WISP1 mediates IL-6-dependent proliferation in primary human lung fibroblasts
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1 WISP mediates IL-6-dependent proliferation in primary human lung fibroblasts Klee S, Lehmann M, Wagner DE, aarsma H, Königshoff M Comprehensive Pneumology Center, Helmholtz Zentrum München, Munich, Germany; Member of the German Center of Lung Research (DZL) and Ludwig-Maximilians-Universität, University Hospital Grosshadern Corresponding author: melanie.koenigshoff@helmholtz-muenchen.de Max-Lebsche Platz 879 Munich Germany Tel: Fax:
2 WISP mrn expression (Fold of control) WISP mrn expression (Fold of control) # ## ## TGFβ (ng/ml) S SC- U6 TNFα (ng/ml) SC- SP6 7-Z- Oxozeaneol Supplementary Figure : TGFβ- and TNFα induce WISP in phlfs via NF-κ. The phlfs were treated for hours with either () ng/ml TGFβ in the absence or presence of the LK inhibitor S ( µm), the IKKβ inhibitor SC- ( µm) or the MEK/ inhibitor U6 ( µm) or () with ng/ml TNFα in the absence or presence of the IKKβ inhibitor SC- ( µm), the JNK inhibitor SP 6 ( µm) or the TK inhibitor 7-Z-Oxozeanol ( nm). (n=;,# p<.; student s t-test)
3 IL6 mrn expression IL6 mrn expression WISP mrn expression WISP mrn expression 8 6 # 6 TGFβ sictrl siwisp TNFα sictrl siwisp C D # TGFβ sictrl siwisp TNFα sictrl siwisp Supplementary Figure : WISP is required for IL6 induction in phlfs derived from IPF patients. The phlfs were treated for hours with either (,C) ng/ml TGFβ or (,D) with ng/ml TNFα in the absence or presence of the sirn targeting WISP (siwisp) or a non-targeting control sirn (sictrl). Effects on the expression of (,C) WISP and (,D) IL6 were analyzed using qpcr (n=;,# p<.; p<.;, p<.; -way nova with Newman-Keuls Multiple comparison testing).
4 IL8 mrn expression (Fold of control) IL-8 Secretion (pg/ml) 8 6 TNFα sictrl siwisp TNFα sictrl siwisp h 8h h 8h Supplementary Figure : Production of IL-8 upon TNFα stimulation is not affected by the absence of WISP in phlfs. The phlfs were treated for and 8 hours with with ng/ml TNFα in the absence or presence of the sirn targeting WISP (siwisp) or a non-targeting control sirn (sictrl). () The expression of IL8 was measured by qpcr and () the secretion of IL-8 was analyzed using a multiplex ELIS (n=; p<.; p<.; -way nova with Newman-Keuls Multiple comparison testing).
5 WST- (% of sictrl) WISP mrn expression (Fold of control) WISP mrn expression.. n.s... control IL-6 sictrl h siwisp sictrl siwisp 8h C sictrl h siwisp sictrl 8h siwisp Supplementary Figure : WISP is not induced by IL-6 stimulation in phlfs. () The phlfs were treated for hours with ng/ml IL-6 or (,C) transfected with a WISP-silencing sirn (siwisp) or a control sirn (sirn) and kept in.% FCS-containing medium according to the stimulation protocol for and 8h. (,) The expression of WISP was measured by qpcr and (C) the metabolic activity of the phlfs was measured by WST- assay (n=7; p<.; -way nova with Newman-Keuls Multiple comparison testing).
6 WST- (% of DMSO control) IL6 mrn expression (Fold of DMSO control) IL6 mrn expression (Fold of DMSO control) DMSO SC TGFβ DMSO SC TNFα C DMSO SC- Supplementary Figure : The IKKβ inhibitor SC- abrogates the expression of IL6 and reduces proliferation in phlf. The phlfs were treated for hours with () ng/ml TGFβ or () ng/ml TNFα in the absence or presence of the IKKβ inhibitor SC- (µm) and the expression of IL6 was measured by qpcr. (C) Moreover, treatment of phlfs with SC- (µm) for 8 hours and proliferation was assessed by WST- assay (n=;, p<., -way nova with Newman-Keuls Multiple comparison testing for () and (); p<., students t-test for (C))
7 DL CO (% predicted) FVC (% predicted) p<. r =. p=. r =.7 6 WISP (arbitrary units) 6 WISP (arbitrary units) Supplementary Figure 6: WISP mrn levels negatively correlate with the diffusion capacity of the lung for carbon monoxide (DL CO ) and the forced vital capacity (FVC) in human patients. The correlation of WISP and () the predicted DL CO (N=68) or () the FVC (N=8) showed a significantly negative linear correlation. (dashed line = 9% CI; Data extracted from the LGRC GSE76 GPL68)
8 TGFβ treatment sictrl (pg/ml) siwisp (pg/ml) sictrl + TGFβ (pg/ml) siwisp + TGFβ (pg/ml) IL-6 IL-8 MCP IFNγ GM-CSF n.d. n.d. n.d. n.d. IL-7 TNFα GCSF IL- MIPb n.d. n.d. n.d. n.d. IL- n.d. n.d. n.d. n.d. IL-7 n.d. n.d. n.d. n.d. IL- n.d. n.d. n.d. n.d. IL- n.d. n.d. n.d. n.d. IL- n.d. n.d. n.d. n.d. IL- n.d. n.d. n.d. n.d. IL-b n.d. n.d. n.d. n.d. Supplementary Table : The secretion of different cytokines by phlfs was measured after TGFβ ( ng/ml) stimulation in vitro for hours using a multiplex ELIS kit (io-plex Pro Human Cytokine 7-plex). (n=;,# p<.; -way NOV followed by Neuman-Keuls multiple comparison test)
9 TNFα treatment sictrl (pg/ml) siwisp (pg/ml) sictrl + TNFα (pg/ml) siwisp + TNFα (pg/ml) IL-6 IL-8 MCP IFNγ GM-CSF n.d. n.d. IL-7 TNFα GCSF IL- MIPb n.d. n.d. IL- n.d. n.d. IL-7 n.d. n.d. IL- n.d. n.d. n.d. n.d. IL- n.d. n.d. n.d. n.d. IL- n.d. n.d. n.d. n.d. IL- n.d. n.d. n.d. n.d. IL-b n.d. n.d. n.d. n.d. Supplementary Table : The phlfs were treated with ng/ml TNFα for hours in vitro and different cytokines were measured using a multiplex ELIS kit (io-plex Pro Human Cytokine 7-plex) (n=;,# p<.; -way NOV followed by Neuman-Keuls multiple comparison test)
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