Research Paper Advance Articles

Figure 4. DNMT1 regulates SPINT2 expression via promoter methylation. (A) Schematic representation of SPINT2 promoter constructs used in the luciferase reporter assay, with indicated deletion sites (FL, full-length of −1700 to +300; ΔP1, deletion of −1700 to −839; ΔP1+ΔP2, deletion of −1700 to −268; ΔP2, deletion of −839 to −268). (B) HDFs (DT2) were transfected with pGL4-basic or pGL4-SPINT2 promoter reporter constructs for 4 days, followed by luciferase assay. (C) HDFs (DT2) were transfected with siRNAs targeting negative control (NC) or DNMT1 (siDNMT1) for 24 hours, then transfected with SPINT2 promoter reporter constructs for 2 days. Cell lysates were analyzed for luciferase activity. (D) Schematic representation of CpG islands in the SPINT2 promoter and selected regions used for DNA methylation-specific sequencing (MSS) and methylation-specific PCR (MSP) analyses. (E) MSS analysis of DNA clones from DT2 (early-stage) and DT5 (middle-stage) HDFs. Methylated CpG sites are shown in black, and unmethylated sites in white. Seventeen DNA clones were sequenced per group. (F) Methylation-specific PCR (MSP) analysis of the four CpG methylation hot spots (−445, −366, −363, and −358) identified by MSS. SM indicates DNA size marker. (G) HDFs (DT2) were transfected with siRNAs for negative control (NC) or DNMT1 (siDNMT1) for 3 days and then subjected to MSP analysis against the 4 CpG methylation hot spots.