Fig. 6
LPS promotes HK3 expression through inducing NF-κB/Snail protein complex formation and then binding to HK3 promoter region. a Quantification of the fold change of luciferase reporter assays of HK3 promoter activity. HEK239T cell was pretreated with DMSO or NF-κB inhibitor JSH-23, and then stimulated with LPS. Luciferase reporter gene plasmid containing the promoter region of HK3 was constructed (HK3-luc-WT). Cells were transfected with HK3-luc-WT plasmid DNA vector using LipoD293 transfection reagent. HK3 promoter activity was enhanced with LPS treatment, and NF-κB played an essential role during the process. b Quantification of the fold change of luciferase reporter assays of HK3 promoter in Snail-knockdown (shSnail) and control CRC cells (Scrb) with or without LPS treatment. HK3 promoter activity was enhanced with LPS treatment and Snail played an essential role during the process. c Luciferase reporter assays of HK3 promoter with mutant or deletion in predicted NF-κB binding site of HK3 promoter region. Luciferase reporter gene plasmid HK3-luc-WT contains the promoter region of HK3. A series of luciferase reporter gene plasmids encoding HK3 promoter region deletions (HK3-Luc-deletion) and mutants (HK3-Luc-mut) were constructed. All deletions and mutations of HK3-luc in the expression vector pGL3-basic were created using Mut Express II fast mutagenesis kit. d ChIP assay was carried out using ChIP assay kit to detect whether P65 or Snail could directly bind to HK3 promoter region. Combined HK3 DNA was quantified by real-time PCR. Fold enrichment was calculated relatively to the ChIP IgG control. e The protein interaction between P65 and Snail was confirmed by co-immunoprecipitation. Cells were treated with or without LPS, and cell lysates were prepared in IP lysis buffer. The indicated proteins were detected by western blotting. f The location of LPS-induced P65/Snail protein complex was confirmed by immunofluorescence. g ChIP-Re-ChIP assays were performed in LPS treated or untreated cells to examine whether P65 and Snail jointly regulate HK3 promoter activity. ChIP-Re-ChIP chromatin level was determined by PCR analysis. The PCR products were separated on a 1% agarose gel containing ethidium bromide for visualization and analysis. Data are presented as mean±SEM. *P<0.05, **P<0.01, ***P<0.001