Fig. 3

Fn induces BIRC3 expression through TLR4/NF-kb pathway. a Cells were cocultured with or without Fn for 2 h followed by incubation with an antibody against NF-κB P65 and a Cy3 fluorescein-conjugated secondary antibody, and nuclei were stained with DAPI. b Cells were transfected with specific siRNA (si-P65 or si-NC) for 24 h followed by cocultured with Fn for an additional 6 h or 24 h. Protein levels of BIRC3, p-P65, and P65 were measured by Western blot. c Schematic representation of the location of two predicted NF-κB P65 binding motifs in the BIRC3 promoter region. WT: wild-type; MT1: mutant type 1; MT2: mutant type 2. d pGL3-Promoter reporter plasmids (NC, WT, MT1, MT2) and pcDNA3.1-TF-cDNA vectors (TF-NC, TF-P65) were generated. Dual luciferase reporter assay were carried out according to different combinations. TF: transcription factor; NS: nonsignificant. e Cells were cocultured with or without Fn for 2 h. Then, ChIP assays were carried out with an anti-P65 antibody or rabbit IgG. qPCR was then carried out for the BIRC3 promoter region or the region 5 kb upstream of BIRC3 gene. f Cells were co-cultured with or without Fn for 24 h. The relative mRNA level of TLR4 and MyD88 was detected by qPCR. g Cells were transfected with specific siRNA (si-TLR4 or si-NC) for 24 h followed by co-cultured with Fn for an additional 48 h. Protein levels of TLR4, MyD88, and BIRC3 were measured by Western blot. (***p < 0.001 by unpaired Student’s t-test)