Fig. 3

APLF regulate EMT. a, b Control and APLF-kd MDAMB-231 cells were investigated for the expression of different genes implicated in EMT. mRNA and protein were extracted and analyzed for the expression of genes by qRT-PCR and western blot respectively. c Chromatin Immunoprecipitation (ChIP) analysis was performed with control and APLF-kd MDAMB-231 cells for the recruitment of MACROH2A.1 at EMT-specific gene promoters. Enrichment of chromatin fragments was measured by qRT-PCR using Sybr green fluorescence relative to a standard curve of input chromatin. IgG was used as the negative control [1]. d Co-expression analysis at the mRNA level between APLF and FOXA1 in samples from TCGA study [5]. Co-expression analysis demonstrated maximal negative correlation of APLF with FOXA1 expression supported by a Pearson score of − 0.48. e Protein was extracted from control and APLF-kd MDAMB-231 cells and analyzed for the expression of FOXA1 by western blot. f ChIP analysis was performed with control and APLF-kd MDAMB-231 cells. The plots represent the recruitment of FOXA1 at CDH1 promoter. IgG was used as the negative control. Enrichment of chromatin fragments was measured by qRT-PCR using Sybr green fluorescence relative to a standard curve of input chromatin. g Expression of EZH2 in control and APLF-kd MDAMB-231 cells at protein level was analyzed by western blot. h ChIP analysis was performed with control and APLF-kd MDAMB-231 cells. The plots represent the recruitment of EZH2 at endogenous FOXA1 promoter. IgG was used as the negative control. I. Same set of cells analyzed in H were investigated for the incorporation of H3K27me3 mark at endogenous FOXA1 promoter. The graph represents the fold enrichment with respect to the input. IgG was used as the negative control. j. Model depicting the mechanism responsible for downregulation of mesenchymal genes and upregulation of epithelial gene CDH1 in response to APLF downregulation in TNBC MDAMB-231 cells. Error bar = S.E.M for three independent experiments. Statistical analyses were performed using Student t-Test function, *p < 0.05, **p < 0.01