Fig 2: DDX3 siRNA and DDX3 inhibitors block TGF-ß1–increased NEU3 levels.(A) HLFs were transfected with control or DDX3 siRNA, cultured without (control) or with TGF-ß1, and then stained with anti-DDX3 and NEU3 antibodies. Protein levels were determined by Western blotting. Top image is a Coomassie-stained gel of cell lysates; bottom images are Western blots. Images are representative of 3 independent experiments. (B and C) Quantification of A. (D) Human lung fibroblasts were treated with DDX3 inhibitors, cultured without (control) or with TGF-ß1, and then stained with anti-NEU3 antibody. Protein levels were determined by Western blotting. In B–D, values are mean ± SEM, n = 3. ** P < 0.01, *** P < 0.001 versus control, # P < 0.05, ## P < 0.01, ### P < 0.001 versus TGF-ß1 treated (1-way ANOVA, Dunnett’s test).

Fig 3: The motifs in the NEU3 mRNA are necessary for TGF-β1–increased NEU3 translation, and the group 4 motif is sufficient for TGF-β1–induced translation of S-formylglutathione hydrolase and McKusick-Kaufman syndrome protein.(A) Human lung fibroblasts transfected with the indicated constructs were cultured without (control) or with TGF-β1 and then stained with anti-Myc-tag antibodies. Green indicates Myc-NEU3 staining and blue is DAPI counterstain. Bar is 100 μm. Images are representative of 3 independent experiments. (B and C) Human lung fibroblasts transfected with the indicated constructs were cultured without (control) or with TGF-β1 and then stained with anti–Myc-tag antibodies. Red indicates Myc-ESD or Myc-MKKS staining and blue is DAPI counterstain. Images are representative of 3 independent experiments. (D–F) Quantification of A–C. IF, immunofluorescence. (G) Myc-NEU3 in transfected and nontransfected human lung fibroblasts was confirmed by Western blotting with an anti–Myc-tag antibody. Top image is Coomassie blue–stained gel of cell lysates; bottom image is Western blot. (H and I) Myc-ESD and Myc-MKKS in transfected and nontransfected human lung fibroblasts were verified by Western blotting with an anti–Myc-tag antibody. Images in G–I are representative of 3 independent experiments. (J–L) Quantification of G–I. WB, Western blot. Values in D–F and J–L are mean ± SEM, n = 3. * P < 0.05, *** P < 0.001 (t tests). ESD, S-formylglutathione hydrolase; MKKS, McKusick-Kaufman syndrome protein.