Fig 2: Cytoplasmic PML induces SMAD2/3 phosphorylation, EMT and cell invasion in vitro. (a) Immunoblotting of whole-cell extracts from DU145 and PC3 cells expressing PML mutant constructs using antibodies against phosphorylated forms of SMAD2/3 and β-actin (loading control). (b) Immunoblotting analysis of whole-cell extracts from DU145 and PC3 cells expressing the PML ΔNLS construct, treated with DMSO (control) or TGFβ-RI inhibitor (3 μM for 48 h) using antibodies against HA, phosphorylated forms of SMAD2/3, E-Cadherin and β-actin (loading control). (c) Immunoblotting of whole-cell extracts from DU145 and PC3 cells expressing PML mutant constructs using antibodies against total SMAD2, SMAD3 and β-actin (loading control). (d) Immunofluorescence analysis of DU145 and PC3 cells expressing the PML ΔNLS construct, treated with DMSO (control) or TGFβ-RI inhibitor (3 μM for 48 h) using antibodies against phosphorylated forms of SMAD2/3, E-Cadherin and N-Cadherin. Scale bar=20 μm. (e, f) Wound healing assay (Scratch assay) using DU145 and PC3 cells expressing the PML ΔNLS construct, treated with DMSO (control) or TGFβ-RI inhibitor (3 μM for 48h) and the data relating to the percentages of gap closure at time points 0 and 24 h following scratching. Two-tailed paired test: *P=0.0451 and **P=0.0088.

Fig 3: EW-7197 down-regulates the expression on fibrogenic mediators in diabetic mice. (A) Immunohistochemistry of renal collagen IV and fibronectin, (B) positively stained area. Original magnification is 400× (scale bar=50 μm). (C, D) By Western blot of renal cortex protein expression of col IV and fibronectin. (E, F) The expression of Smad2/3, p-Smad 2/3, vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) as proteins related to fibrosis was confirmed, respectively from diabetic mice (n=4–5/group). aP<0.05 vs. db/m controls; bP<0.05 vs. db/db controls; cP<0.05 vs. db/db+EW5 mg/kg/day.

Fig 4: Effect of TGF-β inhibitor LY2157299 on the levels of VEGF and TNF-α protein expression and Smad2/3 phosphorylation in RPE-choroid complex of mice after Laser-induced CNV formation. (A) The typical result of Western blot showed changes of VEGF and TNF-α protein expressions. The quantitative analysis results demonstrated that the protein expression levels of VEGF (B) and TNF-α (C) were significantly elevated and peaked at 3–4 weeks following CNV induction, but TGF-β chemical inhibitor LY2157299 (intraperitoneal injection, 3.0 μg/g BW) treatment could significantly suppress the increase of VEGF and TNF-α protein expressions in RPE-choroid complex of mice at 1–4 weeks after Laser-induced CNV formation. (D) The representative result of Western blot showed the effect of LY2157299 on Smad2/3 phosphorylation and TGF-β protein expression levels in RPE-choroid complex of Laser-treated mice. The quantitative analysis results indicated that LY2157299 could significantly inhibit the increase of p-Smad2/Smad2 (E) and p-Smad3/Smad3 (F) levels in RPE-choroid complex of mice at 2–3 weeks after Laser-induced CNV formation. *P < 0.05 vs Nor; #P < 0.05 vs corresponding PBS treatment; n = 5 per group.

Fig 5: Smad3 Fancd2-129B6F2 embryos with ERK pathway activity express NHEJ components.(A) Western blot of the lysates from adult bone marrow stroma cells of mice showing that irradiation activates the canonical phosphorylation of Smad3 in bone marrow stroma of WT and Fancd2-/- mice and activates the non-canonical ERK1/2 phosphorylation in Smad3-/- and DKO mice stroma. Quantifications relative to wild type in basal conditions and the loading control are shown below every lane. Pink indicates the irradiated cultures per cell line. (B) Western blot of the lysates from E12.5 fetal liver showing that a subset of Smad3 Fancd2-129B6F2 embryos have higher levels of the phospho-ERK protein, suggesting activation of the non-canonical TGFβ pathway. High phospho-ERK embryos have a reduced expression of CD45 and CD41 (CD45 and CD41 were used as surrogates for indication of active hematopoiesis in the fetal liver). High phospho-ERK embryos have higher expression of NHEJ related proteins, DNA-PK and 53BP1. Two western blots are depicted showing a total of two wild type fetal livers, two Fancd2-/- fetal livers, two Smad3-/- fetal livers, and six Smad3 Fancd2-129B6F2 fetal livers. Quantifications relative to wild type in basal conditions and the loading control per gel are shown below every lane. Red indicates Fancd2-/- fetal livers, pink indicates Smad3 Fancd2-129B6F2 fetal livers with high phospho-ERK levels. (C) Left. Representative scatter plots of variant allele fractions (VAFs) of the 129/Sv germline variants across the genomes of the analyzed embryos (n = 2 wild type, n = 3 Fancd2-/-, n = 2 Smad3-/-, n = 6 Smad3 Fancd2-129B6F2). Long chromosomal segments with known strain origins are clearly observed. Homozygous reference (C57BL/6) segments are those with VAFs around zero (red); homozygous 129/Sv segments are those with VAFs around one (blue); and heterozygous segments of both strains are those with VAFs between zero and one (green). Wild type and Smad3-/- embryos show a balanced heterozygosity with VAFs of heterozygous sites clustering around 0.5. Fancd2-/- and Smad3 Fancd2-129B6F2 embryos show pronounced genomic instability with VAFs of heterozygous sites deviating markedly from 0.5. Right. Green inset summarizes the density of VAFs in the heterozygous 129/Sv germline. (D) Heatmap for gene expression showing that two types of E12.5 Smad3 Fancd2-129B6F2 embryos exist. Half of the studied E12.5 Smad3 Fancd2-129B6F2 embryos have a BaxHigh gene expression profile (suggesting lethality) and the other half have a BaxLow gene expression profile (suggesting survivorship). When profiled for DNA repair related genes, these two groups of E12.5 Smad3 Fancd2-129B6F2 embryos seem to have mutually exclusive gene expression profiles, with down-regulation of alternative error-prone and low fidelity DNA repair genes in the BaxHigh embryos, an effect similar to what has been previously ascribed to pharmaceutical inhibition of the TGFβ pathway. Black arrowhead indicates Prkdc, the gene codifying for DNA-PKcs. Data in (C) are represented as variant allele frequency (VAF). Data in (D) are represented as heatmap. See also S4 Fig.