TGFβ-Dependent Epithelial–Mesenchymal Plasticity in Immortalized Human Atrial Epicardial Cells: An mRNA Profiling Study

Figure A1. (A) Volcano plot of differentially expressed genes in EPDCs treated with SB431542 vs. NoAdditive. (B) Volcano plot of differentially expressed genes in EPDCs treated with TGFβ3 vs. NoAdditive. The boxes highlight genes associated with EPDCs and the epicardium. Volcano plot showing DEGs after 5 days of treatment. Complete differential expression results for the respective comparisons are provided in Supplementary Tables S2 and S3.

Figure 1. Generation and characterization of immortalized human primary EPDCs. (A) Schematic overview of the immortalization strategy. Primary human EPDCs were immortalized by lentiviral transduction with SV40LT. (B) Successful immortalization was confirmed by ICC of SV40LT expression in transduced cells. (C) Representative brightfield image and ICC marker profile of primary human EPDCs showing expression of MSLN, WT1 and CRIP1. (D) Representative brightfield image and ICC marker profile of immortalized human EPDCs showing preserved expression of MSLN, WT1 and CRIP1. (B–D) Nuclei were counterstained with DAPI.

Figure 2. Morphological characterization of iEPDCs after five days of culture with SB431542, NoAdditive and TGFβ3. Phalloidin-F-actin staining illustrated distinct morphological phenotypes of iEPDCs following treatment. TGFβ3-treated iEPDCs exhibited an elongated spindle-shaped morphology, whereas SB431542-treated cells displayed a cobblestone-like appearance. Cells cultured without any additive showed an intermediate morphology with features of both TGFβ3- and SB431542-treated phenotypes. Nuclei were counterstained with DAPI.

Figure 3. TGFβ3 treatment reduces epithelial-like features in iEPDCs. iEPDCs were cultured for five days under SB431542, NoAdditive or TGFβ3 conditions. (A) Immunocytochemical staining of iEPDCs demonstrates lower expression of epithelial-associated markers MSLN and CRIP1 in iEPDCs treated with TGFβ3 for five days compared with SB431542-treated cells, indicating a loss of epithelial characteristics. iEPDC cultures under the NoAdditive condition show intermediate levels of these markers, resembling an expression pattern between SB431542- and TGFβ3-treated cells. Nuclei were counterstained with DAPI. (B) Quantification of nuclear WT1 relative fluorescence intensity confirms significantly lower levels of WT1 in TGFβ3-treated iEPDCs compared to SB431542-treated cells. Relative fluorescence intensities of MSLN and CRIP1, determined by threshold-based analysis, were similarly decreased following TGFβ3 treatment compared to SB431542-treated iEPDCs. All data are shown as mean ± SEM, RM one-way ANOVA with Tukey’s post hoc test; p * < 0.05; p ** < 0.01; n = 3 donors.

Figure 4. TGFβ3 enhances mesenchymal-like features in iEPDCs. iEPDCs were cultured for five days under SB431542, NoAdditive or TGFβ3 conditions. (A) Immunocytochemical staining of iEPDCs shows that VIM and CD105/Endoglin expression levels are elevated in iEPDCs treated with TGFβ3 compared with SB431542-treated cells. iEPDCs cultured with NoAdditive show intermediate levels of both markers. The increased abundance and filamentous organization of VIM in TGFβ3-treated iEPDCs reflect cytoskeletal remodeling associated with a mesenchymal-like phenotype. Nuclei were counterstained with DAPI. (B) Quantification of VIM and CD105 relative mean fluorescence intensity confirms higher levels in TGFβ3-treated iEPDCs compared to SB431542-treated cells. All data are shown as mean ± SEM, RM one-way ANOVA with Tukey’s post hoc test; p * < 0.05; p ** < 0.01; n = 3 donors.

Figure 5. Principal component analysis (PCA) of iEPDCs and related cell populations. PCA of iEPDCs cultured for five days under SB431542-, NoAdditive- or TGFβ3-conditions, together with hCSCs and hPSC-derived epicardial cells, reveals that all cell populations cluster independently.

Figure 6. Differential gene expression in TGFβ3- versus SB431542-treated iEPDCs after five days of treatment. Volcano plot of differentially expressed genes shows upregulation of 287 genes in TGFβ3-treated iEPDCs and upregulation of 293 genes in SB431542-treated iEPDCs. The boxes highlight genes associated with EPDCs and the epicardium. The complete list of DEGs is provided in Supplementary Table S1.

Figure 7. Heatmap of the top 200 differentially expressed genes in iEPDCs treated with SB431542, NoAdditive and TGFβ3 for five days. The dashed box indicates genes that are mainly upregulated in TGFβ3-treated iEPDCs and downregulated under SB431542 conditions, whereas the solid box highlights genes that are mainly upregulated in SB431542-treated iEPDCs and mainly downregulated in TGFβ3-treated cells. A version of the heatmap displaying all gene labels is provided in Supplementary Figure S4.

Figure 8. KEGG and GO enrichment analyses of TGFβ3-treated vs. SB431542-treated iEPDCs after five days of treatment. (A) Upregulated KEGG terms in TGFβ3-treated iEPDCs compared with SB431542-treated cells. (B) Upregulated KEGG terms in SB431542-treated iEPDCs compared with TGFβ3-treated cells. Upregulated GO terms in TGFβ3-treated iEPDCs compared with SB431542-treated in (C) cellular component, (D) molecular function and (E) biological process. (F) Upregulated biological process GO terms in SB431542-treated iEPDCs compared with TGFβ3-treated cells. For each analysis, the top 15 significantly enriched pathways/terms are displayed.

Figure 9. TGFβ3-treated mesenchymal-like iEPDCs show a higher relative closure ratio than SB431542-treated cells after 8 h. iEPDCs were cultured for five days under SB431542, NoAdditive or TGFβ3 conditions before scratching. (A) Representative images of a scratch after 0 h and 8 h. (B) Relative closure ratio indicates a higher migration rate of TGFβ3-treated iEPDCs compared to SB431542-treated cells. Data are shown as mean ± SEM, RM one-way ANOVA with Tukey’s post hoc test; p * < 0.05; p ** < 0.01; n = 5 donors.