Fig. 5

C12orf59 activates CDH11 expression through NF-κB signaling. a Luciferase reporter assay was performed in C12orf59 overexpressed GC cells and control cells, respectively (right). Remarkable activation of NF-κB signaling pathway is observed after C12orf59 was overexpressed. b Western blotting showed increased expression level of nuclear NF-κB p65 after overexpression of C12orf59 in GC cells. c Schematic illustration of CDH11 promoter. One potential NF-κB p65 binding sites was shown. d ChIP assay showed the binding to NF-κB p65 to CDH11 promoter in vivo. The promoter region of CDH11 was amplified from the DNA recovered from the immunoprecipitation complex using a specific antibody for p65. The following PCR primers for ChIP assays were used: 5′- GTCCTTGGCGTGATTCCTAA-3′, 5′- TACGGGAGAAAGGGCCTAAT-3′. e Luciferase reporter assay of CDH11 promoter showed remarkable activation is observed when C12orf59 was overexpressed. f Western blotting showed decreased expression level of CDH11 after transfection of sip65 in HGC-27/C12orf59 cells. g Wound-healing assays showed that sip65 transfection inhibited the migratory ability of C12orf59-overexpressing HGC-27 cell. h Transwell analysis indicated that enhanced the migratory and invasive abilities of C12orf59-overexpressing HGC-27 cell were largely compromised after treatment with sip65. i Western blotting shows that after sip65 transfection in HGC-27/C12orf59 cells, the levels of E-cadherin and α-catenin increased, whereas the levels of fibronectin and vimentin decreased. j IF staining demonstrates an upregulated expression of E-cadherin and a downregulated expression of vimentin in HGC-27/C12orf59 cells, after treatment with sip65. k and l. sip65 transfection largely attenuate the enhanced effects of C12orf59 overexpression on the abilities of capillary tube formation (k) and in vitro migration (l) of HUVECs