Research Paper Volume 2, Issue 7 pp 415—431

miRNAs regulate SIRT1 expression during mouse embryonic stem cell differentiation and in adult mouse tissues

Figure 5. Inhibition of miR-9 prevents downregulation of SIRT1 during mESC differentiation.. (A-C) mESCs were differentiated and transfected at d4 and d7 with LNA probes. Protein and RNA were isolated on indicated days. (A) qRT-PCR of miR-9 shows the expected upregulation during differentiation and 35% inhibition when embryoid bodies were transfected with LNA-miR-9 but not with LNA-SCR. (B) qRT-PCR show no significant change in SIRT1 mRNA levels. Data are mean ± s.d. for four samples and representative of three experiments. (C) Western blot analysis shows that the downregulation of SIRT1 protein during mESC differentiation was specifically inhibited in cells transfected with LNA-miR-9 but not by transfection of LNA-SCR or untransfected controls. Data are representative of four experiments. (D-F) EBs were dissociated and transfected at d6 with LNA probes. Protein and RNA were isolated on d11. (D) Western blot analysis shows upregulation of SIRT1 protein in EBs transfected with LNA-miR-9 but not LNA-SCR. (E) qRT-PCR analysis shows inhibition of miR-9 in EBs transfected with LNA-miR-9, but not with LNA-SCR, and no significant change in SIRT1 mRNA levels (F). Data are mean ± s.d. for four samples.