Smad2/3 pathway activation

Full-thickness 3D tissue skin equivalents (EpiDermFT, MatTek) were treated with 10 ng/ml and 100 ng/ml rGDF11 or 50 ng/ml TGFβ1 (R&D Systems, Minneapolis, MN) added into proprietary growth factor-free media provided by the manufacturer for 2 hours. The treated tissues were placed on ice, washed with ice-cold PBS, lysed in 600μl T-PER Tissue Protein Extraction Reagent (ThermoFischer Scientific), and reacted with anti-P-Smad3 clone (EP823Y, Abcam, Cambridge, MA) and anti-S6 clone (5G10, Cell Signaling, Danvers, MA). The P-Smad2/3 and ribosomal S6 levels were assessed by western blot analysis. Ribosomal S6 was used as a loading control (Fig 3A). Primary human dermal microvascular endothelial cells (HDMEC, PromoCell, Heidelberg, Germany) were grown in Endothelial Growth Cell Medium MV with supplements (PromoCell, Heidelberg, Germany) and seeded for experiment at density of 100,000 cells/5ml into each well of a 6-well plate (triplicate for each treatment). After overnight incubation, cells were transferred into media without supplement and treated with 10 ng/ml, 50 ng/ml, and 100 ng/ml rGDF11 or 50 ng/ml TGFβ1 for 60 minutes. The treated cells were placed on ice, washed with ice-cold PBS, and lysed in 200 μl M-PER Mammalian Protein Extraction Reagent (ThermoFischer Scientific). The P-Smad2/3 and ribosomal S6 levels were analyzed as above (Fig 3B).

(a) Human full thickness skin equivalents were treated with vehicle, rGDF11 (10ng/ml or 100ng/ml), or TGF-β1 (50ng/ml) for 2 hours, lysed, and Smad2/3 phosphorylation was analyzed by immunoblotting. Ribosomal protein S6 was used as a loading control. Semiquantitative analysis of signal intensity was performed on the original TIFFs files in Photoshop. Background-subtracted signal was normalized to vehicle treatment and converted to a fold change. * represents p<0.05 as calculated by T-test. (b) Primary human dermal microvascular endothelial cells were stimulated with rGDF11 (10ng/ml, 50ng/ml, or 100ng/ml) or TGF-β1 (50ng/ml) for 60 minutes, lysed, and P-Smad2/3 and ribosomal S6 levels were analyzed.