Induction of mesenchymal markers by CSE is HIF1α dependent.

<p>Relative E-cadherin (A), PAI1 (B) and vimentin (C) mRNA levels after 48 hours of CSE stimulation (A549 2.5% CSE and BEAS2B 1.0% CSE) compared to control in A549 and BEAS2B cells stably transfected with PLKO.1 empty vector and shRNA HIF1α construct. Western blots and quantification of A549 en BEAS2B cells stably transfected with PLKO.1 empty vector and shRNA HIF1α construct after stimulation with for 48 hours for E-cadherin (D–E), vimentin and fibronectin (F–G) with GAPDH as loading control. Collagen in the medium, measured using sircoll assay (H). Adhesion in A549(I) and BEAS2B cells (J). Data are expressed as mean+SD, * indicates p<0.05 compared to control, ** indicates p<0.05 compared to empty vector.</p

The TGF-β signaling pathway is not involved in CSE-induced EMT.

<p>Submerged grown A549 cells were treated for 24 h with 2.5% CSE. Levels of TGF-β mRNA (A), SMAD reporter luciferase activity corrected for levels of β-galactosidase activity (B), SMAD2 phosphorylation as analyzed and corrected for total SMAD2 by Western blot (after stimulation with 2.5% CSE or 10 ng/mL TGF-β) (C) and SMAD7 mRNA (D) are shown. Effect of inhibition of the TGF-β signaling was studied using the receptor blocker SB431542 which was pre-incubated at a dose of 10 µM for 30 minutes followed by CSE stimulation for 48 hours. E-cadherin (E) and PAI1 (F) mRNA levels were measured by qPCR. Data are expressed as mean+SD, * indicates p<0.05 between CSE stimulated and untreated control.</p

CSE increased mesenchymal markers in lung epithelial cells.

<p>Cells were treated for 48 hours with respectively 2.5% CSE for A549 cells and 1.0% CSE for BEAS2B cells. mRNA levels of PAI-1 and vimentin were measured in A549 cell submerged (A), A549 cells at ALI (B) and BEAS2B cells (C) via qPCR. Data are expressed as mean+SD, * indicates p<0.05 compared to untreated controls. SNAIL staining of untreated control A549 cells (D) and A549 cells stimulated with CSE for 48 hours (E) as detected by immunofluorescence (red). Nuclei are counterstained using DAPI. Vimentin and fibronectin Western blot and quantification of untreated controls and 48 hours CSE treated A549 cells (F) and BEAS2B cells (G) in triplicate using GAPDH as a loading control. Collagen in the medium, measured using sircoll assay (H), Adhesion of A549 and BEAS2B cells (I) and leakage of A549 cells grown ALI after CSE exposure (J).</p

Primer sequences.

<p>Primer sequences.</p

Hypoxia induces mesenchymal markers.

<p>A549 cells were cultured under normoxic or hypoxic conditions (4% O₂) for 24 or 48 hours. Vimentin (A) and PAI1 (B) mRNA levels were measured by qPCR and are expressed as mean+SD, * indicates p<0.05 compared to control. C57BL/6J mice were exposed to normoxia (n = 8) or hypoxia (n = 7) for 21 days and additionally a pair-fed group (n = 8) was used. Lung fibronectin (C) and PAI1 (D) mRNA levels were determined by qPCR. Data are expressed as mean, * indicates p<0.05.</p

CSE decreased epithelial markers in lung epithelial cells.

<p>Cells were treated for 48 hours with respectively 2.5% CSE for A549 cells and 1.0% CSE for BEAS2B cells. mRNA levels of E-cadherin and Keratin 18 were measured in A549 cell submerged (A), A549 cells at ALI (B) and BEAS2B cells (C) via qPCR. Data are expressed as mean+SD, * indicates <i>p</i><0.05 compared to untreated controls. ZO-1 staining (red) of untreated A549 cells (D) and A549 cells treated for 48 hours with CSE (E) detected by immunofluorescence. Nuclei are counterstained using DAPI. E-cadherin and ZO-1 Western blots and quantification of untreated controls and A549 cells treated for 48 hours with 2.5% CSE (F) and BEAS2B with 1.0% CSE (G) in triplicate using GAPDH as a loading control.</p