Dynamics of TCF7L2 expression in the stromal compartment during skeletal muscle regeneration and repair. (A) Representative western blots showing TCF7L2, β-catenin, PDGFRα, MyHC fast (myosin heavy chain MyHC-2x), Myogenin and GAPDH in TA muscle after damage with glycerol at different time points (0, 3, 7 and 14 d). Ponceau red was used as the loading control. (B) Quantification of TCF7L2, β-catenin, PDGFRα, MyHC fast, and Myogenin protein expression. n=3. (C) z-stack confocal images showing the localization of TCF7L2⁺ cells in diaphragm muscle sections of adult wild-type and mdx mice. Laminin-α2 (red) and nuclei (Hoechst, blue) are also stained. Boxes indicate regions also shown as magnified images (zoom in). Asterisks indicate TCF7L2⁺ cells in the magnified images. Scale bars: 50 μm and 10 μm. (D) Pie charts showing the increase in TCF7L2⁺ cells in dystrophic diaphragms compared to wild-type diaphragms. n=4. (E) Quantification of fluorescence intensity of TCF7L2 in stromal TCF7L2⁺ cells. n=4. ***P<0.001 (two-tailed Student's t-test). Horizontal bars indicate the first, second and third quartiles. (F) Representative confocal image showing the localization of phosphorylated-SMAD3⁺ cells in diaphragm muscle sections of adult wild-type and mdx mice. Laminin-α2 (LN-α2, red) and nuclei (Hoechst, blue) are also stained. Scale bar: 50 μm. (G) Quantification of the percentage of phospho-SMAD3⁺ cells in fibers and stromal cells of wild-type (WT) and mdx mice. n=4. (H) Representative confocal image showing the increase of ECM collagen type 1 immunostaining in diaphragm of mdx compared to wild-type mice. Nuclei (Hoechst, blue) are also stained. n=3. Scale bar: 50 μm. Data in B and G are mean±s.e.m.

TGF-β signaling downregulates the expression of the Wnt TF TCF7L2 in mesenchymal progenitors and fibroblasts. (A) Representative western blots showing TCF7L2, fibronectin, β1-integrin, CCN2 (CTGF), and αSMA (ACTA2) expression levels in C3H/10T1/2 MSCs after treatment with different concentrations of TGF-β1 for 24 h. Tubulin was used as the loading control. (B) Quantification of TCF7L2 protein expression in the experiment described in A. n=4. **P<0.005; *P<0.05 (one-way ANOVA with Dunnett's post-test). (C,E) Representative western blots showing TCF7L2, fibronectin, CCN2 (CTGF), and αSMA (ACTA2) expression levels in (C) C3H/10T1/2 MSCs and (E) NIH-3T3 fibroblasts after treatment with TGF-β1 (5 ng/ml) at different time points (0, 2, 8, 24, 48 h). Tubulin was used as the loading control. (D,F) Quantification of TCF7L2 protein expression in the experiments described in C and E, respectively. n=4. ****P<0.0001; ***P<0.001; *P<0.05; n.s, not significant (one-way ANOVA with Dunnett's post-test). (G) Representative western blots showing TCF7L2 and fibronectin expression levels after treatment with 5 ng/ml TGF-β1 (24 h) in PDGFRα-EGFP⁺ FAPs. Tubulin was used as the loading control. (H) Quantification of TCF7L2 protein expression in the experiment described in G. n=3. **P<0.005 (two-tailed Student's t-test). (I) Tcf7l2 mRNA expression levels were analyzed by quantitative PCR in C3H/10T1/2 MSCs after 2, 8 and 24 h of treatment with TGF-β1 (5 ng/ml). n=3. ****P<0.0001; **P<0.005 (one-way ANOVA with Dunnett's post-test). (J) Representative western blots showing TCF7L2 and PDGFRα expression levels after treatment with actinomycin D for different time periods (0, 2, 3, 4, 7 h). GAPDH was used as the loading control. (K,L) Heat maps showing the expression changes of several validated TCF7L2-target genes that are repressed or induced by TGF-β in lung fibroblasts (K) and cardiac fibroblasts (L). Data in B,D,F,H and I are mean±s.e.m.

Extracellular TGF-β reduces the expression of TCF7L2 TF through TGFβR1 activation. (A) Representative western blots showing TCF7L2, β1-integrin, tubulin, GAPDH, and c-Jun expression levels in control and TGF-β1-treated C3H/10T1/2 cells. Ho, whole cell lysate; Cyto, cytoplasmic lysate; Nuc, nuclear lysate. (B) z-stack confocal images showing localization of TCF7L2 (green) and αSMA (red) in control and TGF-β1-treated (36 h) C3H/10T1/2 MSCs. Nuclei (Hoechst, blue) are also stained. Scale bars: 50 μm. (C) Quantification of the TCF7L2 fluorescence intensity in NIH-3T3 and EGFP⁺ FAPs. (a.u., arbitrary units). Each dot represents a single cell quantified where a region of interest area was previously defined. Bars indicate mean±s.e.m. Data are pooled from n=3 experiments. ***P<0.001 (two-tailed Student's t-test). (D) z-stack confocal images showing localization of TCF7L2 (green) and αSMA (red) in control and TGF-β1-treated (24 h) C3H/10T1/2 MSCs. Nuclei (Hoechst, blue) are also stained. White lines indicate sections quantified in E. Scale bars: 50 μm. (E) Label-distribution graphs showing the fluorescence intensity of TCF7L2 and Hoechst (Nuclei) along the cell axis as shown in D. Distance is shown in pixels; a.u., arbitrary units. Dotted lines show the nucleus–cytoplasm boundary. (F) Representative western blots showing TCF7L2 and β1-integrin expression levels in wild-type PDGFRα⁺ FAPs co-treated for 24 h with TGFBR1 inhibitor SB525334 (5 µM) and TGF-β1 (5 ng/ml). Tubulin was used as the loading control and the ratio of TCF7L2 to tubulin signal is shown. (G) Representative western blots showing TCF7L2, β-catenin, β1-integrin, and CCN2 (CTGF) expression levels in C3H/10T1/2 MSCs co-treated for 24 h or 48 h with the TGFBR1 (ALK-5) inhibitor SB525334 and TGF-β1. Tubulin was used as the loading control. Expression levels of TCF7L2 relative to tubulin expression are indicated as percentages.

Histone deacetylases participate in TGF-β-mediated repression of TCF7L2. (A) Representative western blots showing TCF7L2, β1-integrin, and CCN2 (CTGF) protein levels after TGF-β1 and SB525334, SIS3, SB203580, SP600125 and UO126 pharmacological co-treatments. Tubulin was used as the loading control and the ratio of TCF7L2 to tubulin signal is shown. (B) Quantification of TCF7L2 protein expression in the experiment described in A. n=5. ****P<0.0001; ***P<0.001; **P<0.005; *P<0.05 (one-way ANOVA with Dunnett's post-test). (C) Representative western blots showing TCF7L2 and PDGFRα protein levels after TGF-β1 (5 ng/ml) and trichostatin A (TSA; 10 µM) treatments (8 h) of MSCs. Tubulin was used as the loading control. (D) Quantification of TCF7L2 protein expression in the experiment described in C. n=3. ***P<0.001; **P<0.005 (one-way ANOVA with Dunnett's post-test). (E) Representative western blots showing TCF7L2 and PDGFRα protein levels after TGF-β1 and TSA co-treatments (8 h) in MSCs. Tubulin was used as the loading control. (F) Quantification of TCF7L2 protein expression in the experiment described in E. n=3. ****P<0.0001; **P<0.005; *P<0.05 (one-way ANOVA with Dunnett's post-test). (G) Heat map showing Tcf7l2 expression (given as reads per kilobase per million mapped reads, RPKM) is substantially repressed by TGF-β and reversed by the pan-HDAC inhibitor pracinostat (HDACi). Known ECM pro-fibrotic mediators [e.g. genes encoding collagens, CCN2 (CTGF), fibronectin and integrins] that are significantly increased by TGFβ and reversed by pracinostat are also shown. Each row is normalized to the within row variation. Each column, per treatment condition, represents an individual IPF lung fibroblast donor, n=3. Data in B,D and F are mean±s.e.m.

TGF-β impairs TCF7L2 protein stability via the UPS. (A) Representative western blots showing TCF7L2 protein levels after treatment with cycloheximide (CHX, 30 μg/ml) for different time periods (0, 3, 6, 9, 12 h) in C3H/10T1/2 cells. Ponceau was used as the loading control. (B) Quantification of three independent experiments showing TCF7L2 protein levels (as the mean±s.e.m. percentage of the 0 h level) after CHX treatment. (C) Representative western blots showing TCF7L2 protein levels after treatment with CHX for different time periods (0, 3, 6, 9 h) in wild-type muscle PDGFRα⁺ FAPs. (D) BioGRID interactome analysis of the human TCF7L2 protein. Black circles mark the protein–protein interactions between TCF7L2 and RNF4, RNF43, RNF138, NLK, UHRF2, UBE2I, UBE2L6, USP4, UBR5 and XIAP. (E) In silico prediction of TCF7L2 ubiquitination sites, showing the amino acid sequence of human TCF7L2 protein variant 1. Potential TCF7L2-ubiquitinated lysine residues were ranked and are shown in red. (F) Representative western blots showing TCF7L2 and CCN2 (CTGF) protein levels after TGF-β1 (1 ng/ml) and MG132 (15 μM) treatments (9 h). Tubulin was used as the loading control. (G) Quantification of TCF7L2 protein levels in the experiment described in F. n=6. ***P<0.001; **P<0.005; *P<0.05 (one-way ANOVA with Dunnett's post-test). (H) Western blots representative of three independent experiments that evaluate total levels of TCF7L2 following USP7 small-molecule inhibitor HBX 41108 (10 μM) and CHX treatments for different time periods (0, 4, 6, 8 h) in C3H/10T1/2 MSCs. Tubulin was used as the loading control and the ratio of TCF7L2 to tubulin signal is shown.

The expression of TCF7L2 remains unchanged in response to TGF-β treatment in myoblasts. (A) Western blots representative of three independent experiments, evaluating TCF7L2 and β-catenin protein levels in proliferating C3H/10T1/2 MSCs and C2C12 myoblasts. GAPDH was used as the loading control. (B) Quantification of TCF7L2 protein levels in the experiment described in A. n=3. **P<0.005 (two-tailed Student's t-test). (C) Representative western blots showing TCF7L2, fibronectin, and CCN2 (CTGF) protein levels after treatment with different concentrations of TGF-β1 for 24 h. (D) Quantification of TCF7L2 protein levels in the experiment described in C. n=3. n.s, not significant (two-tailed Student's t-test). (E) Tcf7l2 and Ccn2 mRNA expression levels were analyzed by quantitative PCR in C2C12 myoblasts after 8 h of treatment with TGF-β1 (5 ng/ml). n=3. ***P<0.001; n.s, not significant (two-tailed Student's t-test). (F) Representative z-stack confocal images showing localization of TCF7L2 (green) and F-actin (red) in control and TGF-β1-treated (24 h) C2C12 myoblasts. Nuclei (Hoechst, blue) are also stained. Scale bar: 50 μm. (G) Quantification of the TCF7L2 fluorescence intensity (a.u., arbitrary units) in C2C12 myoblasts. Each dot represents a single cell quantified where a region of interest area was previously defined. Data are pooled from n=3 experiments. Horizontal bar indicates the mean. n.s, not significant (two-tailed Student's t-test). Data in B,D and E are mean±s.e.m.