Fig. 5

Characterization of STET in CBX5 regulation. a Down-regulation of STET mRNA levels by morpholino transfection. MDA-MB-231 cells were transfected with morpholino’s directed against the STET intron and exon boundary or control morpholino. Relative expression was calculated using RT-qPCR using GAPDH expression for normalization. Neg, mock transfected cells; Mo-con, morpholino control transfected cells; and Mo-STET, STET-specific morpholino transfected cells. b STET morpholino transfection has no significant effect on HP1α-V3 expression. MDA-MB-231 cells were transfected with morpholino’s as described in A. A4, A5, and A6 represents amplicons in CBX5 intron 1 described in Fig. 4. Relative expression was calculated using RT-qPCR using GAPDH expression for normalization. c Transient transfection analysis of CBX5 and hnRNPA1 bi-directional promoter activity without and with STET exon insert in dual reporter minigenes in MCF7 and MDA-MB-231 cells. 48 h after transfection RT-qPCR was used to detect relative expression levels of the spliced minigene derived reporter fusion transcripts. Expression of the vector co-expressed neomycin marker was used for normalization for transfection efficiency. Fold changes in expression ratio are shown in the right section. d Genomic transposition analysis of the CBX5 and hnRNPA1 bi-directional promoter activity without and with STET exon insert in dual reporter minigenes in MCF7 and MDA-MB-231 cells. Stable cell lines generated by sleeping-beauty transposition of minigenes were analyzed by RT-qPCR to determine the expression levels of the spliced minigene derived reporter fusion transcripts. Because of copy integration number differences per transposition only the ratio of expression which was copy number independent is displayed. Fold changes in expression ratio are shown. For all panels, bars represent mean values with standard deviations