Fig. 8

Thrombin stimulates YAP and p65 formation in A549 cells. A A549 cells were transfected with pGL2-ELAM-κB-Luc (0.5 μg) and pBK-CMV-lacZ (0.5 μg), control (con siRNA), or DCLK1 siRNA, or YAP siRNA for 48 h before being incubated for 16 h with thrombin (10 U/ml). Cells were harvested for a luciferase activity assay (n = 3, mean ± SEM, *p < 0.05 vs thrombin-treated cells). B A549 cells were collected and immunoprecipitated with anti-NF-κB p65 antibody after being stimulated for 30 min with thrombin (10 U/ml). Immunoblotting was performed to detect the immunoprecipitated complex with anti-YAP and anti-NF-κB p65 antibodies. The y7immunoprecipitated complex was identified using immunoblotting with anti-YAP and anti-NF-κB p65 antibodies (n = 3). C A549 cells were transfected with control (con siRNA) or DCLK1 siRNA for 48 h and subsequently collected and immunoprecipitated with anti-NF-κB p65 antibody after being stimulated for 30 min with thrombin (10 U/ml). Immunoblotting was performed to detect the immunoprecipitated complex with anti-YAP and anti-NF-κB p65 antibodies. The immunoprecipitated complex was identified using immunoblotting with anti-YAP and anti-NF-κB p65 antibodies (n = 3). D A549 cells were treated with 10 U/m thrombin for 30 min. Cell lysates were used for ChIP analysis with antibodies against YAP or p65. PCR was used to detect YAP or p65 binding on IL-8/CXCL8 promoter. Input, a positive control. IgG, a negative control (n = 4). E A549 cells were transfected with control (con siRNA) or DCLK1 siRNA for 48 h and subsequently the cell lysates were used for ChIP analysis with antibodies against YAP or p65. PCR was used to detect YAP or p65 binding on IL-8/CXCL8 promoter. Input, a positive control. IgG, a negative control (n = 3). F RhoA, YAP, and p65 mRNA expression levels after transfection of A549 cells with control (con siRNA) or DCLK1 siRNA (n = 3–4, mean ± SEM, *p < 0.05 vs control)