Compound A inhibits IL-5 promoter activity and cytokine production.

<p><i>A,</i> EL4 cells were cotransfected with 9 µg of IL-5-Luc reporter plasmid plus 9 µg of GR and GATA-3 expression vectors. After 16 h, cells were stimulated for 5 h with CpdA (10 µM) and cAMP. Also, EL4 cells were pretreated during 1 h with p38 inhibitor, SB203580 (10 µM). n = 6, * p<0.001 vs. GATA-3 without cAMP and CpdA, ** p<0.001 vs. GATA-3 with cAMP and without CpdA, *** p<0.001 vs. GATA-3 with cAMP and CpdA, # p<0.001 vs. GATA-3 with cAMP and SB203580 and without CpdA. <i>B,</i> EL4 cells were cotransfected with 9 µg of the wild-type or GATA-3 mutated-binding site on the IL-5 promoter (IL-5-Luc-WT and IL-5-Gm-Luc) plus 9 µg of GR and GATA-3 expression vectors. After 16 h, cells were stimulated for 5 h with CpdA and cAMP. n = 6, * p<0.001 vs. GATA-3 without cAMP and CpdA on IL-5-Luc WT, ** p<0.001 vs. GATA-3 with cAMP and without CpdA on IL-5-Luc WT, *** p<0.001 vs. GATA-3 with cAMP and CpdA on IL-5-Luc WT, # p<0.05 vs. GATA-3 with cAMP and without CpdA on IL-5-Gm-Luc. <i>C,</i> A20 cells were cotransfected with 9 µg of IL-5-Luc reporter plus 9 µg of GR and GATA-3 expression vectors. After 16 h, cells were stimulated for 5 h with CpdA and cAMP. n = 6, * p<0.001 vs. basal, ** p<0.001 vs. GATA-3, *** p<0.001 vs GATA-3 with cAMP. <i>D,</i> Purification of T cells was achieved as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035155#s4" target="_blank">Materials and Methods</a>. Cells were activated with PMA (P), Ionomycin (I) and cAMP during 24 h and then with CpdA for 5 h. Supernatants were used to measure mouse IL-5 by ELISA. n = 4, * p<0.05 vs. basal without P+I, ** p<0.001 vs. P+I without cAMP, *** p<0.001 vs. P+I and cAMP, of one representative experiment of three independent experiments with similar results. For the transfections experiments, results, as folds, normalized to β-galactosidase activity, are expressed as mean ± SEM, averaged from three independent experiments.