Fig. 3.

Effects of Stattic, a STAT-3 inhibitor, on the induction of inflammatory mediators in Beas2B cells. Beas2B cells were incubated with medium [control (Ctrl)] or medium containing 5 µM Stattic for 1 h and then treated with or without dust extract (DE, 0.25%) for 3 h. A: levels of mRNAs were determined by real-time quantitative RT-PCR and normalized to 18S rRNA levels. Levels of mRNAs in DE-treated cells were arbitrarily considered as 100, and relative levels in treated cells are shown. Data shown are means ± SE (n = 4). **P < 0.01 compared with cells treated with DE alone according to one-way analysis of variance using Tukey’s multiple-comparison test. CCL2, chemokine (C-C motif) ligand 2; TLR4, Toll-like receptor 4. B–E: effects of Stattic on the protein levels of prostaglandin G/H synthase 2 (PTGS2), ICAM-1, and pro-IL-1β were determined by Western blot analysis, and protein levels were normalized to actin. Protein levels in control cells were arbitrarily considered as 1, and relative levels in treated cells are shown. Data shown are means ± SE (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant, according to one-way analysis of variance using Tukey’s multiple-comparison test. F–H: IL-8, IL-6, and TNF-α protein levels in cell medium were determined by ELISA. Data shown are means ± SE (n = 5 for IL-8 and TNF-α; n = 4 for IL-6). *P < 0.05 and **P < 0.01 according to one-way analysis of variance using Tukey’s multiple-comparison test. I: Beas2B cells were transfected with IL-8 promoter plasmid containing −133/+44 bp of human IL-8 promoter sequence linked to luciferase reporter gene. Transfected cells were incubated first with medium (Ctrl) or Stattic (5 µM) for 1 h and then treated with or without DE (0.25%) for 6 h. Luciferase activities in cell extracts were normalized to total cell protein. Luciferase activity in control cells was arbitrarily considered as 1, and relative levels in treated cells are shown. Data shown are means ± SE (n = 3). ****P < 0.0001 according to one-way analysis of variance using Tukey’s multiple-comparison test. J: effects of Stattic on cell viability of DE-treated Beas2B cells. Beas2B cells were incubated with medium or medium containing 5 µM Stattic for 1 h and then treated with 0.25% DE for 3 h. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Absorbance in control cells was arbitrarily considered as 100, and relative levels in treated cells are shown. Data shown are means ± SE (n = 5); ns, not significant according to one-way analysis of variance using Tukey’s multiple-comparison test.