Figure 3

TPUF represses endogenously expressed VEGFA gene in HEK293 cell line. (a) Mutations and binding sequences of TPUFs designed for VEGFA 3′ UTR recognition. Black, WT modules and corresponding RNA bases. Red, mutant modules and corresponding RNA bases. Blue, a mismatch in the recognition sequence. Ct, C-terminus, Nt, N-terminus of the protein. (b) The graph demonstrates inhibition of hypoxia-induced VEGFA expression in cells transfected with engineered TPUFs VEGF3 and VEGF7. In hypoxic (+) cultures, VEGFA expression was induced with 500 μM CoCl₂ 24 hours after transfection and then cultivated for 24 hours. Secreted VEGFA levels measured by ELISA were normalized to total protein amounts from lysed cells measured by Bradford Assay. Data represented as mean ± SD: n.s., not significant, *P ≤ 0.05, **P ≤ 0.01, (n = 3, t test). (c) The graph demonstrates inhibition of DHB-induced VEGFA expression in cells transfected with TPUFs WT, VEGF1, VEGF3, and VEGF7. HEK293 cells with the integrated V24P-GS60 transcriptional activator of endogenous VEGFA promoter were treated with 100 nM DHB 24 hours after TPUF transfection and then cultivated for 24 hours. ELISA and normalization to total protein amounts as in the previous panel. Data represented as mean ± SD: n.s., not significant, *P ≤ 0.05. (d) Western blot of effector proteins using anti-Flag antibody shows greater expression of TPUFs VEGF3 and VEGF7 compared with TPUF (WT) and other mutants. Anti-α–tubulin antibody was used as a loading control.