FIGURE 5.

NO modulates SVCT-2 expression and function. Cultures were preincubated with (A) actinomycin D (1 μg/ml) or (C) anisomycin (150 μm) for 10 min. Then, cells were incubated with SNAP (100 μm) in the presence or absence of the mentioned compounds for 80 min and prepared for Western blotting. Data in A and C represent the mean ± S.E.M. of independent experiments; SNAP (n = 9), SNAP + AcD (n = 5), AcD (n = 5), SNAP + Aniso (n = 3), and Aniso (n = 3). Cells were incubated with SNAP (100 μm) for 80 min and harvested in TRIzol, total RNA was extracted, cDNA was synthesized, and real-time qRT-PCR was performed (B). The results represent the mean ± S.E.M. of 3 independent experiments. Cultures were also preincubated with (D) actinomycin D (1 μg/ml) and (E) anisomycin (150 μm) or CHX (350 μm) for 10 min. Then, cells were incubated with SNAP (100 μm) in the presence or absence of the mentioned compounds for an additional 10 min. Next, cells were pulsed with [¹⁴C]ascorbate (0.1 μCi/ml) for 40 min in the presence of SNAP (100 μm), lysed with 5% TCA (w/v), and the radioactivity was measured; SNAP (n = 17), SNAP + AcD (n = 3), AcD (n = 3), SNAP + CHX (n = 3), CHX (n = 3), SNAP + Aniso (n = 3), Aniso (n = 3). **, p < 0.01 (relative to control); ***, p < 0.001 (relative to control). AcD, actinomycin; CT, control.