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1.
Figure 6

Figure 6. PDCD4 and α-SMA immunofluorescence of TGF-β-incubated EMCs.. From: miR-21 Promotes Fibrogenic Epithelial-to-Mesenchymal Transition of Epicardial Mesothelial Cells Involving Programmed Cell Death 4 and Sprouty-1.

EMCs were pre-incubated for 20h in TGF-β, then co-transfected with anti-miR-21 (or anti-miR control) and/or PDCD4-specific siRNA (or siRNA control; “α-CTL”), and stained after 24h. Control for immunofluorescence is in Figure 4A.

Hasse Brønnum, et al. PLoS One. 2013;8(2):e56280.
2.
Figure 7

Figure 7. Concepts of cardiac fibrogenic EMT.. From: miR-21 Promotes Fibrogenic Epithelial-to-Mesenchymal Transition of Epicardial Mesothelial Cells Involving Programmed Cell Death 4 and Sprouty-1.

Adult EMCs undergo EMT to generate fibroblast-like cells through up-regulation of miR-21, which in turn directly targets, and hence suppresses, PDCD4 and SPRY1. On the other hand, PDCD4 and SPRY1 can regulate various EMT biomarkers through miR-21-dependent mechanisms.

Hasse Brønnum, et al. PLoS One. 2013;8(2):e56280.
3.
Figure 1

Figure 1. Clonogenic cultures of adult rat EMCs are homogenous with respect to epicardial marker expression.. From: miR-21 Promotes Fibrogenic Epithelial-to-Mesenchymal Transition of Epicardial Mesothelial Cells Involving Programmed Cell Death 4 and Sprouty-1.

A, Phase pictures of isolated and expanded EMCs in culture (500 µm and 100 µm scale, respectively). B and C, Expression of epicardial markers (Wilms Tumor protein homolog (WT1), E-cadherin, Tbx18, Raldh2, and Islet-1) and the cardiomyogene marker Nkx2.5 in cultured EMCs. Relative qRT-PCR data (means+SD, n = 3) were normalized against GAPDH and RPL13A, and depicted relative to an adult (Ad.), embryonic (Em.), or neonatal (Neo.) rat heart. Rat ventricular fibroblasts were used as negative controls for immunocytochemistry (data not shown).

Hasse Brønnum, et al. PLoS One. 2013;8(2):e56280.
4.
Figure 4

Figure 4. PDCD4 and SPRY1 are miR-21 targets in TGF-β-mediated EMT of EMCs.. From: miR-21 Promotes Fibrogenic Epithelial-to-Mesenchymal Transition of Epicardial Mesothelial Cells Involving Programmed Cell Death 4 and Sprouty-1.

A, TGF-β stimulated EMC cultures were examined after 48 h for expression of PDCD4/α-SMA or SPRY1/α-SMA by immunofluorescence. qRT-PCR detection of PDCD4 (B), SPRY1 (C), and RECK (D) following TGF-β as well as pre-miR-21 and anti-miR-21 (“α-miR-21”) transfection, the latter performed during TGF-β incubation. Expression values (means+SD, n = 3) are displayed relative to the corresponding treatment controls (medium, pre-miR scramble, or anti-miR scramble) as visualized by the dotted lines intersecting “1”. Expression data were normalized against GAPDH and RPL13A, and statistical significance was tested by a two-tailed t-test. *P<0.05, **P<0.01 vs. the control. E, PDCD4 protein amounts (mean+SD, n = 3) were evaluated by Western blotting, and band intensities were quantified and standardized with corresponding bands from a Coomassie staining (total protein; not shown).

Hasse Brønnum, et al. PLoS One. 2013;8(2):e56280.
5.
Figure 2

Figure 2. Fibrogenic EMT of EMCs is induced differentially by IL-1β, TNF-α, and TGF-β.. From: miR-21 Promotes Fibrogenic Epithelial-to-Mesenchymal Transition of Epicardial Mesothelial Cells Involving Programmed Cell Death 4 and Sprouty-1.

Non-confluent EMC cultures were stimulated with IL-1β, TNF-α, or TGF-β, and examined after 48 and 96h by A, phase contrast microscopy for morphology, B, immunofluorescence of epicardial cell markers (Wilms Tumor protein homolog (WT1)) and C, fibrogenic markers (S100A4 and α-smooth muscle actin (α-SMA)). qRT-PCR for expression of the EMT biomarkers D, α-SMA, E, Slug and F, E-cadherin. For immunofluorescence, fibroblasts and antibody isotype controls (not shown) were used to assess non-specific staining. qRT-PCR data (means+SD, n = 3) were normalized against GAPDH and RPL13A, and statistical significance was tested by one-way ANOVA and treatment effects by Tukey’s post test. *P<0.05, ***P<0.001 vs. the control (CTL) at indicated time-points.

Hasse Brønnum, et al. PLoS One. 2013;8(2):e56280.
6.
Figure 5

Figure 5. PDCD4 and SPRY1 regulate biomarkers of fibrogenic EMT in EMCs, partly via miR-21.. From: miR-21 Promotes Fibrogenic Epithelial-to-Mesenchymal Transition of Epicardial Mesothelial Cells Involving Programmed Cell Death 4 and Sprouty-1.

TGF-β-incubated (20h) EMCs were co-transfected with an anti-miR control or an anti-miR-21 along with siRNAs against PDCD4 or SPRY (or siRNA-control; “α-CTL”), and analyzed after 24h for E-cadherin expression (A), α-SMA expression (B), TIMP-1 (C) in the culture medium (ELISA) as well as cell number (D), mean cell diameter (E), and cell motility (F) by the relative wound size closure in a scratch assay. qRT-PCR data were normalized against GAPDH and RPL13A. All data are presented as means+SD, n = 3 relative to the control treatment (α-CTL+anti-miR control). Statistical significance was tested by one-sample t-tests for each treatment and one-way ANOVAs followed by Tukey’s post test compared treatment effects within the PDCD4- and SPRY1-specific experiments. *P<0.05, **P<0.01, ***P<0.001.

Hasse Brønnum, et al. PLoS One. 2013;8(2):e56280.
7.
Figure 3

Figure 3. miR-21 is highly up-regulated during fibrogenic EMT.. From: miR-21 Promotes Fibrogenic Epithelial-to-Mesenchymal Transition of Epicardial Mesothelial Cells Involving Programmed Cell Death 4 and Sprouty-1.

Differentially expressed miRNAs were assessed in EMC cultures stimulated for 48h with IL-1β, TNF-α, or TGF-β. A, Cluster analysis and heatmap of regulated miRNAs (fold change (FC) above 2 or under −2) as detected by microarrays and compared to non-stimulated control cells. B, Validation of miR-21 expression after 48h of EMT induction in EMCs by qRT-PCR (means+SD, n = 3; standardized to the control (CTL)). Statistical significance was tested by one-way ANOVA and treatment effects by Tukey’s post test. miR-21 up-regulation was further validated by qRT-PCR in vivo using both a model of C, pressure-overload (transverse aortic constriction; TAC) and D, myocardial infarction (ligation of left anterior descending artery; LAD). These data were acquired on biological triplicates (means+SD) or a pool of 1–4 hearts, respectively, and analyzed against sham treated animals by two-way ANOVA. All miRNA qRT-PCR data were normalized against miR-17 and miR-195. **P<0.01, ***P<0.001 vs. control.

Hasse Brønnum, et al. PLoS One. 2013;8(2):e56280.

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