Effect of LY294002 on the stability of mRNA and transcriptional rate in acid SMase and GCS. KHYG-1 cells were treated with (LY294002 + IL-2⁺) or without (IL-2⁺) 30 μM LY294002 in the presence of IL-2 (100 U/mL) after IL-2 deprivation. A newly synthesized mRNA was blocked by addition of 10 μg/mL actinomycin D 10 minutes before IL-2 supplementation, and then the cells were harvested at the indicated time (0, 1, 2, 3, and 4 hours). Northern blot analysis was performed to detect the changes of acid SMase (A) and GCS mRNA levels (B) as described in “Materials and methods.” The amount of 18 S ribosomal RNA was visualized by bromide staining under UV illuminator to confirm the equal amounts of loading (C). KHYG-1 cells were also cultured in the presence (D) or absence (E) of IL-2 (100 U/mL) for 20 hours or rescued with IL-2 supplementation (100 U/mL) after 12 hours of deprivation and further cultured for 8 hours without (F) or with (G) 30 μM LY294002. LY294002 was added 1 hour before IL-2 supplementation. The nuclear run-on assay was performed to assess the transcriptional rate of acid SMase, GCS, and β-actin genes as described in “Materials and methods.” The results were representative of 2 independent experiments. Dot intensity was measured by NIH image, and the average of relative intensity of acid SMase and GCS to β-actin was plotted under each panels. The bars indicate 1 SD.