Fig. 4

HOXD9 regulates RUFY3 transcriptional activity by binding to the RUFY3 promoter. a The vector and HOXD9 plasmid, or Src siRNA and HOXD9 siRNA, were transfected into GC cells. The HOXD9, RUFY3, FOXK1, Vimentin, FHL2 and Cyr61 expression levels were detected in GC cell lines using Western blot analysis. b Deletion or selective mutation analysis identified a responsive transcription factor HOXD9 binding site in the RUFY3 promoter. Truncated and mutated RUFY3 promoter constructs were cotransfected with HOXD9, and relative luciferase activities were determined. ■, wildtype site and □, mutated site. *, P > 0.05, ***, P < 0.01 and ****, P < 0.001. c A ChIP -qPCR assay demonstrated the direct binding of HOXD9 to the RUFY3 promoter in GC cells. Gene enrichment was quantified relative to input controls by qPCR using primers specific for the promoter regions of RUFY3. Results are shown as a fold change of qPCR value over IgG. P > 0.05, **, P < 0.05 and ***, P < 0.01