Fig. 8: Tfeb silencing in MECs sensitized them to lower concentrations of TGFβ1.

a Tfeb and Tgif1 silencing caused a stronger upregulation of the Acta2 transcript in response to low doses of TGFβ1. MECs were infected with a lentivirus coding for a shRNA for Tfeb or Tgif1 or control shRNA. Cells were treated with 0.05, 0.5, 1, 5, and 10 ng/ml TGFβ1 for 24 h. The Acta2 mRNA quantity was assessed by real-time PCR analysis. Values are shown as the mean ± SEM, n = 4. Two-way ANOVA Bonferroni correction p values are shown in the plots. b Effect of Tfeb silencing on TGIF1 protein levels in MECs treated with low (0.05 ng/ml) and high (10 ng/ml) doses of TGFβ1 for 15 min. Images are shown in Supplementary Fig. 9b. Experiment was repeated three times, six images for each experimental point were analyzed. Box plot shows the quartiles, the 5th and 95th percentiles (whiskers). Student’s two-tailed test p values are shown in the plot. c Tfeb knock out in epicardium favors premature EPDCs differentiation onto vSMC and fibroblast precursors at E13.5. Immunofluorescence analysis for PDGFRα, PDGFRβ (green) and cTnT (magenta) in E13.5 Gata5⁺; Tfeb ^flox/flox and control embryos (top) and its quantification (bottom). Scalebar is 10 μm. Five embryos of each genotype, at least four images for embryo were used for quantification in ImageJ. Total immunofluorescence signal of a marker in the image was normalized to the length of the myocardium surface in the image. Box plots show the quartiles, the 5th and 95th percentiles (whiskers). Student’s two-tailed test p values are shown in the plots. d Tgif1 is downregulated in the epicardium of Gata5⁺; Tfeb ^flox/flox mice. Immunostaining for TGIF1 (green) and cTnT (magenta); nuclei are stained with DAPI (magenta) in Gata5⁺; Tfeb ^flox/flox and control embryos at E12.5 (top) and its quantification (bottom). The dashed line indicates the myocardium surface. The scale bar is 50 μm. The TGIF1 immunofluorescence signal was quantified in nuclei selected by DAPI with ImageJ software. Four embryos for each genotype and at least 10 images for an embryo were used for quantification (graph on the right). Box plot shows the quartiles, the 5th and 95th percentiles (whiskers). Student’s two-tailed t test p value is reported in the plot. e Epicardial explants from Gata5⁺; Tfeb ^flox/flox embryos produced more αSMA in response to low (0.5 ng/ml) and high (10 ng/ml) concentrations of TGFβ1 than control explants. Gata5⁺; Tfeb ^flox/flox and control hearts at E11.5 were explanted in DMEM with 10% FCS for 24 h. Then, the hearts were removed, and the explants were cultured for 48 h in DMEM with 10% FCS and 0.5 ng/ml or 10 ng/ml TGFβ1 and immunostained for αSMA (representave images in top panel). The αSMA immunofluorescence signal was quantified in ImageJ software, and five explants and at least four images for an explant were analyzed for an experimental point (plot in bottom panel). Box plot shows the quartiles, the 5th and 95th percentiles (whiskers). Student’s two-tailed t test p values are reported in the plot. f A proposed model for the role of TFEB and TGIF1 in the regulation of TGFβ1 signaling. Under basal conditions, TFEB expressed in epicardial cells is required to establish an appropriate quantity of TGIF1 protein. TGFβ1 stimulation rapidly initiates proteosome-dependent TGIF1 degradation; however, a low concentration of TGFβ1 is not sufficient to remove TGIF1 repression, thus creating a dose-dependent response. Source data are provided as a Source Data file.