Fig. 4

CAFs activated the transcription of HOTAIR by SMAD2/3/4. a Western blotting analysis of the effect of recombinant TGF-β1, CAF-CM, SB or PFD treatment on the p-SMAD2, p-SMAD3 and p-SMAD4 in MCF-7 and MDA-MB-231 cells. b The HOTAIR expression upon treatment of siRNA against SMAD2, SMAD3, and SMAD4 was determined by real time PCR. c SMAD2/3/4 were enriched in the promoter of HOTAIR in CAF-CM-treated MDA-MB-231 and MCF-7 cells, as determined by a CHIP assay. d A bioinformatics assay showed the predicted binding sites of SMAD2/3/4 within the HOTAIR promoter region. e A mutational analysis of HOTAIR promoter activity. The wild-type and mutant promoter constructs were co-transfected with SMAD2, 3, or 4 plasmids into MCF-7 and MDA-MB-231 cells. The promoter activity was determined by a luciferase assay