Supplementary Material
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Supplementary material ERJ-00386-2017_Supplement
Figure S1. Mechanical stretch machine. a) Top-down schematic representation of the ex vivo model to apply mechanical stretch to lung strips. b) Photograph of the ex vivo model to apply mechanical stretch. ERJ-00386-2017_Figure_S1
Figure S2. Circulating HSP90α is upregulated in IPF patients and in AdTGF-β1 induced lung fibrosis. a) Serum level of HSP90β measured by ELISA on IPF patients and respective aged-matched healthy volunteers (Left panel). Results are presented as mean±SEM, n=9 and n=31 for controls and IPF patients respectively. IPF patients have been clustered in 2 groups (right panel): moderate IPF (FVC % predicted >60%) and severe IPF (FVC % predicted <60%). Results are presented as mean±SEM. ***p<0.001, n=9, n=25 and n=6 for controls, moderate IPF and severe IPF patients respectively. b) Western blot analysis of HSP90 expression in BALF of rats treated with AdTGF-β1 (AdDL=control). WB presented is representative of 3 independent experiments. c) Correlation curves between the serum and BALF level of HSP90α in rats treated with AdTGF-β1 and Ashcroft scores of corresponding lungs. ERJ-00386-2017_Figure_S2
Figure S3. Mechanical stretch and tissue stiffness induce HSP90α secretion. A. Western blot analysis of HSP90α and HSP90β expression in cells’ supernatant of untreated control/IPF primary lung fibroblasts and A549 treated or not with rTGF-β1. WB presented are representative of 3 independent experiments. Ponceau staining served as loading control. b) Correlation curves between the levels of HSP90α released by fibrotic lung strips after stretch and Youngâs modulus (stiffness) of corresponding lung strips. ERJ-00386-2017_Figure_S3
Figure S4. HSP90α does not induce pro-fibrotic markers in IPF fibroblasts Western blot analysis (and corresponding densitometry) of collagen 1A, p-ERK, ERK, p-Smad2, Smad2 and α-SMA on human primary IPF fibroblasts treated with rHSP90α or vehicle (ctrl) for 48 h at 10 μM. GAPDH staining served as loading control. Results are presented as mean±SEM, n=4. ERJ-00386-2017_Figure_S4
Figure S5. rHSP90α does not interact with TGFβRI. a) Immunoprecipitation (IP) of HSP90α performed on plasma membrane extracted proteins followed by immuno-detection of TGFβRI and HSP90α. IP was performed with prior crosslinking of extracellular/membrane proteins. IP performed on IPF fibroblasts treated with or without rHSP90α for 48 h at 10 μM. IgG: non-relevant antibody; input: non-immunoprecipitated extracts. ERJ-00386-2017_Figure_S5
Figure S6. HSP90α signaling in IPF fibroblasts is independent of TGF-β1 pathway. a) Western blot analysis of TGF-β1, α-SMA, p-ERK and ERK expression on human primary control fibroblasts treated with rHSP90α for 48 h at 10 μM and with or without SD-208, an ALK5 inhibitor at 30 μM. GAPDH staining served as loading control. WB presented is representative of 3 independent experiments. b) Total and active TGF-β1 levels measured by ELISA on control fibroblasts treated with rHSP90α for 48 h at 10 μM and with or without SD-208, an ALK5 inhibitor at 30 μM. Results are presented as mean±SEM, **p<0.01, ***p<0.001, n = 5. c) Total and active TGF-β1 levels measured by ELISA on control fibroblasts treated with rHSP90α for 48 h at 10 μM and with or without HS-30 at 1 mM. Results are presented as mean±SEM, **p<0.01, ***p<0.001, n = 5. ERJ-00386-2017_Figure_S6