Fig. 3: ARSD exhibits lower expression in MDA-MB-231 cells due to that it is still subjected to XCI.

A Xist expression levels of MCF-7 and MDA-MB-231 cells are tested by qRT-PCR. B Validation of Xist knockdown efficiency by qRT-PCR. C ARSD mRNA and protein expression levels are tested after Xist knockdown. D ARSD promoter occupancy in MCF-7 and MDA-MB-231 cells is detected using ChIP-qPCR with D-a EZH2, D-b 5-mC, D-c H3K27me1, D-d H3K27me2, and D-e H3K27me3 specific antibodies, respectively. Light blue and dark blue bars indicate loci in MCF-7 and MDA-MB-231 cells, respectively. Experiments were duplicated and two data sets are concordant (R2 = 0.92). The ratios of enrichment of EZH2, 5mC, H3K27me, H3K27me2, and H3K27me3 were higher in MDA-MB-231 cells than that in MCF-7 cells. Error bars are ±SEM (*p < 0.05, **p < 0.01, ***p < 0.001). E A negative correlation exists between ARSD mRNA and ARSD methylation (HM27). F The probes Cg3949008, Cg 23547143, Cg13324949, and Cg04710661 were used to evaluate and compare the methylation levels of ARSD gene promoter between normal and tumor tissue (http://www.bioinfo-zs.com/smartapp/). G ARSD promoter is analyzed by using Methyl Primer Express Software v. 1.0. H MSP analysis of the methylation status of the ARSD promoter. “U” indicates unmethylated amplification, and “M” indicates methylated amplification. Lower panel shows MDA-MB-231 cells were, respectively, treated with 5-Aza or RG108 for 5 days prior to DNA isolation. I Synopsis of ARSD gene subjected to XCI in MDA-MB-231 cells instead of that in MCF-7 cells. Data are presented as the means ± SD of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student’s t-test) as compared to control cells.