Fig 1: Thbs3 enhances secretory pathway activity but reduces membrane integrins. a Time course of intracellular trafficking fluorescence changes in cultured neonatal ventricular cardiomyocytes (NRVMs) infected with a GalNac-T2-RFP baculovirus and the indicated adenoviruses. The data show a quantitative time course of GalNac-T2-RFP recovery in the Golgi network after FRAP to measure ER-to-Golgi vesicular trafficking. *P < 0.05 versus Adßgal infected cells. b Quantitative time course of loss of VSVG-eGFP fluorescence in the Golgi after iFRAP as a measurement for Golgi-to-membrane (Golgi exit) vesicular trafficking. *P < 0.05 versus Adßgal infected cells. Statistical analysis was performed using two-way ANOVA and Turkey multiple comparisons test. c Western blots for ECM proteins using heart extracellular matrix extracts from tTA, Thbs3 DTG and Thbs4 DTG mice at baseline or with TAC stimulation. A silver-stained gel is shown with a non-specific (n.s.) band as a loading control. d, e Quantitative time-course of the loss of a5 integrin-GFP fluorescence at the Golgi after iFRAP as a measure of Golgi-to-plasma membrane vesicular trafficking of a5 integrin. The data were generated by iFRAP of a5 integrin-GFP from COS-7 cells d transfected with the plasmids harboring the cDNAs shown or e treated with recombinant Thbs3 or Thbs4 proteins, or bovine serum albumin (BSA) as a control. *P < 0.05 versus Adßgal transfected cells; #<0.05 versus Thbs3 transfected cells. Statistical analysis was performed using two-way ANOVA and Turkey multiple comparisons test. Results were summed from four independent experiments and error bars are +/- standard error of the mean
Fig 2: THBS4 is a soluble dermal inflammatory signal that activates the fibroblast migration for skin regeneration and wound healing. See section “Discussion” for closer explanation.
Fig 3: THBS4 promotes fibroblast migration. (A) Transwell migration assay with fibroblasts stimulated with recombinant THBS4 and quantification of the number of migrating cells in a field of view. (B) Transwell invasion assay through Matrigel-coated chambers and quantification of the number of migrating cells in a field of view. (C) Representative images of the in vitro scratch wound healing assay with fibroblasts stimulated with recombinant THBS4 and quantification of the relative wound closure in time. Scale bar is 200 μm. The graphs depict the averages of at least 3 independent replicates ± standard deviation, * indicates a statistically significant (P < 0.05) difference compared to cells stimulated with control medium.
Fig 4: Thbs3 reduces surface integrin levels and destabilizes the sarcolemma. a Representative Western blots of sarcolemma protein extracts from hearts of tTA control, Thbs3 DTG, and Thbs4 DTG mice, assayed for integrin proteins and ß-dystroglycan. Laminin2a2 and Cacna1c serve as loading controls. Quantitation is shown in Supplementary Figure 3a-k. The red box shows integrin proteins specifically reduced on the sarcolemma by Thbs3. b Western blots for input of microsomal protein extracts before immunoprecipitation for Thbs3 and ß1D integrin and Cacna1c from NRVMs infected with Adßgal or AdThbs3 (left panel). The microsomal fraction was then immunoprecipitated (IP) for ß1 integrin and blotted for the indicated proteins (right panel). Mouse IgG was used as a control for the IP. c Regimen of Evans blue dye (EBD) and isoproterenol (Iso, 300 mg/kg) injection to measure membrane permeability in the hearts of mice. d Representative histological heart sections from 3 tTA control and 3 Thbs3 DTG mice, imaged for WGA (green fluorescence) and leakage of EBD into the cardiomyocytes by red fluorescence subjected to the experimental regimen shown in c. Scale bars are 1 mm. e Regimen of Evans blue dye (EBD) injection after TAC surgery to measure membrane permeability in adult mice. Mice were sacrificed (Sac) 24 h after EBD injection, 1 week after TAC surgery. f, g Representative cardiac histological images of tTA control and Thbs3 DTG hearts 1 week after TAC surgery stained with WGA (green) and intracellular leakage of EBD (red) according to the experimental scheme shown in e, while g shows quantitation of EBD positive cells in the hearts of these mice. Scale bars are 100 µm. *P < 0.05 versus tTA control sham; two-tailed students T-test. Data are represented as percentage EBD positive cells (=3000 cells from =8 animals). Error bars are +/- standard error of the mean and number of mice used is shown in the graph as individual data points
Fig 5: THBS4 promotes keratinocyte but not fibroblast proliferation in vitro. (A) Representative images of primary human fibroblasts cultured in the presence of recombinant THBS4 protein and the quantification of Ki67+ positive cells, n = 3 (B). (C) Representative images of primary human keratinocytes cultured in the presence of recombinant THBS4 protein and quantification of Ki67+ positive cells. (D) Scale bar is 200 μm. The graphs depict the averages of at least 3 independent replicates ± standard deviation, * indicates a statistically significant (P < 0.05) difference compared to cells stimulated with control medium.
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