Research Paper Volume 7, Issue 12 pp 1143—1155

Figure 3. Methylation by NSun2 is functional for regulating the translation of p27. (A) Schematic representation of the pGL3-derived reporter vectors used for reporter gene assays. (B) HeLa cells were transfected with each of the reporter vectors described in Fig. 3A together with a pRL-CMV control reporter. Twenty-four hours later, cells were further transfected with NSun2 siRNA and cultured for an additional 48 h. Firefly luciferase activity against Renilla luciferase activity was analyzed. Data represent the means ± SD from 3 independent experiments; significance was analyzed by Student's t test. (C) In vitro methylated (Met) or unmethylated (Unmet) Luciferase (Luc), luc-5′UTR, and luc-5′UTRΔ reporter transcripts were used for in vitro translation assays. Firefly luciferase activity was measured to determine the translation efficiency. Data represent the means ± SD from 3 independent experiments; significance was analyzed by Student's t test. (D) Cells described in Fig. 1A were used for isolating the polysomal and non-polysomal fractions. RNA prepared from the fractions was subjected to RT-qPCR analysis to assess the presence of p27 mRNA in the polysomal and non-polysomal fractions. Data represent the means ± SD from 3 independent experiments; significance was analyzed by Student's t test.