Promoter Methylation Analysis of S100A6 Gene

We conducted a promoter methylation assay as previously described (Yang et al., 2016 (link)). In brief, the putative promoter region of the S100A6 gene was polymerase chain reaction (PCR)‐amplified, followed by digestion with the restriction enzyme HindIII and cloning into the pGL4.21 luciferase expression vector (Promega). The vector was then subjected to in vitro methylation using M. SssI, M. Hhal, and M. Hpall methyltransferase enzymes (Invitrogen), which recognize the sequence patterns CG, CGCG, and CCGG, respectively. These three enzymes catalyze in vitro cytosine methylation at the recognized sequence pattern. Finally, a luciferase assay was conducted with the 293T cells using a Dual‐Glo luciferase reporter assay system kit (Promega) 24 h after transfection.