Fig. 4

Thrombin induces IL-8/CXCL8 promoter activity via DCLK1 in A549 cells. A A549 cells were transfected with IL-8/CXCL8 wt-Luc (0.2 μg) and pBK-CMV-lacZ (0.1 μg), control (con siRNA), or DCLK1 siRNA for 48 h before being incubated for 16 h with thrombin (10 U/ml). Cells were collected for a luciferase activity assay (n = 6, mean ± SEM, *p < 0.05 vs thrombin-stimulated cells). B A549 were transfected with DCLK1 siRNA or control (con siRNA) for 48 h. We performed qPCR analysis to determine the mRNA expression of DCLK1 (n = 3, mean ± SEM, *p < 0.05 vs control group). C A549 cells were transfected with pcDNA or DCLK1 KD or DCLK1 WT plasmid for 48 h, and the level of IL-8/CXCL8 release was verified (n = 3, mean ± SEM, *p < 0.05 vs DCLK1 KD group, vs pcDNA group). D A549 cells were transfected with pcDNA or DCLK1 KD or DCLK1 WT plasmid for 48 h. Immunoblotting was performed to determine the expression level of DCLK1 (n = 3, mean ± SEM, *p < 0.05, vs pcDNA group, vs DCLK1 KD group). E A549 cells were induced by thrombin (10 U/ml) for 0–60 min. Levels of phospho-DCLK1 Ser30 were evaluated using Western blot (n = 5, mean ± SEM, *p < 0.05 vs non-stimulated cells)