Figure 4. IKKβ-IκBα and p38 signaling axes mediate CXCL5 induction via p65 nuclear translocation–dependent and –independent mechanism, respectively.

(a,b) HMEECs were treated with IKKβ inhibitor (0.5 μM), SB203580 (10 μM) or both for 1 h, followed by stimulation with NTHi for (a) 5 h, and CXCL5 mRNA expression was measured, (b) 12 h, and CXCL5 protein levels in cell culture supernatants was measured by ELISA. (c) HMEECs were pre-treated with CAPE (5, 10 or 25 μg/ml) for 1 h, followed by stimulation with NTHi for 5 h, and CXCL5 mRNA expression was measured. (d) HMEECs were transfected with NF-κB luciferase vector. Cells were pre-treated with CAPE (25 μg/ml) for 1 h, followed by NTHi stimulation for 5 h. NF-κB promoter activity was measured by luciferase assay. (e) HMEECs were transfected with control siRNA or p65 siRNA. Cells were stimulated with NTHi for 5 h, and CXCL5 mRNA expression was measured. Knockdown of p65 protein by siRNA was confirmed by western blot. (f,g) HMEECs were transfected with Mock or p65. Cells were (f) stimulated with NTHi for 5 h, (g) pre-treated with SB203580 (20 μM) for 1 h, followed by stimulation with NTHi for 5 h; and CXCL5 mRNA expression was measured. (h,i) HMEECs were transfected with (h) NF-κB luciferase vector alone, (i) NF-κB luciferase vector and Mock, p38α–DN, p38β–DN or both (p38α–DN and p38β–DN) plasmids. Cells were (h) pre-treated with SB203580 (20 μM) for 1 h and (h,i) stimulated with NTHi for 5 h. NF-κB promoter activity was measured by luciferase assay. (j) HMEECs were pre-treated with CAPE (25 μg/ml) or SB203580 (20 μM) for 1 h, followed by NTHi stimulation for 1 h. p65 translocation was visualized by immunofluorescence by FITC staining. DAPI, nuclear stain. Magnification: 400x. Data are mean ± s.d. (n = 3). (a–c,f,g,i) *p < 0.05, ANOVA (Tukey’s post-hoc). (d,e,h) *p < 0.05, t-test. Displayed immunoblots are cropped images from full-length blots, presented in Supplementary Fig. S2. Data are representative of three or more independent experiments.