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Figures
- Fig. 1.
Dynamic mRNA and protein expression changes during ATII-to-ATI cell trans-differentiation in vitro. (A) Immunofluorescence staining of pmATII cells on coverslips for epithelial cell marker expression at day 2 after isolation. Fluorescent images represent a 400× magnification. The scale bar represents 50 µm. (B) mRNA expressions of epithelial cell markers during the culture of pmATII cells over a period of 5 days. mRNA levels were measured by quantitative RT-PCR (qRT-PCR) and normalized to Hprt as housekeeping gene. Data represent means of ΔCt values+s.e.m. of at least three independent experiments. (C) Protein expression of epithelial markers in cultured pmATII cells. Cells were lysed at the indicated time points and 15 µg of total protein per sample was subjected to immunoblot analysis. β-actin expression served as loading control. A representative experiment and a densitometric analysis of at least three independent experiments are shown. Means at indicated time points were compared to day 1 (d1) using one-way ANOVA, followed by Dunnett's post-hoc test. Significance: *P<0.05; **P<0.01; ***P<0.001.
- Fig. 2.
CBR2, ENO1 and PDIA3 are differentially expressed during ATII-to-ATI cell trans-differentiation. pmATII cells were isolated and cultured for the indicated time points (days). (A) Protein profile of primary mouse alveolar epithelial cells cultured for 1, 3 or 5 days was generated by subjecting 30 µg total protein to a 2D-PAGE. Circles mark protein spots which were identified and quantified by MALDI-TOF mass spectrometry and are differently expressed at the indicated time points. A representative image of three independent experiments for each time point is shown. (B) mRNA expression of Cbr2, Eno1 and Pdia3 was determined by qRT-PCR and normalized to Hprt. Data represent means of ΔCt values+s.e.m. of at least three independent experiments. (C) Protein expression was determined by subjecting 15 µg of total protein per sample to immunoblot analysis. β-actin expression served as loading control. A representative experiment and a densitometric analysis of at least three independent experiments are shown. Means at indicated time points were compared to day 1 (d1) using one-way ANOVA, followed by the Dunnett's post-hoc test. Significance: *P<0.05; **P<0.01; ***P<0.001.
- Fig. 3.
Wnt/β-catenin pathway is activated during ATII-to-ATI cell trans-differentiation. pmATII cells were isolated and cultured for the indicated time points (days). (A,B) Cells were lysed using T-Per lysis buffer containing protease inhibitors and 15 µg of total protein per sample was subjected to immunoblot analysis. β-actin expression served as loading control. A representative experiment and a densitometric analysis of at least three independent experiments is shown. ABC, active β-catenin. (C) mRNA expression of Wnt/β-catenin target genes. (D) mRNA expression of canonical Wnt ligands. mRNA expression was measured by qRT-PCR and normalized to Hprt. Data represent means of ΔCt values+s.e.m. of at least three independent experiments. Means at indicated time points were compared to day 1 (d1) using one-way ANOVA, followed by the Dunnett's post-hoc test. Significance: *P<0.05; **P<0.01; ***P<0.001.
- Fig. 4.
β-catenin inhibition alters ATII-to-ATI cell trans-differentiation along with CBR2, ENO1 and PDIA3 expression. (A) pmATII were treated with PKF115-584 (1 µM) or DMSO as control at day 1 after isolation until day 3 and day 5, respectively. Treated cells were lysed and subjected to immunoblot analysis. β-actin expression served as loading control. A representative experiment is shown. (B) Densitometric analysis of at least three independent experiments using PKF115-584 treatment. Means of the indicated groups were compared to time-matched treatment controls using one-way ANOVA, followed by Bonferroni multiple comparison test. Significance: **P<0.01; ***P<0.001; ns, not significant. (C) pmATII cells were transfected using an siRNA pool targeting Ctnnb1 and a scrambled (siScr) control sequence, respectively. Non-transfected cells served as additional control. At day 5 cells were lysed and subjected to immunoblot analysis. A representative experiment is shown. (D) Quantification of at least three independent experiments of Ctnnb1 siRNA treatments. Means were compared to time-matched transfection control (siScr), using one-way ANOVA, followed by Bonferroni multiple-comparison test. Significance: *P<0.05; ***P<0.001.
- Fig. 5.
ENO1, PDIA3 and CBR2 expression is altered in injured pmATII cells. Mice were instilled with either PBS or bleomycin (BLEO) (5 U/kg body weight). At day 7 or day 14 after instillation, mice were sacrificed and lungs of four PBS- and four bleomycin-treated mice were pooled for pmATII cell isolation. (A) Freshly isolated pmATII cells (day 0) were analyzed for mRNA expression of Cbr2 and Sftpc. (B) Correlation analysis of mRNA expression of Cbr2 and Sftpc using a linear regression model. Data points represent ΔCt values for the respective genes. The corresponding regression line is indicated in red. 95% confidence intervals are depicted by a gray scattered line. r2 and P-value is given in the graph for the compared variables. (C) Eno1, Pdia3 and T1α mRNA expression is shown using qRT-PCR. Means of the indicated groups were compared using one-way ANOVA, followed by Bonferroni multiple-comparison test. Significance: *P<0.05; **P<0.01; ***P<0.001; ns, not significant. (D) Protein expression of the indicated proteins in freshly isolated pmATII cells from PBS-treated or BLEO-treated mice at day 14 after isolation. β-actin expression served as loading control.
- Fig. 6.
The siRNA-mediated knockdown of ENO1 and PDIA3 inhibits ATII-to-ATI cell trans-differentiation. pmATII cells were transfected at day 2 using an siRNA pool targeting Eno1, Pdia3 and a scrambled (siScr) control sequence, respectively. Non-transfected cells served as additional control. At day 5 cells were lysed and subjected to immunoblot analysis. Knockdown efficiency at day 5 was determined by detection of ENO1 and PDIA3 protein, respectively. A representative experiment and a quantification of three independent experiments of T1α expression upon (A) ENO1 and (B) PDIA3 knockdown are shown. Means were compared to time-matched transfection control (siScr) using one-way ANOVA followed by Bonferroni multiple-comparison test. Significance: *P<0.05; ***P<0.001. (C) Viability of pmATII cells transfected with siScr, siEno1 or siPdia3, respectively. Analysis was performed at day 3 and day 5 using the WST-1 assay. Data were measured in triplicates and were normalized to untreated control cells at the respective time points.
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