Pyrosequencing analysis for ARHI methylation. (a) Design of pyrosequencing primers for ARHI CpG islands I and II. The vertical bars indicate CpG sites in PCR products. COBRA indicated the restriction enzyme cutting site. (b) Pyrosequencing results for ARHI CpG island II methylation in MB-MDA-231 cells using two detection primers (Pyro F1 and F2). Methylation levels ranged from 65% to 100%, with a mean of 84%. Gray highlighting marks the CpG sites tested and the percentage reading of C and T. COBRA, combined bisulfite restriction analysis.