Fig. 1

The down regulation of ALX4 in breast cancer is caused by promoter methylation. a Analysis of ALX4 mRNA expression levels in normal breast tissues and breast cancer cells using RT-PCR and qPCR. ALX4 was down regulated in breast cancer cells compared with normal breast tissues. b The protein expression of ALX4 was detected in normal breast tissues and breast cancer cells. c Down regulation of ALX4 in breast cancer patients was further confirmed using TCGA database. ALX4 mRNA expression in the TCGA breast cancer RNAseq (IlluminaHiSeq; n = 1208, normal tissue = 111, breast cancer tissue = 1097) data sets. Error bars indicate s.d. ** P < 0.01. d ALX4 promoter methylation was analyzed by MSP method. ALX4 gene promoter was methylated in breast cancer cell lines, unmethylated in all normal breast tissues and frequently methylated in breast tumor tissues. M: PCR product amplifed by methylated -specific primers; U: PCR product amplifed by unmethylated specific primers; N: normal tissue; T: tumor tissues; PC: positive control, including fully unmethylated or fully methylated control; NC: negative control. e Methylation status of ALX4 promoter region of normal breast tissue and breast cancer cell lines was analyzed by BSP method. CpG islands were represented as open dots (unmethylated) or filled dots (methylated). f Re-expression of ALX4 in MDA-MB-231 and MCF-7 cell lines after treatment by pharmacologic demethylation. −: DMSO control; +: 5-aza-dC