Validation of SOX9 methylation. (A) Lollipop diagrams of bisulphite sequencing of SOX9 promoter. Black lollipops are methylated CpGs; white lollipops are unmethylated CpGs. A minimum of five clones were sequenced for each cell culture. (B) mRNA expression of SOX9 normalized to the housekeeping gene RPL28 across 10 melanoma cell cultures. Results are presented as mean +/− s.d., n = 3. Statistical significance of differential expression between the proliferative and invasive phenotype cell cultures was determined by Student’s t-test. (C) Western blot for SOX9 in 10 melanoma cell cultures. GAPDH served as loading control. (D) Western blot for SOX9 of five proliferative melanoma cell cultures treated with 5-aza-2-deoxycytidine (Aza).