Figure 6

Effect of hGBP-1 expression on VEGF-A synthesis. (A) The Tet-regulated hGBP-1-expressing TS/A cell clones 14 and 18, which were used for generation of subcutaneous tumors in Balb/c mice, were analyzed for VEGF-A synthesis applying a mouse-specific ELISA. As a control, cells harboring the parental vector pUHD were used. Cells (duplicates) were cultured for 96 h in the presence of doxycycline (1 and 2.5 µg/mL), and the respective cell culture supernatants were analyzed. Obtained values (mean ± SD) were normalized to cell numbers (1 × 10⁶) and set in relation to values obtained with nontreated cells (0 µg/mL dox). Corresponding cell extracts were analyzed for hGBP-1 expression by Western blotting (insert). (B) Quantitative Western blot analysis of VEGF-A in TS/A cell-derived tumor samples. Frozen tissue samples (n = 6) of tumors grown in mice that had received doxycycline for induction of hGBP-1 expression (TS/A-GBP⁺) or were injected with saline (TS/A-GBP⁻) were subjected to Western blot analysis to detect murine VEGF-A protein. GAPDH was stained as an internal control to monitor the amount of loaded protein. Quantification of band intensities was performed with the ImageQuant software. Background corrected single values were normalized to measurements of GAPDH-specific bands and summarized, and the mean values (± SEM) were compared for statistically significant differences by applying an unpaired Student t test. The calculated P value is indicated.