Fig. 2.

Glutamine (Gln) downregulated Sp3 expression under normoxia and hypoxia/reoxygenation conditions. A: glutamine suppressed Sp3 mRNA expression in a dose-dependent fashion. After incubation in serum-free media overnight, cells were treated with indicated concentrations of glutamine for 24 h, and quantitative real-time PCR was performed for evaluation of Sp3 mRNA expression. B: glutamine suppressed Sp3 mRNA expression in a time-dependent fashion. After incubation in serum-free media overnight, cells were treated with 10 mM glutamine for indicated time points, and quantitative real-time PCR was performed for evaluation of Sp3 mRNA expression. C: glutamine suppressed Sp3 protein expression. After incubation in serum-free media overnight, cells were treated with 2 mM or 10 mM glutamine for 24 h, and Western blot was performed for evaluation of Sp1 and Sp3 protein expression. D: glutamine suppressed Sp3 protein expression in a dose-dependent fashion. After incubation in serum-free media overnight, cells were treated with indicated concentrations of glutamine for 24 h, and Western blot was performed for evaluation of Sp3 protein expression. E: glutamine suppressed Sp3 protein expression in a time-dependent fashion. After incubation in serum-free media overnight, cells were treated with 10 mM glutamine for indicated time points, and Western blot was performed for evaluation of Sp3 protein expression. F: glutamine also suppressed Sp3 expression after hypoxia/reoxygenation. After incubation in serum-free media with 2 mM or 10 mM glutamine overnight, cells were subjected to a hypoxic chamber with 1% O₂-5% CO₂-94.5% N₂ for 4 h and then reoxygenated for 4 h, and Western blot was performed for evaluation of Sp3 protein expression. The densitometric analysis for Western blot corresponds to the means ± SE of 3 independent experiments.