Figure 6.

Characterization of TGFbeta-1-mediated upregulation of FAP mRNA, FAP protein, and FAP enzymatic activity in human glioma cells. (A) Time-dependent upregulation of FAP mRNA expression in U87 glioma cells during cultivation without and with 10 ng⋅mL⁻¹ of human recombinant TGFbeta-1. (B) Time-dependent upregulation of FAP protein in U87 glioma cells during cultivation without and with 10 ng⋅mL⁻¹ of human recombinant TGFbeta-1. A complete blocking of exogenous and partial blocking of endogenous FAP protein upregulation by co-treatment with the TGFbeta type I receptor inhibitor A8301 (1 µM). (C) Time-dependent upregulation of FAP enzymatic activity in U87 and U251 glioma cells during cultivation without and with 10 ng⋅mL⁻¹ of human recombinant TGFbeta-1. (D) Upregulation of both FAP protein and FAP enzymatic activity in U87 glioma cells after cultivation with 10 ng⋅mL⁻¹ of human recombinant TGFbeta-1 for 72 h and its complete blocking by co-treatment with the TGFbeta type I receptor inhibitors A7701 and A8301 (both at 1 µM), which also reduced the baseline FAP protein and FAP enzymatic activity levels. (E) Time course of secretion of endogenous TGFbeta-1 into serum-free media by cultured glioma cells U87 and U251 and human brain vascular pericytes (HBVP). The mean increase in secreted TGFbeta-1 level between the 2nd and 72nd hour of cultivation is indicated for each examined cell line. (F) Time course of increase in the baseline level of FAP enzymatic activity in cultured U87 glioma cells and its marked decrease by treatment with A8301 and A7701 (both at 1 µM). The results in (A) are presented as the mean ± SD from three parallel cell cultures measured in triplicate. Results in (B–F) are presented as the mean ± SD from five or six parallel cell cultures measured in triplicate. In A, * p < 0.05, one way repeated measures ANOVA. In (B,C,F), * p < 0.05, Friedman repeated measures ANOVA on ranks. In (D), * p < 0.05, one way ANOVA, Tukey’s post hoc test.