Additional file 1: of Impaired diversity of the lung microbiome predicts progression of idiopathic pulmonary fibrosis

Impaired Diversity of the Lung Microbione Predicts Progression of Idiopathic pulmonary Fibrosis. Table S1. Overview of DNA sequencing data. Figure S1. Comparison of the total bacterial genes between the AE and non-AE groups. Figure S2. Rarefaction curves were calculated, which suggests that all expected OTUs have been obsreved. Figure S3. the concentration of SP-A in BALF correlates well with the relative abundance of Veillonellaceae, but not serum SP-A. Figure S4. The relative abundance of family Streptococcaceae also correlated with 6MWD. Figure S5. Histopathological assesment and BAL cell counts in mice with the bleomycin-induced lung fibrosis. (PDF 7772 kb

The life span of the <i>Da-GAL4>UAS-TG IR</i> flies.

<p>(A) The life span of the RNAi flies was compared with those of the control flies, <i>Da-GAL4>+</i>, <i>Da-GAL4>UAS-lacZ IR</i> and <i>+>UAS-TG IR</i>. Sixty adult flies were collected and maintained at 25°C. The number of surviving flies was recorded daily. The means ± S. D. of four independent experiments were plotted. <i>Da</i>, <i>Da-GAL4</i>. (B) Phenotypes of <i>Tub-GAL80^ts; Da-GAL4>UAS-TG IR</i>. <i>Tub-GAL80^ts; Da-GAL4</i> flies were crossed with the <i>UAS-TG IR</i> flies in 20 vials and maintained at 18°C. The suppression of <i>TG</i> by RNAi was triggered by increasing the temperature to 29°C. The ratios flies with abnormal wings (square) and abnormal abdominal cuticles (circle) to total adult flies are indicated (upper panel). The number of adult flies born from each vial is indicated (lower panel).</p

Identification of TG substrates associated with cuticle formation.

<p>The wings of wild-type and <i>Da-GAL4>UAS-TG IR</i> flies were collected at indicated times after eclosion. Wing proteins were extracted and subjected to SDS-PAGE. TG antigen was detected by Western blotting (upper panels). Loaded proteins were stained with Coomassie Brilliant Blue R-250 (lower panels). <i>Da, Da-GAL4.</i></p

Phenotypes of TG substrate RNAi flies.

<p>Phenotypes of the <i>MS1096-GAL4>UAS-Cpr97Eb IR</i> (A), <i>MS1096-GAL4>UAS-Clect27 IR</i> (B), <i>Da-GAL4>UAS-LSP2 IR</i> (D) and <i>Da-GAL4>UAS-Cpr76Bd IR</i> (E) flies. The control flies, <i>MS-GAL4</i>>+ (C) and <i>Da-GAL4</i>>+ (F), are also indicated. Each fly was laid at 25°C. <i>MS, MS1096-GAL4; Da, Da-GAL4</i>.</p

The effect of wounding on <i>TG</i> expression.

<p>Wild-type flies were injured using a steel pin. Flies were collected at the indicated times and homogenized. (A) TG antigens at the indicated times were detected by Western blotting (upper panel). β-tubulin was detected by Western blotting as control (lower panel). (B) The amount of TG after the wounding was determined by enzyme-linked immunosorbent assay. The means ± S. D. of three independent experiments were plotted. A significant difference (versus 0 h) is indicated by asterisk (<i>P</i><0.05 after Bonferroni correction). (C) TG activities were measured by the monodansylcadaverine incorporation at 1 and 4 h after wounding. The means ± S. D. of three independent experiments were plotted. A significant difference (versus 0 h) is indicated by asterisk <i>(P</i><0.05).</p

Phenotypes of <i>TG</i>-RNAi flies.

<p>(A) TG antigen from whole body extract of adult <i>TG</i>-RNAi flies was detected by Western blotting. <i>w¹¹¹⁸</i>, <i>Da-GAL4>+</i> and <i>Da-GAL4>UAS-LacZ IR</i> were used as controls. <i>Da</i>, <i>Da-GAL4</i>. (B) Phenotypes of <i>TG</i>-RNAi flies for the wing (left panels) and abdominal cuticle (right panels) were classified into three grades depending on the extent of observed abnormality. The ratios of abnormal flies to total adult flies are indicated. Each fly was laid at 25°C. (C) Scanning electron microscopy of <i>TG</i>-RNAi fly. Scale bar = 200 µm.</p

Stage-specific expression of <i>TG</i>.

<p>(A) The wild-type flies were collected at indicated developmental stages and homogenized. TG antigens were detected by Western blotting (upper panel). β-tubulin was detected by Western blotting as a control (lower panel) with a mouse anti-tubulin antibody. (B) TG activity was assayed by the incorporation of Bi-PA into <i>N, N'</i>-dimethylcasein. The means ± S. D. of three independent experiments were plotted.</p

Acid-extractable wing proteins from <i>TG</i>-RNAi flies identified by mass spectrometry.

<p>RFABP, retinoid and fatty acid binding protein; NP, no phenotypic difference observed.</p

Binding of Cpr97Eb and Clect27 proteins to chitin.

<p>Cpr97Eb and Clect27 proteins were mixed with chitin, and unbound (UB) and bound (B) fractions were subjected to SDS-PAGE. The bound fraction was eluted by 2% SDS. Z2A was used as negative control for chitin binding. Proteins were detected by Coomassie Brilliant Blue R-250 staining (Clect27 and Z2A) or by anti-6×His tag antibody (Cpr97Eb).</p

TG-dependent incorporation of Bi-PA to Cpr97Eb and Clect27 proteins.

<p>Cpr97Eb (A) and Clect27 (B) proteins were incubated with or without Bi-PA in the presence of TG. The incorporation of Bi-PA was detected with biotinylated streptavidin-HRP (upper panel). Z2A (C) is a recombinant version of horseshoe crab β-1,3-D-glucan-binding protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013477#pone.0013477-Ueda1" target="_blank">[43]</a>, which was used as negative control for the TG-dependent incorporation. Loaded recombinant proteins were detected by Western blotting with an anti-6×His tag antibody or anti-Z2A antibody (lower panels).</p