FIGURE 1.

Regulation of IL-6 and p38 by sphingolipid salvage enzymes in MCF-7. A, PKC inhibitors Gö6976 (3 μm) and bisindoleilamide (Bis; 3 μm) were added to subconfluent MCF-7 cells for a 1-h pretreatment followed by PMA (100 nm) treatment for 18 h. IL-6 was measured by ELISA in cultured media as described under “Experimental Procedures” (n = 2). B, MCF-7 cells were treated with 20 nm siRNA for the indicated enzymes for 48 h and then stimulated with PMA for 18 h; IL-6 in cultured media was measured by ELISA (two-way analysis of variance, Bonferroni's post-test. **, p < 0.01; ***, p < 0.001 compared with control (Con) siRNA + PMA, n = 3). C, MCF-7 cells were treated with 20 nm siRNA for 48 h and stimulated with PMA (100 nm) for 30 min. Whole cell lysates were prepared, and equal amounts of protein were subjected to Western blotting for phospho-p38 and p38. Results are representative of two experiments. D, MCF-7 cells were cotreated with siRNA for the above enzymes for 48 h, followed by PMA treatment for 18 h. -Fold change was calculated to either control siRNA or ACD siRNA alone for each group (n = 2). E and F, MCF-7 cells were treated with 20 nm various siRNAs as labeled for 48 h. Cells were harvested either for qRT-PCR (E) or immunoblotting (F) as described under “Experimental Procedures.” Equal amounts of protein were loaded for siRNA validation for GBA and ACD (F). Error bars, S.E.