Fig. 5

BRD4 transcriptionally regulates Snail via Gli1. Cells were transfected with si-NC or si-BRD4 for 24 h, the mRNA expression of Snail was checked by qRT-PCR; a Cells were transfected with pGL3-Basic-Snail-luc reporter and pRL-TK plasmid for 24 h and then treated with JQ1 (1 μM) for increasing time periods. Results were expressed as the ratio between the activity of the reporter plasmid and pRL-TK; b Cells were treated with JQ1 (1 μM) for increasing time periods. Pre-mRNA of Snail was checked by qRT-PCR; c MDA-MB-231 cells were treated with JQ1 (1 μM) for 4 h. The expression of transcription factors regulating Snail transcription was checked by qRT-PCR; d MDA-MB-231 cells were treated with JQ1 (1 μM) for increasing time periods. Protein of Gli1 was checked by western blot analysis; e Cells were transfected with siNC or si-BRD4 for 24 h. The mRNA expression of Gli1 was checked by qRT-PCR; f Cells were transfected with siNC or si-BRD4 for 24 h. The Gli1 in subcellular fractions was checked by western blot analysis; g The expression of Gli1 in paraffin-embedded sections obtained from xenografts was measured by IHC (left) and quantitatively analyzed (right); h The expression of Gli1 in paraffin-embedded sections obtained from xenografts was measured by IHC (left) and quantitatively analyzed (right); i, j MDA-MB-231 cells were transfected with pcDNA (vector) or pcDNA/Gli1 for 24 h and then treated with or without JQ1 (1 μM) for 24 h. i Protein expression was checked by western blot analysis. j Wound healing was recorded (left) and quantitatively analyzed (right); k Position in relation to the transcription start site and sequence homology to the Gli1 consensus binding sequence for putative Gli‐binding sites in the human SNAI1 promoter. Red bases represent those different from the Gli‐binding consensus sequence; l Binding between Gli1 transcriptional factor and the promoter of SNAI1 at the potential binding site “a” to “f” or negative site “BLK” was checked by ChIP-PCR; m Schematic representation of the mutated promoter in pGL3-Basic-Snail-luc reporter to investigate the role of Gli1 in Snail expression; n MDA-MB-231 cells were transfected with pGL3-Snail-WT-Luc, pGL3-Snail-eMut-Luc, pGL3-Snail-fMut-Luc, and pRL-TK plasmid for 24 h and then treated with or without JQ1 (1 μM) for 12 h. Results were expressed as the ratio between the activity of the reporter plasmid and pRL-TK. Data are expressed as mean±SD and similar results were obtained from three independent experiments