Figure 3

NF-κB is capable of binding to the KCNN4 gene promoter. A) Western blotting for KCNN4 of TAMs that were pretreated with or without BAY11-7082. Bars correspond to the mean ± SD, **p < 0.01, compared with no BAY11-7082 treatment. B and C) siRNAs were used to specifically inhibit p50 and p65. Bars correspond to the mean ± SD, **p < 0.01, compared with Control (non-transfected). D) Western blotting for KCNN4 of TAMs cocultured with LoVo-P cells (Control), and TAMs pretreated with Lipo2000(Lipo), p50-siRNA (p50-si), p65-siRNA (p65-si) before coculturing with LoVo-P cells. Bars correspond to the mean ± SD, **p < 0.01, compared with Control (non-transfected) and Lipofectamine 2000. E) Luciferase reporter assay demonstrated the influence of NF-κB on KCNN4 promoter activity. TAMs were cotransfected with KCNN4-promoter-luciferase plus pRL-TK-luciferase; KCNN4-promoter (mut) plus pRL-TK-luciferase; or pGL-3-basic plus pRL-TK-luciferase. Luciferase activity in cell extracts was analyzed by the Dual-Luciferase Reporter Assay System and normalized using pRL-TK-luciferase activity in each sample. Bars correspond to the mean ± SD, *p < 0.05, **p < 0.01, compared with TAMs transfected with pGL-3-basic. F) ChIP-qPCR assay confirmed that the transcription factor NF-κB can specifically bind to the regulatory region of KCNN4 in TAMs. Bars correspond to the mean ± SD, **p < 0.01, compared with isotype-matched IgG control (IgG).