FIGURE 4.

Regulation of p38/IL-6 by ASM in HeLa cells. A, HeLa cells were treated with control or ASM siRNA for 48 h, followed by 18 h of PMA treatment. IL-6 in cultured media was measured by ELISA. B and C, HeLa (B) or MDA-MB-231 cells (C) were treated with 50 μm desipramine or PBS control (Con) for 1 h and then stimulated with PMA and TNFα for 3 h. IL-6 in cultured media was measured as above (n = 2). D, HeLa cells were treated with the indicated siRNA for 72 h and then stimulated with TNFα (20 ng/ml) for 15 min. Whole cell lysates were subjected to Western blotting for phospho-p38 (p-p38) and p38. Equal amounts of protein were loaded (representative of two experiments). E, HeLa cells were pretreated with 25 μm desipramine for 1 h and treated with TNFα (20 ng/ml) for 15 min. Whole cell lysates were prepared, and equal amounts of protein were subjected to Western blotting for phospho-p38 and p38 (representative of three experiments). F, HeLa cells were pretreated with PBS or desipramine (50 μm) for 1 h and then stimulated with PMA for 90 min. Next, cDNA was synthesized, and qPCR was performed for IL-6 mRNA and normalized to actin as described under “Experimental Procedures” (n = 3). G, HeLa cells were treated with ASM siRNA for 48 h. RNA and cDNA were harvested and prepared, and qPCR was performed for ASM mRNA and normalized to actin (n = 3). H, HeLa cells were transfected as previously for 48 h, and then lysates were harvested, and acid sphingomyelinase activity was measured as described under “Experimental Procedures” (n = 3). Error bars, S.E.