Effect of SK-216 on fibroblast to myofibroblast differentiation induced by TGF-β1 in human primary lung fibroblasts.

<p>After pre-incubation in serum free medium for 24 hours, ILD-derived and normal human primary lung fibroblasts were pretreated in the presence or absence of SK-216 (50, 150 μM) for 1 hour followed by stimulation with TGF-β1 (5 ng/ml). Real-time quantitative RT-PCR analysis and western blotting analysis were performed after incubation for 24 and 48 hours, respectively. (A) mRNA expression of α-SMA, fibronectin, and COL1A1 in ILD-derived human primary lung fibroblasts evaluated using real-time quantitative RT-PCR. Results are expressed as fold change from the control and reflect the mean ± SEM of 4 experiments. (B-C) Western blot analysis of α-SMA, fibronectin, and type I collagen in (B) ILD-derived and (C) normal human primary lung fibroblasts. Data normalized to β-actin levels are shown as fold change from the control and reflect the mean ± SEM of 3 independent experiments. *p <0.05, **p <0.01 vs untreated cells, ^#p <0.05, ^##p <0.01 vs cells treated with TGF-β1.</p

Effect of SK-216 on mRNA expression of epithelial or mesenchymal markers in LA-4 cells undergoing TGF-β-induced EMT.

<p>The cells were pre-incubated in the presence or absence of SK-216 (50, 100 μM) for 1 hour and then co-incubated with TGF-β1 (5 ng/ml). After incubation for 24 hours, the cells were harvested and total RNA was extracted. The mRNA expression shows E-cadherin as an epithelial marker, and fibronectin and α-SMA as mesenchymal markers using real-time quantitative RT-PCR. Results are expressed as fold change from the control and reflect the mean ± SEM of 4 experiments. *p <0.05, **p <0.01 vs untreated cells, ^#p <0.05, ^##p <0.01 vs cells treated with TGF-β1.</p

Effect of SK-216 on morphological transformation and the expression of epithelial or mesenchymal markers in A549 cells undergoing TGF-β-induced EMT.

<p>Cells were pre-incubated in the presence or absence of SK-216 (50, 100 μM) for 1 hour and then co-incubated with TGF-β1 (5 ng/ml). All assays were performed after 48 hours of incubation. (A) Phase contrast images of A549 cells. A549 cells untreated with TGF-β1 maintained epithelial cobblestone-shape with cell-cell contacts. The treatment with TGF-β1 transformed the cells into mesenchymal spindle-shape cells with loss of cell-cell adhesion. The co-incubation with SK-216 reversed these TGF-β-induced morphologic alterations. Magnification: ×200. Scale bar = 100 μm. (B) Proliferation of A549 cells after treatment with SK-216 and TGF-β1. (C) A549 cells stained for E-cadherin (green), vimentin (red) and nuclei (blue). Magnification: ×200. Scale bar = 100 μm. (D) Fluorescence intensities for E-cadherin and vimentin. Results are expressed as Box plot of at least 6 fields. (E) Western blot analysis of E-cadherin, vimentin, and N-cadherin. Data normalized to β-actin levels are shown as fold change from the control, and reflect the mean ± SEM of 3 independent experiments. (F) Measurement of total soluble collagen in the culture medium. Data are normalized to the amount of total protein in the cell lysate. Results are expressed as the mean ± SEM of 4 independent experiments. *p <0.05, **p <0.01 vs untreated cells, ^#p <0.05, ^##p <0.01 vs cells treated with TGF-β1.</p

Effect of SK-216 on BLM-induced pulmonary fibrosis in mice.

<p>Mice were allocated into three groups: PBS+DW group, BLM+DW group, BLM+SK-216 group. Mice were intratracheally instilled with PBS or PBS containing BLM (1.5 mg/kg body weight) on day 0. PBS+DW group; PBS intratracheally instilled and orally administered with distilled water, BLM+DW group; BLM intratracheally instilled and orally administered with distilled water, BLM+SK-216 group; BLM intratracheally instilled and orally administered with distilled water on days from 0 to 8 and distilled water containing SK-216 (1000ppm) on days from 9 to 21. (A) mRNA expression levels of PAI-1, α-SMA, and COL1A1 in lung. Left lung harvested on day 11 was analyzed using real-time quantitative RT-PCR. Results are expressed as fold change from the control. (B) Levels of active PAI-1 and TGF-β1 in BALF. BALF was collected on day 11. (C) The degrees of pulmonary fibrosis were analyzed by measuring hydroxyproline contents in the whole lungs on day 21. (D) Histological and immunohistochemical analysis of bleomycin-injured lung. Right lungs were excised and sectioned on day 21. Scale bar = 200 μm. Results are presented as the mean ± SEM of 6 mice per group. *p <0.05, **p <0.01 vs PBS+DW group, ^#p <0.05, ^##p <0.01 vs BLM+DW group.</p

Additional file 6 of Nestin and Notch3 collaboratively regulate angiogenesis, collagen production, and endothelial–mesenchymal transition in lung endothelial cells

Additional file 6. Figure S5

Effect of SK-216 on fibroblast to myofibroblast differentiation induced by TGF-β1 in MRC-5 cells.

<p>After pre-incubation in serum free medium for 24 hours, MRC-5 cells were pretreated in the presence or absence of SK-216 (50, 150 μM) for 1 hour followed by stimulation with TGF-β1 (5 ng/ml). Real-time quantitative RT-PCR analysis was performed after incubation for 24 hours. Proliferation assay, immunofluorescence staining, and western blotting analysis were performed after incubation for 48 hours. (A) Proliferation of MRC-5 cells after treatment with SK-216 and TGF-β1. (B) mRNA expression of α-SMA, fibronectin, and COL1A1 in MRC-5 cells evaluated using real-time quantitative RT-PCR. Results are expressed as fold change from the control and reflect the mean ± SEM of 4–5 experiments. (C) MRC-5 cells stained for α-SMA labeled with Zenon Alexa Fluor 488 (green) and nucleus labeled with DAPI (blue). Magnification: ×400. Scale bar = 50 μm. (D) Fluorescence intensities for α-SMA in MRC-5 cells. Results are expressed as Box plot of at least 6 fields. (E) Western blot analysis of α-SMA, fibronectin, and type I collagen. Data normalized to β-actin levels are shown as fold change from the control and reflect the mean ± SEM of 3 independent experiments. *p <0.05, **p <0.01 vs untreated cells, ^#p <0.05, ^##p <0.01 vs cells treated with TGF-β1.</p

Additional file 5 of Nestin and Notch3 collaboratively regulate angiogenesis, collagen production, and endothelial–mesenchymal transition in lung endothelial cells

Additional file 5. Figure S4

PAI-1 expression (A-B) and production of endogenous TGF-β1 (C) in A549 cells when TGF-β-induced EMT was blocked by SK-216.

<p>(A-B) The cells were pre-incubated in the presence or absence of SK-216 (50, 100 μM) for 1 hour and then co-incubated with TGF-β1 (5 ng/ml). After incubation for 24 hours, the cells and the supernatants of cell cultures were collected. (A) Active and total PAI-1 levels in the supernatants were measured using active and total PAI-1 ELISA kit. Results are expressed as the mean ± SEM of 4 samples. (B) PAI-1 mRNA expression in the cells was measured using real-time quantitative RT-PCR. Results are expressed as the mean ± SEM of 5 experiments. (C) The cells were pre-incubated in the presence or absence of SK-216 (100 μM) for 1 hour and then co-incubated with TGF-β1 (5 ng/ml). After incubation for 24 hours, the culture medium was replaced with serum-free medium. The supernatants of cell cultures were collected at 12, 24, and 48 hours after the medium change. Endogenous TGF-β1 was analyzed by measuring the concentration of TGF-β1 in the supernatants using TGF-β1 ELISA kit. Results are expressed as the mean ± SEM of 4 experiments. *p < 0.05, **p < 0.01, ***p < 0.001.</p

Additional file 4 of Nestin and Notch3 collaboratively regulate angiogenesis, collagen production, and endothelial–mesenchymal transition in lung endothelial cells

Additional file 4. Figure S3