Figure 6.

The VEGF 5′UTR, coding region, and 3′UTR do not individually confer hypoxic stabilization on a reporter mRNA. (A) Time courses of degradation from cells expressing the f5′V-GH-3′GH, the f164–3′GH, or the f164–3′V mRNAs grown under normoxia (open symbols) or hypoxia (closed symbols) for 24 h. The graph shows the data as mean ± SEM for at least two independent experiments, normalized with respect to GAPDH. The levels of serum induction observed for the f5′V-GH-3′GH reporter gene were 9.6-, 4.4-, and 8.6-fold under normoxia and 6.2- and 6.6-fold under hypoxia. For the f164-GH-3′GH reporter gene the serum induction was 23-, 16-, and 11-fold under normoxia and 12- and 6.2-fold under hypoxia. For the fGH-3′V reporter gene the serum induction was 7.5-, 7.5-, 4.4-, and 14-fold under normoxia and 7.8-, 5.0-, and 6.6-fold under hypoxia. The maximal levels of induction (expressed as a ratio to the GAPDH level) for the f5′V-GH-3′GH reporter gene were 0.30 and 0.29 under normoxia and 0.23 and 0.18 under hypoxia. The maximal levels of induction for the f164–3′GH reporter gene were 0.23 and 0.22 under normoxia and 0.24 and 0.28 under hypoxia. The maximal levels of induction for the fGH-3′V reporter gene were 0.32, 0.49, and 0.22 under normoxia and 0.26, 0.16, and 0.16 under hypoxia. (B) RNase protection gel of the endogenous VEGF mRNA and the neo mRNA from serum stimulated cells incubated under hypoxia for 24 h before serum stimulation and for the duration of the time course after serum stimulation. RNA from cells grown under normoxic conditions before serum stimulation is shown for comparison. The neo mRNA was used for normalization because the gene is expressed from the SV40 early promoter, the transcription of which is unaffected by hypoxia (Semenza and Wang, 1992).