Fig. 1: Multifunction of TβR2 binding peptide PTβR2I in different cells.
a Binding affinity between PTβR2I and TβR1/TβR2/TβR3/IgG, tested by the binding assay (n = 8). b–e Kd values of PTβR2I in binding with b TβR2, c TβR1, d TβR3, and e IgG measured by an ELISA-like binding assay (n = 8). f PTβR2I binding specificity was tested by staining the HDFs with an anti-TβR2 antibody. The PTβR2I treated group showed evident expression of FITC, and no unbound TβR2 was observed. Scale bar =50 µm. g The competitive binding affinity to TβR2 was also examined with a total treatment time of 48 h. Scale bar = 50 µm. h The competitive binding between PTβR2I and TGFβ1 was determined using fluorescence intensity measurements by a plate reader (excitation/emission = 485/535 nm). The non-PTβR2I -treated group served as a control. The total time for all treatments was 48 h (n = 10). p > 0.05 for any pair of the groups with the addition of PTβR2I. i Schematic showing how PTβR2I binds to TβR2 on HDF and how it surpasses TGFβI in binding ability. j dsDNA of HaCaT treated with TGFβ1 and PTβR2I at day 3 (n = 4). k dsDNA of dermal fibroblasts at day 3 (n = 5). l dsDNA of endothelial cells at day 3 (n = 6). m Representative images and quantification of migration using a scratch assay for HaCaT at 0 and 48 h (n = 4). n Representative images and quantification of migration for dermal fibroblasts at 0 and 72 h (n = 7). o Representative images and quantification of migration for endothelial cells at 0 and 36 h (n = 7). p Representative images of endothelial cell lumen formation (lumens are indicated by white arrows) 24 h post-treatment. Cytoskeleton was stained by F-actin. Scale bar = 50 µm. q Quantification of lumen density based on the images (n = 6). r Folds of change for PDGFBB, VEGFA, and HGF in endothelial cells. s Folds of change for IL6, TNFA, PDGFBB, and VEGFA expressed from macrophages. t Folds of change for IL1B, IL6, TNFA, PDGFBB, and IGF1 in dermal fibroblasts. u Folds of change for IL1B, IL6, TNFA, PDGFBB, and HGF in HaCaT treated with no TGFβ1, TGFβ1, or PTβR2I and TGFβ1. v, w Immunoblotting of p-p38 and p-Smad2/3 derived from dermal fibroblasts. GAPDH was used as a loading control (n = 3). The cells were treated with no TGFβ1, TGFβ1, and PTβR2I with TGFβ1 under high glucose. All data demonstrated as mean ± standard deviation. Data were analyzed by one-way ANOVA with Bonferroni post-test (n.s. p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001).
