Figure 4. miRNAs post-transcriptionally regulate the 3'-UTR of SIRT1 mRNA.. (A) Luciferase assays were performed 24 h after transfection of the full-length 1.6 kb SIRT1 3'-UTR downstream of luciferase (SIRT1 3'-UTR) or constructs with 4 bp in the seed-binding regions mutated (SIRT1 3'-UTR 181mt, left panel; SIRT1 3'-UTR 9mt, right panel) and control, miR-181a, b, and c miRNA mimics (left panel) or pSuper and pSuper miR-9 expression constructs (right panel). Data are mean ± s.d. for eight experiments. (B-C) mESCs were transfected with individual miRNA expression constructs; protein and RNA were isolated 48 h later. (B) Repression of SIRT1 protein was analyzed by western blotting. Data are representative of six experiments. (C) qRT-PCR analysis of SIRT1 mRNA levels and mature miRNA levels. Data are mean ± s.d. for four samples.