Fig. 2

FOXP3 induces the transcriptional activity of miR-200c and miR-141 in breast cancer cells through KAT2B. a Location of human miR-200c and miR-141 on chromosome 12. Forkhead consensus motifs are indicated as black stars, and PCR-amplified DNA fragments are depicted as red boxes below black stars. b Amounts of DNA precipitated, expressed as a percentage of the total input DNA, as determined by chromatin immunoprecipitation (ChIP) analyses of FOXP3-binding sites in the promoter region of miR-200c and miR-141 in FOXP3/GFP-Tet-off MCF7 cells without doxycycline (Dox) at 0 and 48 h. A FOXP3-binding locus up to 20 kb upstream of miR-200c/141, as shown in our ChIP-seq data (Additional file 1: Figure S3B), was used as a positive control for the FOXP3 ChIP assay. c Heat map depiction of alterations in FOXP3 target gene expression in FOXP3/GFP-Tet-off MCF7 cells without Dox at 0 and 48 h generated from Affymetrix Human U133 Plus 2.0 microarrays. The microarray data were submitted to EMBL-EBI (accession number E-MTAB-73). d, e mRNA expression of FOXP3 target genes (measured by quantitative (q)PCR) as a percentage of GAPDH expression in FOXP3/GFP-Tet-off MCF7 cells without Dox at 0, 24, and 48 h. Data are presented as means ± SD. *p < 0.05 vs. 0-h group (one-way analysis of variance (ANOVA) followed by the protected least significant difference test). f Western blot analyses showing protein expression of FOXP3 and its target genes before and after FOXP3 induction in FOXP3/GFP-Tet-off MCF7 cells. g Western blot analyses showing protein expressions of KAT2B and PITX2 in scramble (Scr) or siRNA-transfected cells. h Relative quantification of miR-200c and miR-141 (by qPCR) as percentages of RNU6B in FOXP3/GFP-Tet-off MCF7 cells with scramble or siRNA of FOXP3 target genes at 0, 24, and 48 h. All data in each group were normalized to the scramble control at 0 h. Data are presented as the means ± SD of triplicates. *p < 0.05 vs. 0-h group (one-way ANOVA followed by the protected least-significant difference test). All experiments were repeated three times