Fig 2: AF9 Interacts with Snail and Hampers Snail Transcriptional Activity(A) Transcription factor (TF) enrichment of AF9 target genes demonstrated that most genes were potentially regulated by SNAI1 transcription factor, with q value less than 0.0001 considered statistically significant. Description of this method is provided in the Materials and Methods section. (B) Mass spectrometry analyses of AF9-associated proteins in MDA-MB-231 cells. The data are presented by a volcano plot. Proteins with a p value <0.0001 and Log2 (FLAG-AF9 versus FLAG) >2.0 were regarded as candidate proteins that significantly interact with AF9. The enlarged red point indicates Snail; other significant proteins are marked as green points. All significant proteins are listed in the larger circle. (C) In MDA-MB-231-FLAG-AF9 OE cells, M2 FLAG beads was used to immunoprecipitate the AF9 associated complex. Western blot analysis was performed using the indicated antibodies. (D) HA-Snail and FLAG-AF9 (WT or Y78A) were cotransfected into MDA-MB-231 cells. Antibody against FLAG was used to immune-precipitate the FLAG-AF9 associated complex. Western blot analysis was performed to test the interaction between Snail and AF9 using the indicated antibodies. (E) Separate deletion of three Snail regions (N-terminal, middle domain, and C-terminal), as shown in the upper panel. FLAG-AF9 and HA-Snail (including WT, dN ter, dM, and dC ter) were cotransfected into MDA-MB-231 cells. Antibody against HA was used to immunoprecipitate HA-Snail full length and truncated Snail associated complex. Ter, terminal; M, middle. (F) Snail mRNA expression and protein levels were tested in MDA-MB-231-Vehicle and MDA-MB-231-AF9 OE (WT or Y78A) cells. (G) Snail transcriptional activity was determined in MDA-MB-231-Vehicle and MDA-MB-231-AF9 OE (WT or Y78A) cells by a dual luciferase assay. These cells were cotransfected with the pGL3-CDH1 promoter (-57 to +149 relative to TSS) and Renilla control plasmid pRL-TK for 48 h. The relative luciferase activities were normalized to those of the cells expressing vehicle and then those of the Renilla control. The data represent the mean ± SD of three independent experiments. (H) ChIP assay was performed in MDA-MB-231-AF9 OE (WT or Y78A) cells with indicated the antibodies. Primers against the promoter regions of CDH1, CLDN3, VDR, and CXADR were used to perform quantitative real-time PCR to measure the binding affinity of Snail. The calculation of the relative binding affinities of Snail to the target regions is described in the methods section. (F–H) Two-tailed Student’s t test, **p < 0.01, ***p < 0.001. ns, not significant. Error bars represent the standard deviation (SD).