Figure 5.

WT and mutant PAM promoter activity. A 5 kb PAM promoter-5’UTR WT sequence was cloned upstream of a luciferase reporter. Six mutant constructs were created by site-directed mutagenesis. The constructs were transiently transfected into HEK-293AD cells together with a Renilla luciferase reporter for normalization. Firefly and Renilla luciferase activities were measured 24 h post-transfection. Two SNVs, c.-361G>A and c.-133T>C, have significantly lower transcriptional activity than the WT hybrid transcript. Mock, empty pRMT-Luc vector. Differences between experimental groups were analyzed by one-way ANOVA with Dunnett’s post hoc test, using WT as the control group. RLU, relative luciferase activity. *p = 0.0443; **p = 0.0019; ***p = 0.0005.