Fig. 4: TS1 acts on the canonical TGFβ pathway via Drd3, inhibiting differentiation of both murine and human lung fibroblasts.

A HPLC analysis shows compound stability of solubilised dopamine and TS1-HCl at room temperature over 48 h, data normalized over the initial time point (T0). B Murine lung fibroblasts were treated ex vivo with non-targeting siRNA (siNT) or pools of two siRNAs targeting either Drd3 or Drd4. After 48 h, cells were treated with TGFβ and TS1-HCl for an additional 48 h prior to fixation. Representative images are shown (Hoechst in blue, αSMA-RFP in red). C Quantification of the number of αSMA positive cells (shown as fold over control). D HEK293T cells stably expressing the TGFβ-sensitive (CAGA)12-luciferase reporter were treated with TGFβ and Dopamine or TS1-HCl to evaluate the effect of the compounds on the activation of the canonical TGFβ-Smad pathway. Data is the Firefly-luciferase intensity normalized over Renilla-luciferase intensity. Computational model of TS1 docked to murine Drd3: side (E) and top (F) view. Colour code: missense mutation with respect to the human receptor (yellow), residues interacting with TS1 with hydrogen bonds (green), and other interacting residues: carbons (cyan), nitrogens (blue), oxygens (red), hydrogens (white). TS1 atoms are represented with their van der Waal spheres (space-filling). G Human adult primary lung fibroblasts were treated ex vivo with TGFβ and TS1-HCl for 48 h prior to fixation and staining of αSMA. Quantification of the number of αSMA positive cells (shown as fold over control). H Representative images are shown (Hoechst in blue, αSMA in red). All data are shown as mean ± SD. Statistical significance was determined using a one-way ANOVA followed by Dunnett’s multiple comparison test, *P < 0.05, **P < 0.01, n = 3.