FIGURE 9.
IL-6-neutralizing Ab, PGF2α analog, and FP prostanoid receptor antagonist AL-8810 attenuate CLA CM-induced IL-8 gene expression and P-STAT3 levels. A, AD0 and AD50 cultures treated with BSA or CLA CM at a 1:1 ratio of CM to AM-1 for 3 h were analyzed for IL-6 receptor (R), IL-6 signal transducer (ST), GAPDH, and PGF2α receptor expression via qPCR. B, AD50 cultures were pretreated with 0.01 μg/ml IL-6 Ab or 50 μm AL-8810 for 30 min and then treated with 50 μm 10,12-CLA or BSA vehicle for 24 h after which IL-8 mRNA levels were measured. Next, AD0 cultures were treated with AD50 CM for 3 h, and IL-8 mRNA levels were measured. C, AD50 cultures were pretreated with 0.01, 0.1, and 1 μg/ml IL-6 Ab or 1 and 10 μm AL-8810 for 30 min. Then cultures were treated with 50 μm 10,12-CLA or BSA vehicle for 24 h after which CM was collected and added to AD0 cultures for 1 h. Cultures were treated with 0.1 μg/ml recombinant human IL-6 and 10 μm PGF2α (PG) for 30 min as positive controls. D, AD0 cultures were untreated (NT) or pretreated with 0.01, 0.1, 1, or 10 μg/ml IL-6 Ab or 0.5, 5, or 50 μm AL-8810 for 30 min and then treated with 0.1 μg/ml recombinant human IL-6 or 10 μm PGF2α for 30 min, respectively. C and D, cells were harvested and analyzed for levels of phosphorylated and total STAT3, ERK, or JNK via immunoblotting. Means ± S.E. (A, n = 3; B–D, n = 2–3) not sharing a common superscript (a–b) differ significantly (p < 0.05). The data in all panels are representative of three independent experiments. NT, no treatment; PG, PGF2α.