FIGURE 2.
The role of ASM in the regulation of IL-6 RNA in MCF-7. A, MCF-7 cells were treated with 20 nm siRNA for 48 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.” -Fold change was calculated based on the untreated, control (Con) siRNA group (n = 3). B, MCF-7 cells were treated with siRNA as before, stimulated with PMA for 90 min, and then treated with 5 μg/ml actinomycin D for a 3-h time course, including time points of 0, 1, 2, and 3 h. RNA was harvested, cDNA was prepared, and IL-6 mRNA was measured by qPCR. The remaining amount of mRNA was calculated by normalizing each group to the values at t0 of actinomycin D. Values represent calculated t½ for each sample group (n = 2). C, MCF-7 cells were treated with 20 nm siRNA for 48 h and then stimulated with PMA for 90 min. As in A, cDNA was prepared, but qPCR was performed utilizing primers specific for IL-6 hnRNA (n = 3). D, MCF-7 cells were treated with either vehicle or PMA for 90 min, cDNA was prepared, qPCR was performed for IL-6, and CCL5/RANTES mRNA was expressed as -fold change from control group (n = 5). E, MCF-7 cells were treated with the indicated agents for 90 min, and RNA was harvested for qRT-PCR with primers specific to IL-6 (n = 3). F, MCF-7 cells were treated with BIRB796 and PMA for 90 min, and whole cell lysates were prepared. Equal protein was loaded in each lane of a 4–12% SDS-polyacrylamide gel and probed for total p38 or phospho-p38 (p-p38), as indicated. Error bars, S.E.