIQLGNY B. Galectin-3 Sequence IGF-II Mean ratio IGF-II vs control Quantified in (out of 3 replicates) IALDFQR 0.77 3 Sequence: IALDFQR

4 Mean ratio IGF-vs control Quantified in (out of 3 replicates)
C. TGFig-h3 Sequence IGF-II Mean ratio IGF-vs control Quantified in (out of 3 replicates) EGVYTVFAPTNEAFR 2.50 3 GDELADSALEIFK 1.73 LTLLAPLNSVFK 1.58 2 STVISYECCPGYEK 3.04 VLTDELK 2.65 Sequence: EGVYVTVFAPTNEAFR

5 Mean ratio IGF-II vs control Quantified in (out of 3 replicates)
D. IGFBP-5 Sequence IGF-II Mean ratio IGF-II vs control Quantified in (out of 3 replicates) AVYLPNCDR 1.55 3 FVGGAENTAHPR 2.10 GVCLNEK 1.29 HMEASLQELK 1.35 IISAPEMR 1.58 QESEQGPCR 1.51 2 Sequence: AVYLPNCDR Supplementary Figure 4. Representative examples of SILAC data for identification and quantification of peptides from media of responder cells. The examples shown are A) decorin, b) galectin-3, C) TGFig-h3 and D) IGFBP-5. In each case, tryptic peptides shown underlined on a yellow background were quantified in at least 2 out of 3 replicate samples; representative spectra for one peptide are shown, together with a Table of the mean ratio of abundance in IGF-II treated samples compared with control.

6 A. Preprosecretogranin II
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atient 1, shotgun (8 tryptic peptides). Patient 2, shotgun (8 tryptic peptides). B. Preproenkephalin MARFLTLCTW LLLLGPGLLA TVRAECSQDC ATCSYRLVRP ADINFLACVM ECEGKLPSLK IWETCKELLQ LSKPELPQDG TSTLRENSKP EESHLLAKRY GGFMKRYGGF MKKMDELYPM EPEEEANGSE ILAKRYGGFM KKDAEEDDSL ANSSDLLKEL LETGDNRERS HHQDGSDNEE EVSKRYGGFM RGLKRSPQLE DEAKELQKRY GGFMRRVGRP EWWMDYQKRY GGFLKRFAEA LPSDEEGESY SKEVPEMEKR YGGFMRF Patient 1, shotgun (1 hemi-tryptic peptide) Patient 2, shotgun (2 tryptic peptides, I hemi-tryptic) Patient 3, SILAC ± IGF (2 tryptic peptides)

7 C. Peptidyl α-amidating mono-oxygenase
MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF GDQEPVQGFV MNFSNGEIID IFKPVRKHFD MPHDIVASED GTVYIGDAHT NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA EAVVETKMEN KPTSSELQKM QEKQKLIKEP GSGVPVVLIT TLLVIPVVVL LAIAIFIRWK KSRAFGDSEH KLETSSGRVL GRFRGKGSGG LNLGNFFASR KGYSRKGFDR LSTEGSDQEK EDDGSESEEE 970 YSAPLPALAP SSS Patient 3, SILAC ± IGF (5 tryptic peptides)

8 D. Preproadrenomedullin
MKLVSVALMY LGSLAFLGAD TARLDVASEF RKKWNKWALS RGKRELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL Patient 1, shotgun (1 hemi-tryptic peptide) Patient 3, SILAC ± IGF (2 tryptic peptides) MLSCRLQCAL AALSIVLALG CVTGAPSDPR LRQFLQKSLA AAAGKQELAK YFLAELLSEP NQTENDALEP EDLSQAAEQD EMRLELQRSA NSNPAMAPRE RKAGCKNFFW KTFTSC E. Preprosomatostatin 271, shotgun (1 tryptic, 1 hemi-tryptic peptide) Supplementary Figure 5. Examples of neuroendocrine markers identified in the secretomes of responder myofibroblasts. The sequences of A) preprosecretogranin II, B) preproenkephalin, C) peptidyl α-amidating mono-oxygenase (PAM), D) preproadrenomedullin (ADM), and E) preprosomatostatin are shown; in each case, quantified peptides are indicated by horizontal arrows. The data are from three different responder secretomes (patients 1 and 2 taken from Holmberg et al., J.Proteome Res., 2103; patient 3, this study). Validation of the expression of SGII and proenkephalin is provided in Fig 5. Validation of PAM, adrenomedullin and somatostatin localisation is shown by immunohistochemistry using Texas Red labelled secondary antibody, with SGII identified by FITC labelled secondary (PAM, ADM). Signal peptides in italics. Scale bars 10 m.

9 A B SGII SGII GAPDH GAPDH SGII-siRNA SGII-EP Supplementary Figure 6. Knockdown and overexpression of SGII in gastric myofibroblats. A) Representative Western blots showing decreased SGII abundance after siRNA knockdown. B) Representative Western blots showing increased SGII abundance after transfection with SGII expression plasmid (SGII- EP).

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