Fig. 5

ACTN2 is a direct target gene of the NF-κB signaling in GC cells. A c-Fos, NFATc1, RelA, Smad3 or Smad4 was cotransfacted with the ACTN2 promoter (-1918/ + 2455) reporter gene (ACTN2 Luc-R) into SNU-16 cells for 12 h and dual-luciferase reporter assays were performed. B ACTN2 promoter 5′ sequential deletion constructs. Fragments of different lengths of the ACTN2 promoter with the same 3′ end were cloned into pGL3-Basic. RelA was co-transfected with each of the ACTN2 promoter-based reporters for 12 h and dual-luciferase reporter assays were performed. C Mutation of putative RelA binding site at the ACTN2 promoter. SNU-16 cells were transfected with wild type or mutant ACTN2 Luc-R for 12 h and dual-luciferase reporter assays were performed. The statistical analysis information in A-C: The bars indicate the SD. The results are expressed as the mean ± SD of three independent experiments. *P < 0.05 using Ordinary one-way ANOVA. D NAGC and HAGC tissue sections obtained after infusion were immunostained for α-Actinin-2 (green), Ser276 phosphorylated RelA (p-RelA, red) and nucleus (blue). E Differences in expression of α-Actinin-2 and p-RelA between HAGC and NAGC, and relationship of overall survival proportion of patients based on univariate analyses. *P < 0.05 using the two-sided Pearson chi-squared tests. F Overall survival proportion of patients with HAGC and NAGC stratified by α-Actinin-2 and p-RelA simultaneous high expression based on Kaplan–Meier survival analyses. HAGC-yes: α-Actinin-2 and p-RelA were simultaneously highly expressed in HAGC sample. HAGC-no: at least one marker in α-Actinin-2 and p-RelA had low expression in HAGC sample. Same to NAGC-yes and NAGC-no. *P < 0.05 using Kaplan–Meier plots and compared with the log-rank test. G and H SUN-16 cells were transfected with or without RelA for 24 h, then the cell nuclear extracts were subjected to ChIP analysis using the p-RelA antibody. G Western blotting analysis with the p-RelA antibody demonstrates the IP specificity and efficiency. H DNA isolated and purified from immunoprecipitated material was amplified with primers spanning the RelA binding site (Fs/Rs) or primers far away from the RelA binding site (Ff/Rf) of the ACTN2 gene promoter