Fig. 5

HBXIP co-activates HOXB13 to stimulate IL-6 transcription. a Relative mRNA levels of HBXIP and IL-6 in 34 ER+ clinical breast tumor tissues examined by qRT-PCR assay. The correlation between HBXIP and IL-6 mRNA levels was determined by Pearson’s correlation coefficient. b, c The luciferase reporter gene assay of IL-6 promoter activity in MCF-7 and T47D cells (b) and MCF-7-HBXIP and BT474 cells (c). The cells were transiently transfected with the indicated plasmids or siRNA. The luciferase activities were measured after transfection for 24 h. d ChIP assay in BT474 cells immunoprecipitated with anti-HBXIP antibody or control IgG after being transiently transfected with the si-control or si-HOXB13#2. The lower panel shows the quantitative enrichment data of the IL-6 promoter analyzed by qPCR and normalized against the input. e A similar assay as in d but immunoprecipitated with the anti-HOXB13 antibody or control IgG after being transiently transfected with the si-control or si-HBXIP#1. f Interaction of Flag-HOXB13 with HBXIP was analyzed by Co-IP assay in MCF-7 cells. The cells were transiently transfected with pCMV or pCMV-HOXB13 along with pcDNA-HBXIP. g Interaction of endogenous HBXIP with HOXB13 was examined by Co-IP assay in BT474 cells. h Co-localization of endogenous HBXIP and HOXB13 in BT474 cells was examined by confocal microscopy. Scale bar, 20 μm. i Luciferase reporter gene assay of IL-6 promoter activity in MCF-7 cells transiently transfected with pCMV or pCMV-HBXIP along with the indicated si-control or si-HOXB13#2. j Luciferase reporter gene assay of IL-6 promoter activities in MCF-7 cells. The cells were transiently transfected with pCMV or pCMV-HBXIP along with the IL-6 promoter (WT) or constructs with mutated binding sites of HOXB13-site1 (− 270/− 251, called H13-1-M) or HOXB13-site2 (− 135/− 121, called H13-2-M). All experiments were repeated at least three times. Error bars represent ± SD. **P < 0.01 and ***P < 0.001 by two-tailed Student’s t test