Supplementary Material
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Supplementary methods ERJ-04361-2020.Methods
Supplementary figure S1. a) GelMa substrates (5, 10 and 15% GelMa wt/vol) were subjected to compressive modulus using texture analyser to determine the relative stiffness. 5% GelMa was approximately equivalent to the described stiffness of tracheal smooth muscle [22] and so was denoted as 1x stiffness. b) TGFB1 mRNA was determined by qRT-PCR. Data is expressed as median fold change versus mean of the non-asthmatic group. Non-parametric Mann Whitney test was used. c) TGFB2 mRNA was determined by qRT-PCR. Data is expressed as median fold change versus mean of the non-asthmatic group. Non-parametric Mann Whitney test was used. d) TGFB3 mRNA was determined by qRT-PCR. Data is expressed as median fold change versus mean of the non-asthmatic group. Non-parametric Mann Whitney test was used. ERJ-04361-2020.Figure_S1
Supplementary figure S2. a) Basal traction force was determined using traction force microscopy. Data is presented as median root mean square traction (n) across all donor cell lines tested. Non-parametric Mann Whitney test was used. b) ASM cells were subjected to stretch (15%, 0.3Hz (S)) or left unstretched (U) for 4 h. PSmad2, total Smad2/3 and GAPDH were measured by western blotting. Figure shown is representative of n=6 non-asthmatic and n=6 asthmatic donor cell lines. Densitometrical analysis of all blots was performed and data is expressed as ratio PSmad2:tSmad2/3. Kruskall Wallis non-parametric test with Dunn’s multiple comparison test was used. c) Data from figure 2e are expressed here in a format that allows comparison of the effect of ECM on basal TGF-β activity for both non-asthmatic and asthmatic ASM cells. Mann Whitney was used to test for a difference between the ECM effect delta between non-asthmatic and asthmatic cells (p=0.0159). ERJ-04361-2020.Figure_S2
Supplementary figure S3. a) TGM2 mRNA in ASM cells was determined by qRT-PCR. Data is expressed as median fold change versus the mean data from the non-asthmatic group. Non-parametric Mann Whitney test was used, b) LOX mRNA in ASM cells was determined by qRT-PCR. Data is expressed as median fold change versus the mean data from the non-asthmatic group. Non-parametric Mann Whitney test was used. c) LOXL1 mRNA in ASM cells was determined by qRT-PCR. Data is expressed as median fold change versus the mean data from the non-asthmatic group. Non-parametric Mann Whitney test was used. d) LOXL4 mRNA in ASM cells was determined by qRT-PCR. Data is expressed as median fold change versus the mean data from the non-asthmatic group. Non-parametric Mann Whitney test was used. e) Densitometrical analysis of LOXL3 western blots (figure 3c). Data is expressed as median ratio LOXL3:GAPDH. Non-parametric Mann Whitney test was used. ERJ-04361-2020.Figure_S3
Supplementary figure S4. a) Starting body mass data from mice in the in vivo model of asthma. Non-parametric Kruskall-Wallis with Dunn’s multiple comparison test was used. b) The median percentage of eosinophils within the inflammatory cell population of BALF (figure 6b) was determined by differential cell count. Non-parametric Kruskall-Wallis with Dunn’s multiple comparison test was used. c) The median percentage of neutrophils within the inflammatory cell population of BALF (figure 6b) was determined by differential cell count. Non-parametric Kruskall-Wallis with Dunn’s multiple comparison test was used. d) The median percentage of lymphocytes within the inflammatory cell population of BALF (figure 6b) was determined by differential cell count. Non-parametric Kruskall-Wallis with Dunn’s multiple comparison test was used. e) The median percentage of macrophages within the inflammatory cell population of BALF (figure 6b) was determined by differential cell count. Non-parametric Kruskall-Wallis with Dunn’s multiple comparison test was used. f) Representative negative control image for α-SMA immunofluorescence staining in figure 7b. g) Representative images of picro-Sirius red stained mouse lung tissue from the ovalbumin model. Tissue from animals treated with vehicle control was imaged using polarised light microscopy (i) and under brightfield light (ii). Similarly, tissue from animals treated with the LOXL2 inhibitor (PAT1251) was imaged using polarised light microscopy (i) and under brightfield light (ii). ERJ-04361-2020.Figure_S4
Supplementary table S1. Table detailing materials used in the manuscript, including suppliers and clone details of all antibodies used. ERJ-04361-2020.Table_S1
Supplementary table S2. Table describing primer sequences for qRT-PCR. All primers were designed using Primer3 online software. ERJ-04361-2020.Table_S2