Control + SDS + 2BME. Control + SDS. Control

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Hillary Douglas
6 years ago
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Supplementary Figure 1 2BME:2-mercaptoethanol Control Control + SDS Control + SDS + 2BME Control Control + SDS Control + SDS + 2BME Control Control + SDS Control

1 Supplementary Figure 1 2BME:2-mercaptoethanol Control Control + SDS Control + SDS + 2BME Control Control + SDS Control + SDS + 2BME Control Control + SDS Control + SDS + 2BME Ephrin-B2 Oligomers Ephrin-B2 Dimer Ephrin-B2 Monomer Ephrin-B2 Ab Ephrin-B2 Ab + Rc Ephrin B2 (1 ug) Ephrin-B2 Ab + Rc Ephrin B2 (5 ug)

2 Supplementary Figure 2

3 Supplementary Figure 3 a c Ephrin B2 Ephrin B2 Healthy fibroblasts IPF fibroblasts Lung fibroblasts Lung macrophages GAPDH Beta-ac=n b d PBS Bleomycin Ephrinb2 floxed 552 bp Wild type 450 bp 150 Alpha SMA Type I Collagen Cre 8 bp Beta- Ac=n

4 Supplementary Figure 4 a PBS Bleomycin Ephrin B2 GAPDH GAPDH PBS Bleomycin b Recombinant Ephrin-b2 sephrin B2

5 Supplementary Figure 5 a Control BAL NaCve-PAGE Bleo BAL c Ephrin-B2 Oligomer 150 Alpha SMA Type I Collagen 242 Ephrin-B2 Dimer b Native-PAGE Control Bleo BAL BAL Betaac=n SDS + 2ME Ephrin-B2 Oligomer Ephrin-B2 Monomer d 150 Alpha SMA Type I Collagen Betaac=n

6 Supplementary Figure 6 a NT sirna ADAM10 sirna TGF-β ADAM10 sirna - + b Low exposure Alpha SMA Lower exposure Alpha SMA Higher exposure GAPDH GAPDH Higher Exposure c TGF-β TGF-β GFP WT Δ GFP Δ182/194 WT ΔJuxta GFP WT Δ GFP Δ182/194 WT Δ Juxta 80 HA 80 HA Lower exposure Higher Exposure GAPDH

7 Supplementary Figure 6 d GFP Ephrin-B2-WT Δ182/194 Δ197/ TGF-β Alpha SMA GAPDH TGF-β GFP Ephrin-B2-WT Δ182/194 Δ197/ e TGF-β Alpha SMA GAPDH

8 Supplementary Figure 7 a Bleomycin Day 0 Day 1 Day 3 Day 7 Day ADAM10 Beta-acCn b PBS PBS BLM BLM + GI25 Alpha SMA Beta-acCn

Supplementary Figure 8 Control BAL IPF BAL 1 2 3 4

9 Supplementary Figure 8 Control BAL IPF BAL Recombinant Ephrin-b2 60 sephrin B2

10 Supplementary Figure Legends Supplementary Figure 1. Effect of denaturing and reducing conditions on sephrin-b2 oligomerization. BAL samples were run in triplicates on non denaturing gels (native PAGE) with or without SDS or SDS + 2-mercaptoethanol. Individual membranes were then incubated with a) ephrin-b2 antibody, b) Ephrin-B2 antibody preincubated with 1 µg of recombinant mouse ephrin-b2 or c) Ephrin-B2 antibody pre-incubated with 5 µg of recombinant mouse ephrin-b2. Supplementary Figure 2 sirna transfection efficiency. lung fibroblasts were transfected with non-targeting control sirna or ON-TARGETplus sirna against MMP1, MMP2, MMP7, MMP14, ADAM9, ADAM10, ADAM12, ADAM17, ADAM19, EPHB3 and EPHB4. ON-TARGETplus sirna uses a validated pool of 4 individual sirnas per target gene. Cells were transfected with either 10 nm or 50 nm sirna and gene expression levels tested by qpcr 48 hours post-transfection. Data are presented as mean ± SD. P < 0.05, Student s t-test. Supplementary Figure 3 Uncropped blot images from Figure 1. (a). Uncut blots for Figure 1b. (b) Uncut gels for Figure 1d. (c) Uncut blots for Figure 1e. (d). Uncut blots for Figure 1i. Supplementary Figure 4. Uncropped blot images from Figure 2. (a) Uncut blots for Figure 2a. (b) Uncut blots for Figure 2b. Supplementary Figure 5. Uncropped blot images from Figure 3. (a). Uncut blots for Figure 3h. (b) Uncut gels for Figure 3i. (c) Uncut blots for Figure 3l. (d). Uncut blots for Figure 3p.

11 Supplementary Figure 6. Uncropped blot images from Figure 4. (a). Uncut blots for Figure 4h. (b) Uncut gels for Figure 4j. (c) Uncut blots for Figure 4n. (d). Uncut blots for Figure 4p. (e) Uncut blots for Figure 4s. Supplementary Figure 7. Uncropped blot images from Figure 5. (a). Uncut blots for Figure 5a. (b) Uncut gels for Figure 5d. Supplementary Figure 8. Uncropped blot images from Figure 6. (a). Uncut blots for Figure 6i.

12 Supplementary Table 1: IPF and Control Subject Characteristics. IPF (n = 30) Controls (n = 30) Age (yrs) 68.5 (7.0) 68.3 (7.0) Sex (M/F) 21/9 21/9 Smoking 0/26/4 0/26/4 (current/ever/never) FVC predicted (%) 66.8 (14.9) N/A DLCO [Hb] predicted (%) 44.0 (18.2) N/A Data are presented as mean +/- SD as appropriate. N/A refers to data that was not available or collected. Pulmonary function data (FVC/DLCO [Hb] the long forms of this should be defined) was performed within 15 days of plasma collection and was available on 27/30 subjects.

13 Supplementary Table 2 Gene Species Sequence (5 to 3 ) Collagen type I α1 chain (col1α1) α-smooth muscle actin (α-sma) Glyceraldehyde 3- phosphate dehydrogenase (GAPDH) Ephrin A1 Ephrin A2 Ephrin A3 Ephrin A4 Ephrin A5 Ephrin B1 Ephrin B2 Ephrin B3 EphB1 F: GAACGCGTGTCATCCCTTGT R: GAACGAGGTAGTCTTTCAGCAACA F: TTCAATGTCCCAGCCATGTA R: GAAGGAATAGCCACGCTCAG F: CCCATGTTCGTCATGGGTGT R: TGGTCATGAGTCCTTCCACGATA F:CGGAATGAGGACTACACCATACATGTG CAGC R:AAGCAGCGGTCTTCATGCTGGTGGATG GGTT F: CTACACGGTGGAGGTGAGCA R: ACAGCATTGGGAGGCGTGGCA F: ACTACATCTCCACGCCCAC R: TCCCGCTGATGCTCTTCTC F: TTGTACATGGTGGACTGGCC R: AGAACTCTCTGGAGTGGGCACC F: CCAGAAGATAAGACTGAGCGC R: CCATTATCTGGGATTGCAGAGG F: CCAATGCTGTGACGCCTGAG R: CGAACAATGCCACCTTGGAGTTG F: GGAAGAAGTTCGACAACAAGTCC R: TTCAGCAAGAGGACCACCAGCGT F: TGGAGCCTGGGAAGGAGAACC R: CAGGGTGGCGACTCTCCGAAG F: TTCACTTCAGCCAGCGAC R: TTTCCCTCCTCTCCTTCCC

14 EphB2 EphB3 EphB4 EphB6 MMP1 MMP2 MMP3 MMP7 MMP8 MMP9 MT1- MMP/MMP14 ADAM8 ADAM9 ADAM10 ADAM12 ADAM15 F: ATGGCGCCCCTCTCCTCTGGCATCA R: ACCGCTTGGTTCTTCCCGTG F: GCATCGCCTCCACAGTGACC R: ACGAAGACAAGCCCAGCTGTA F: TTCGGCCAGGAACATCACAG R: CCGATGAGATACTGTCCGTG F: TACCTGTCCAGCTTTGCC R: TCTCCATAACTCATCACTTCCC F: GGTCTCTGAGGGTCAAGCAG R: AGTTCATGAGCTGCAACACG F: ACAGCAGGTCTCAGCCTCAT R: TGAAGCCAAGCGGTCTAAGT F: GCAGTTTGCTCAGCCTATCC R: GAGTGTCGGAGTCCAGCTTC F: GAGTGCCAGATGTTGCAGAA R: AAATGCAGGGGGATCTCTTT F: TCTGCAAGGTTATCCCAAGG R: CTTGCTGGAAAACTGCATCA F: TCTGCAAGGTTATCCCAAGG R: CTTGCTGGAAAACTGCATCA F: CAGAGAAGGCACACAAACGA R: CACTGGTGAGACAGGCTTGA F: ACCCTTCCCAGTTCCTGTCT R: CTCCTTGCTTCCTCTGGATG F: GTTCCTGTGGAGCAAAGAGC R: CCAGCGTCCACCAACTTATT F: AGCAACATCTGGGGACAAAC R: CCCAGGTTTCAGTTTGCATT F: GCCTCTGTCTTCTGGACTGG R: TGGAAGGCAAACCAAGATTC F: AGCCTCAAAAAGGTGCTTCA R: CCCTGGTAGCAGCAGTTCTC

15 ADAM17/TACE ADAM19 ADAM28 Collagen type I α1 chain (col1α1) α-smooth muscle actin (α-sma) Glyceraldehyde 3- phosphate dehydrogenase Ephrin A1 Ephrin A2 Ephrin A3 Ephrin A4 Ephrin A5 Ephrin B1 Ephrin B2 Ephrin B3 EphB1 EphB2 F: TGAGGGCAGTTAACCAAACC R: ATACACCCACACACCCCACT F: CTGAAGGCTGTGGGAAGAAG R: ACCACAGGACCCACACTCTC F: TTCAGTCAGGGGGATCAAAG R: TAGTTTGGTGGCAGGTAGGG F: CCGTGCTTCTCAGAACATCA R: GAGCAGCCATCGACTAGGAC F: CTGACAGAGGCACCACTGAA R: CATCTCCAGAGTCCAGCACA F: AACTTTGGCATTGTGGAAGG R: GGATGCAGGGATGATGTTCT F: CCCACATTACGAGGACGACT R: GTGAAGCGCTGGAATTTCTC F: CTCCGAGACAAAATCCTTGC R: CTCCGAGACAAAATCCTTGC F: CCTGCACTGGAAGTGTCTGA R: GGGGATTCTCTCCCTCAAAG F: GCAAAGTCGGAGGATCTGAG R: GCCCTCAGTGTCTCAAGGAG F: TCCAGAGGGGTGACTACCAC R: CTTGAACCCTTTGGACGTGT F: ATCGCAAGCATACACAGCAG R: CTGGGCCTTCAAACCTTGTA F: ACAGGTGGGAGGTGACTGAC R: GCTGCGCTTTTTATTTCCAG F: TGTTCTCCCCACCTAACCAG R: GCACACTAAAGAGCGGGAAG F: GATTTTGGGGGAAGGTTGAT R: ATGGTTTCTGGGAACACTGC F: ACTATGGCGGCTGTATGTCC R: GCACATCCACTTCTTCAGCA

16 EphB3 EphB4 EphB6 F: CAAGGCTGGCTTCTACTTGG R: GAGACCTCCACAGCGTTAGC F: AATGTCACCACTGACCGTGA R: TCAGGAAACGAACACTGCTG F: CGAGAGGGCCAGTTCAGTAG R: GCGAAGCAAGGAACTTGAAC Genotyping primers Cre mice Cre-1s: GCATTACCGGTCGATGCAACGAGTGATG Cre mice Cre-2as: GAGTGACGAACCTGGTCGAAATCAGTCG Ephrin-B2 flox mice EPHB2_1s: TCATTTCCCAACCACCGCCAGAAA Ephrin-B2 flox mice EPHB2_2as: AGATACCACGCCAGGAGAGCAAAT Supplementary Table 2. Primer pairs used for quantitative real-time PCR. The table shows the specific gene detected, the species (mouse or human) and the corresponding sequence for forward (F) and reverse (R) primers.

WT Day 90 after injections

WT Day 90 after injections Supplementary Figure 1 a Day 1 after injections Day 9 after injections Klf5 +/- Day 1 after injections Klf5 +/- Day 9 after injections BLM PBS b Day 1 after injections Dermal thickness (μm) 3 1 Day 9 after