Figure 3

Sox2 binds with HAMP promoter to regulate its expression. (A) Two putative Sox2 binding sites were indicated with open boxes. (B) Schematic diagram of a series of HAMP promoter luciferase reporter constructs and putative Sox2 binding sites were marked. (C) Huh7 cells were cotransfected with various HAMP promoter luciferase reporter plasmids and the internal control plasmid pRL-TK, together with Myc-Sox2 or not. Forty-eight hours later, the cell lysates were subjected to dual luciferase assay, and the results were expressed as fold induction of luciferase activity relative to the empty vector without Myc-Sox2 expression. The error bar represented three replicates. (D) Huh7 cells transfected with Myc-Sox2 for 48 h were cross-linked, and sonicated to generate chromatin fragments. The sheared chromatins were immunoprecipitated with monoclonal antibodies against Myc or a mouse IgG isotype control followed by qPCR analysis using the primer set flanking the HAMP promoter. The data were expressed as the percentage of immunoprecipitated chromatin DNA versus the total input. The experiments were repeated for three times with similar results and data were shown as means ± SE. Statistical analyses were conducted using a one-way ANOVA with Tukey’s multiple comparison test to determine individual p-values (C) or an unpaired, two-tailed student t-test (D). Significant differences are indicated by *p < 0.05.