Fig. 6: Restoration of C/EBPα rescues epithelial phenotype. | Nature Communications

Fig. 6: Restoration of C/EBPα rescues epithelial phenotype.

From: C/EBPɑ is crucial determinant of epithelial maintenance by preventing epithelial-to-mesenchymal transition

Fig. 6

a Schematic representation of the experimental set up. HMLE cells were treated with 2.5 ng/ml of TGF-β for 15 days, and posteriorly C/EBPα expression was induced upon 200 ng/ml of doxycycline for 64 h. b The results from qRT-PCR showing the expression levels of known EMT markers in EV or CEBPA-inducible HMLE cells treated with TGF-β, doxycycline or both. Data represented as mean ± SD of four independent experiments. p-values were calculated using two-way ANOVA with Tukey’s multiple comparisons test. **p < 0.01, ***p < 0.001, ****p < 0.0001. ns not significant. c Immunoblotting analysis of the expression levels of well-stablished EMT markers in TGF-β-treated HMLE cells in the absence or presence of C/EBPα overexpression. Data are representative of three independent experiments. d Confocal microscopy visualization of MCF10A–epithelial spheroids treated with 5 ng/ml of TGF-β for 4 days. Data are representative of three independent experiments. Scale bar: 45 μm. e Schematic representation of the 3D spheroid-formation assay. MCF10A cells expressing empty vector (EV) or doxycycline-inducible C/EBPα were stimulated with TGF-β (5 ng/ml), doxycycline (200 ng/ml), or both as indicated. f Confocal microscopy visualization of epithelial spheroids using MCF10A cells expressing empty vector or doxycycline-inducible C/EBPɑ in the presence of TGF-β, doxycycline (DOX), or both. Cells were stained for C/EBPa (green) and phalloidin (white). DAPI was used to visualize the nucleus (blue). Data are representative of three independent experiments. Scale bar: 45 μm. g Quantification of the number of spheres formed in MCF10A cells expressing empty vector or doxycycline-induced C/EBPɑ upon the treatment of TGF-β, doxycycline, or both. The number of spheres are relative to empty vector untreated. Data are represented as mean ± SD of three independent experiments. p-values were calculated using two-way ANOVA with Tukey’s multiple comparisons test. **p < 0.01, ****p < 0.0001. β2m beta-2-microglobulin, EV empty vector, UNT untreated.

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