Effect of NO• donors and p38 MAPK inhibition on p21 expression and mRNA stabilization. (A) Differentiated U937 cells (1 × 107) were incubated with NO• donors, S-nitrosoglutathione (GSNO; 400 μM), S-nitroso-N-acetylpenicillamine (SNAP; 400 μM), or DETA-NONOate (1 mM) or their degraded controls. Western blotting was then performed to detect p21 expression after 12 h of incubation. Results were quantified with laser densitometry and expressed as ratios relative to their appropriate degraded control. Data are means ± SE of four independent experiments. (B) Differentiated U937 cells (1 × 107) were incubated with increasing concentrations of the p38 inhibitor SB202190 (0 nM to 25 nM) for 30 min, then exposed to PBS, glutathione (GSH; 400 μM) or GSNO (400 μM) for 12 h. Western blotting was performed to detect p21 expression. Results were quantified with laser densitometry. Data, presented as fold change relative to PBS control values, are means ± SE of three independent experiments. Next, TaqMan® RT-PCR was used to quantify p21 mRNA levels normalized to GAPDH mRNA. (C) Changes in p21 mRNA levels during differentiation of U937 cells (1 × 107) with PMA. Data, presented as fold change relative to mean mRNA level in naïve cells, are means ± SE of three independent experiments. (D) NO• stabilization of p21 mRNA is dependent on p38 MAPK. U937 cells (1 × 107) were differentiated with PMA for 8 h. After 30 min pretreatment with actinomycin D (2.5 μg/ml) without and with SB202190 (0.1 μM), cells were further incubated with GSH (400 μM) or GSNO (400 μM) for 2 to 4 h. At the specific time points, cells were harvested for total RNA extraction. Data, presented relative to mRNA level at 0 h (arbitrarily set to 100%), are means ± SE of three independent experiments.